A non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode
The non-competitive lateral flow immunoassay method using AIE nanoparticle dual-signal mode solves the problem of low sensitivity in colloidal gold lateral flow immunoassay, achieving high sensitivity and high specificity for pesticide residue detection, and is suitable for rapid detection of trace pesticides in agricultural products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF AGRI QUALITY STANDARDS & TESTING TECH HENAN ACAD OF AGRI SCI
- Filing Date
- 2026-04-24
- Publication Date
- 2026-06-26
AI Technical Summary
Existing colloidal gold lateral flow immunoassay methods have low sensitivity, making it difficult to meet the detection requirements of trace pesticide residues. Furthermore, traditional lateral flow immunoassays lack sufficient sensitivity and specificity in complex sample matrices.
By employing the dual-signal mode of AIE nanoparticles, AIE nanoparticles PSNPs@AIE650 were prepared and bound to the anti-immune complex peptide AICP to construct a non-competitive lateral flow immunoassay method, enabling detection by both colorimetric and fluorescence signals.
It significantly improves the sensitivity of pesticide residue detection, with a fluorescence quantitative detection limit as low as 0.23 ng/mL. It has high specificity and rapid and portable detection capabilities, and is suitable for the rapid detection of trace pesticides in complex agricultural product matrices.
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Figure CN122283124A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural product testing technology, specifically to a non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode. Background Technology
[0002] Real-time testing of agricultural products is an important means of ensuring food safety for the people. However, achieving accurate detection of target analytes in complex sample matrices, especially small molecules such as pesticides, still faces many challenges: (1) Compared with non-competitive methods, competitive immunoassays of small molecules usually exhibit lower specificity and reliability; (2) Rapid sample pretreatment processes increase matrix interference, thereby reducing sensitivity and accuracy; (3) A single signal readout mode is difficult to meet diverse application scenarios. Lateral flow immunoassay (LFIA) has attracted increasing attention due to its portability, rapid detection, and low cost. Traditional lateral flow immunoassay usually uses colloidal gold nanoparticles as markers, but its sensitivity often cannot meet the detection requirements of trace residues. Aggregation-induced emission (AIE) materials are materials with weak fluorescence when dispersed, but significantly enhanced emission intensity when aggregated. This characteristic is suitable for LFIA, where the signal enhancement occurs when the signal probe accumulates on the test line (T line), enabling the detection system to achieve low background noise and high signal-to-noise ratio, thereby improving detection sensitivity.
[0003] Compared to competitive immunoassays, non-competitive methods offer significant advantages in sensitivity, linearity, and specificity. Currently, various non-competitive small molecule detection methods have been developed, including those using antibody variable region fragments, anti-conformity antibodies, anti-idiotype antibodies, and anti-immune complex peptides (AICPs). Among these, AICPs are widely used due to their ease of preparation via phage display and their availability in various forms, including phage-displayed peptides, peptides fused to functional proteins, and chemically synthesized peptides. Therefore, developing AICP-based non-competitive lateral flow immunoassays (NLFIA) shows great potential in overcoming the limitations of traditional lateral flow immunoassays. Summary of the Invention
[0004] The purpose of this invention is to provide a non-competitive lateral flow immunoassay method based on the dual signal mode of AIE nanoparticles, so as to solve the problem of low sensitivity of existing colloidal gold lateral flow immunoassays.
[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:
[0006] A non-competitive lateral flow immunoassay method based on dual signal modes of AIE nanoparticles includes the following steps: S1, preparing AIE nanoparticles PSNPs@AIE using an organic solvent swelling method. 650 S2, using PSNPs@AIE 650As a marker, the anti-immune complex peptide AICP was labeled with the streptavidin-biotin system using an active ester method to prepare immune recognition probes PSNPs@AIE. 650 @AICP; S3. Mix the sample or standard solution, the immunorecognition probe and the anti-thiamethoxam monoclonal antibody, and then add the mixture to the lateral flow chromatography strip for chromatography reaction; S4. Perform qualitative or quantitative detection by colorimetric and fluorescence signals to achieve dual-signal mode non-competitive immunoassay.
[0007] A further solution is that the PSNPs@AIE 650 The specific preparation method is as follows: 0.5 mL of polystyrene microspheres (PSNPs) suspension with a particle size of 200 nm and a concentration of 100 mg / mL is mixed with 5 mL of propylene glycol methyl ether and heated to 70 °C; a propylene glycol methyl ether solution containing 5 mg TTMN is added, and the mixture is reacted at 70 °C for 30 min, then cooled to room temperature; the mixture is dialyzed through a 30 kDa dialysis bag for 2 days, centrifuged at 13500 rpm for 15 min, and the precipitate is redispersed in 2.5 mL of pure water to obtain PSNPs@AIE. 650 .
[0008] A further embodiment is that the immune recognition probe PSNPs@AIE 650 The preparation steps of @AICP include: adding 10 mg of PSNPs@AIE 650 50 μL of 5 mg / mL EDC and 50 μL of 5 mg / mL Sulfo-NHS were added to 5 mL of 10 mM MES buffer (pH 6.0) and activated at room temperature for 20 min. The precipitate was collected by centrifugation. After redispersing the precipitate, 100 μL of 1 mg / mL streptavidin was added and reacted at room temperature for 1 h. 1 mL of 5% BSA and 50 μL of 10 mM glycine were added for blocking for 1 h. 50 μL of 2 mg / mL biotinylated AICP was added and reacted for 1 h. The mixture was purified by 100 kDa ultrafiltration and redispersed in 10 mM phosphate buffer containing 0.1% BSA and 3% sucrose to obtain the immunorecognition probe PSNPs@AIE. 650 @AICP.
[0009] In a further embodiment, the amino acid sequence of the biotinylated AICP is: CAVFTDQWWTGC-GGGSK-biotin.
[0010] In a further proposed scheme, the mixing system in S3 consists of: 100 μL of sample or standard solution and 4 μL of PSNPs@AIE. 650 @AICP, 1 μL 0.6 mg / mL anti-thiamethoxam monoclonal antibody; chromatography time: 10 min.
[0011] In a further embodiment, the test line T of the lateral flow chromatography test strip is coated with 1.0 mg / mL goat anti-mouse IgG antibody, and the control line C is coated with 0.5 mg / mL rabbit anti-streptavidin polyclonal antibody.
[0012] A further embodiment is characterized in that the sample or standard solution is prepared using a detection buffer solution containing 10% acetonitrile, 1% bovine serum albumin, and 0.5% Tween 20.
[0013] 8. The analytical method according to claim 1, characterized in that the dual-signal detection performance is as follows: colorimetric mode visual detection limit 1.0 ng / mL, quantitative detection limit 0.45 ng / mL; fluorescence mode visual detection limit 0.25 ng / mL, quantitative detection limit 0.23 ng / mL.
[0014] A further proposed method specifically recognizes thiamethoxam and shows no cross-reaction with acetamiprid, dinotefuran, imidacloprid, chlorpyrifos, or thiamethoxam.
[0015] The above analytical methods are applied to the detection of thiamethoxam residues in agricultural products.
[0016] In a further step, the agricultural product is cowpea, and the sample is homogenized, extracted with acetonitrile, precipitated with sodium chloride, centrifuged, diluted, and then tested.
[0017] The present invention has the following beneficial effects:
[0018] This invention prepares PSNPs@AIE with both colorimetric and fluorescence dual signal properties. 650 AIE nanoparticles, used to construct a non-competitive lateral flow immunoassay system, significantly improve the sensitivity of small molecule pesticide residue detection, with a fluorescence quantitative detection limit as low as 0.23 ng / mL, superior to traditional colloidal gold labeling methods. Simultaneously, the non-competitive mode with anti-immune complex peptides achieves high specificity recognition, with no cross-reactivity to thiamethoxam structural analogs. Combined with an optimized buffer system, interference from complex matrices is effectively reduced, and the entire detection process takes only 10 minutes. It can simultaneously meet the needs of on-site visual qualitative analysis and precise laboratory quantification, possessing outstanding advantages such as ease of operation, rapid portability, good stability, and strong applicability, providing an efficient and reliable new method for the rapid detection of trace pesticide residues in agricultural products. Attached Figure Description
[0019] Figure 1 Chemical structure of TTMN and PSNPs@AIE 650 Preparation diagram;
[0020] Figure 2 For PSNPs@AIE 650 Electron micrographs, particle size, spectra, and fluorescence properties;
[0021] Figure 3 For PSNPs@AIE 650 Schematic diagram of AICP probe fabrication;
[0022] Figure 4 This is a schematic diagram of the principle of dual-signal non-competitive lateral flow immunochromatographic detection.
[0023] Figure 5 A graph showing the optimization of antibody and probe dosages;
[0024] Figure 6 A graph showing the optimization of reaction time;
[0025] Figure 7 Optimization diagram for acetonitrile content;
[0026] Figure 8 For the verification of sensitivity, standard curve, and specificity;
[0027] Figure 9 The results of orthogonal experiments for optimizing goat anti-mouse IgG antibody and rabbit anti-streptavidin polyclonal antibody. Detailed Implementation
[0028] 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.
[0029] Example 1: AIE Nanoparticles PSNPs@AIE 650 Preparation and characterization
[0030] Take 0.5 mL of polystyrene microspheres (PSNPs) suspension with a particle size of 200 nm and a mass concentration of 100 mg / mL, mix thoroughly with 5 mL of propylene glycol methyl ether, place on a magnetic stirrer and heat and stir continuously until the system temperature stabilizes at 70 °C.
[0031] Add a propylene glycol methyl ether solution containing 5 mg of TTMN fluorescent dye to the above mixture, maintain a constant temperature of 70 °C, and continue stirring for 30 min to allow TTMN to be fully loaded into the PSNPs. After stopping heating, allow the reaction system to cool naturally to room temperature.
[0032] The cooled product was placed in a dialysis bag with a molecular weight cutoff of 30 kDa and dialyzed in ultrapure water for 2 days, with the pure water being changed regularly during the period to thoroughly remove unloaded free TTMN and organic solvent residues.
[0033] The dialysis product was centrifuged at 13500 rpm for 15 min, the supernatant was discarded, and the bottom precipitate was collected, which is the AIE nanoparticle PSNPs@AIE. 650 .
[0034] The precipitate was redispersed uniformly in 2.5 mL of ultrapure water and stored at 4 ℃ in the dark for later use.
[0035] As attached Figure 1 The diagram shows the chemical structure of TTMN used in this embodiment and the PSNPs@AIE. 650 Preparation route; as shown in the appendix Figure 2 The transmission electron microscope (TEM) image (A) and scanning electron microscope (SEM) image (B) are shown in the PPSNPs@AIE image. 650 It exhibits a uniform spherical structure and good dispersibility; the hydrated particle size distribution (C) shows an average particle size of 196±5 nm and a hydrated particle size of 210±8 nm; the absorption spectrum (D) and fluorescence spectrum (E) indicate that PSNPs@AIE 650 It exhibits a characteristic absorption peak at 500 nm and a significant fluorescence emission peak at 650 nm; physical images (F) under natural and ultraviolet light visually demonstrate its colorimetric and fluorescence dual-signal characteristics; fluorescence quantum yield (GH) and fluorescence lifetime (I) results indicate that PSNPs@AIE 650 It has a quantum yield of 12.34%, a fluorescence lifetime of 3.06 ns, and excellent optical performance.
[0036] Example 2: Immune recognition probes PSNPs@AIE 650 Preparation of @AICP
[0037] Take 10 mg of PSNPs@AIE prepared in Example 1 650 Add 5 mL of 10 mM MES buffer (pH 6.0), then add 50 μL of freshly prepared 5 mg / mL EDC solution and 50 μL of 5 mg / mL Sulfo-NHS solution, and activate by stirring at room temperature in the dark for 20 min.
[0038] Centrifuge the activated mixture at 13500 rpm for 15 min, discard the supernatant, and collect the activated PSNPs@AIE. 650 The precipitate was then redispersed in 5 mL of MES buffer.
[0039] 100 μL of 1 mg / mL streptavidin (SA) was added to the dispersion, and the mixture was stirred at room temperature for 1 h to allow streptavidin to be stably bound to PSNPs@AIE. 650 surface.
[0040] Add 1 mL of 5% bovine serum albumin (BSA) solution and 50 μL of 10 mM glycine solution, and continue stirring at room temperature for 1 h to block unreacted active groups on the surface of nanoparticles and reduce non-specific adsorption.
[0041] 50 μL of 2 mg / mL biotinylated anti-immune complex peptide (AICP) was added to the blocked system, and the mixture was stirred at room temperature for 1 h to complete the AICP conjugation using the high specific affinity of streptavidin and biotin. The amino acid sequence of the biotinylated AICP is: CAVFTDQWWTGC-GGGSK-biotin.
[0042] The reaction product was purified by centrifugation using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. The product was washed 2-3 times to completely remove any unreacted excess reagent.
[0043] The purified final product was redispersed in 2 mL of 10 mM phosphate buffer containing 0.1% BSA and 3% sucrose, and stored at 4 °C in the dark to obtain PSNPs@AIE. 650 @AICP immune recognition probe.
[0044] As attached Figure 3 As shown, this is the PSNPs@AIE example. 650 The complete process of activation, coupling, blocking, and purification of the @AICP immune recognition probe.
[0045] Example 3: Non-competitive lateral flow immunoassay method based on dual signal mode of AIE nanoparticles
[0046] Detection principle:
[0047] As attached Figure 4 As shown, this invention employs a non-competitive recognition mode: when thiamethoxam is absent from the sample, antibody-antigen immune complexes cannot be formed, PSNPs@AIE 650 @AICP probes cannot be captured by the T line, so there is no signal on the T line; when the sample contains thiamethoxam, the antibody forms an immune complex with thiamethoxam, which is specifically recognized and bound by the probe, and then captured by the T line goat anti-mouse IgG, resulting in colorimetric and fluorescent signals on the T line, and the signal intensity increases with the increase of thiamethoxam concentration; the C line can capture free probes and is used to verify the effectiveness of the test strip.
[0048] Optimized testing conditions:
[0049] Antibody coating concentration optimization: through orthogonal experiments (see attached) Figure 9 It was determined that the optimal coating concentration for the T line was 1.0 mg / mL goat anti-mouse IgG, and the optimal coating concentration for the C line was 0.5 mg / mL rabbit anti-streptavidin polyclonal antibody.
[0050] When the T-line was coated with 1.0 mg / mL goat anti-mouse IgG antibody and the C-line was coated with 0.5 mg / mL rabbit anti-streptavidin polyclonal antibody, the positive sample signal was clear, the negative sample background was the lowest, and the signal contrast was the best. Therefore, this combination was determined to be the optimal coating concentration.
[0051] Optimization of reagent dosage: as shown in the appendix Figure 5 As shown, the optimal dosage of antithiamethoxam monoclonal antibody is 1.0 μL, PSNPs@AIE 650 The optimal dosage of the AICP probe is 4.0 μL.
[0052] Optimization of reaction time: as shown in the appendix Figure 6 As shown, the signal reached a stable plateau after 10 min of chromatographic reaction, and the optimal reaction time was determined to be 10 min.
[0053] Organic solvent content optimization: as shown in the appendix Figure 7 As shown, the signal is optimal when the acetonitrile content in the detection buffer is 10%, and the signal intensity will be significantly reduced when it is higher than 10%.
[0054] Standard testing procedures:
[0055] Prepare the detection buffer solution: containing 10% acetonitrile, 1% bovine serum albumin, and 0.5% Tween-20.
[0056] Take 100 μL of sample solution or thiamethoxam standard solution and add 4.0 μL of PSNPs@AIE. 650 @AICP immune probe and 1.0 μL of 0.6 mg / mL anti-thiamethoxam monoclonal antibody were gently pipetted to mix.
[0057] Add the mixture dropwise to the sample pad of the side-flow chromatography test strip, and perform chromatography along the NC membrane by capillary action. React at room temperature for 10 min.
[0058] Result interpretation: Qualitative detection: Visually observe the signals of the T line and C line. The presence of colorimetric / fluorescence on the T line indicates a positive result, while the absence of signal indicates a negative result. Quantitative detection: Measure the difference in hue value between the T line and the background (∆E), and calculate the thiamethoxam content based on the standard curve.
[0059] Sensitivity and specificity:
[0060] As attached Figure 8 As shown:
[0061] Visual sensitivity (A): detection limit 1.0 ng / mL in colorimetric mode, detection limit 0.25 ng / mL in fluorescence mode;
[0062] Standard curves (B, C): colorimetric limit of quantitation 0.45 ng / mL, fluorescence limit of quantitation 0.23 ng / mL;
[0063] Specificity test (D): This method showed no significant cross-reactivity with acetamiprid, dinotefuran, imidacloprid, chlorpyrifos, and thiamethoxam, and exhibited high specificity for thiamethoxam.
[0064] Example 4: Application of the method of the present invention in the detection of thiamethoxam residues in agricultural products
[0065] Using cowpeas as the actual test sample, the following steps were performed for testing:
[0066] Take a fresh cowpea sample, homogenize it thoroughly using a tissue homogenizer, and accurately weigh 10 g into a 50 mL centrifuge tube.
[0067] Add 5 mL of ultrapure water, 20 mL of acetonitrile, and 3 g of sodium chloride in sequence, and vortex at high speed for 15 min to fully extract thiamethoxam.
[0068] Centrifuge the extract at 5000 rpm for 5 min and collect the upper organic phase.
[0069] The supernatant was diluted to a suitable multiple with the detection buffer solution and tested according to the method in Example 3.
[0070] The spiked recovery experiment results show that the method has high recovery rate and good precision, and is suitable for rapid, accurate and highly sensitive detection of thiamethoxam residues in complex agricultural product matrices.
[0071] 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. A non-competitive lateral flow immunoassay method based on dual-signal mode of AIE nanoparticles, characterized in that, Includes the following steps: S1, AIE nanoparticles PSNPs@AIE were prepared by organic solvent swelling method 650 ; S2, PSNPs@AIE as marker, AICP as target, the preparation of immunorecognition probe PSNPs@AIE 650 S2, PSNPs@AIE as marker, AICP as target, the preparation of immunorecognition probe PSNPs@AIE 650 @AICP; S3. Mix the sample or standard solution, the immunorecognition probe and the anti-thiamethoxam monoclonal antibody, and then add the mixture to the side-flow chromatography strip for chromatography reaction. S4. Qualitative or quantitative detection is achieved through colorimetric and fluorescence signals, realizing dual-signal mode non-competitive immunoassay.
2. The non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode according to claim 1, characterized in that, The PSNPs@AIE 650 The specific preparation method is as follows: Mix 0.5 mL of a polystyrene microsphere (PSNP) suspension with a particle size of 200 nm and a concentration of 100 mg / mL with 5 mL of propylene glycol methyl ether and heat to 70 °C. Add a propylene glycol methyl ether solution containing 5 mg TTMN, react at 70 °C for 30 min, and then cool to room temperature; After dialysis with a 30 kDa dialysis bag for 2 days, the precipitate was centrifuged at 13500 rpm for 15 min, and then redispersed in 2.5 mL of pure water to obtain PSNPs@AIE. 650 .
3. The non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode according to claim 1, characterized in that, The immune recognition probe PSNPs@AIE 650 The preparation steps of @AICP include: 10 mg PSNPs@AIE 650 Add 50 μL of 5 mg / mL EDC and 50 μL of 5 mg / mL Sulfo-NHS to 5 mL of 10 mM MES buffer (pH 6.0), activate at room temperature for 20 min, and centrifuge to collect the precipitate; After redispersing the precipitate, add 100 μL of 1 mg / mL streptavidin and react at room temperature for 1 h. Add 1 mL of 5% BSA and 50 μL of 10 mM glycine for blocking for 1 h; Add 50 μL of 2 mg / mL biotinylated AICP and react for 1 h; Purified by 100 kDa ultrafiltration, the immunorecognition probes PSNPs@AIE were redispersed in 10 mM phosphate buffer containing 0.1% BSA and 3% sucrose to obtain the immunorecognition probes PSNPs@AIE. 650 @AICP.
4. The non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode according to claim 3, characterized in that, The amino acid sequence of the biotinylated AICP is: CAVFTDQWWTGC-GGGSK-biotin.
5. The non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode according to claim 1, characterized in that, The mixing system in S3 is: 100 μL sample or standard solution, 4 μL PSNPs@AIE 650 @AICP, 1 μL 0.6 mg / mL anti-thiamethoxam monoclonal antibody; chromatography time: 10 min.
6. The non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode according to claim 1, characterized in that, The test line T of the lateral flow chromatography test strip is coated with 1.0 mg / mL goat anti-mouse IgG antibody, and the control line C is coated with 0.5 mg / mL rabbit anti-streptavidin polyclonal antibody.
7. The non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode according to claim 1, characterized in that, The sample or standard solution is prepared using a detection buffer solution containing 10% acetonitrile, 1% bovine serum albumin, and 0.5% Tween 20.
8. The non-competitive lateral flow immunoassay method based on AIE nanoparticle dual-signal mode according to claim 1, characterized in that, The dual-signal detection performance is as follows: in colorimetric mode, the visual detection limit is 1.0 ng / mL and the quantitative detection limit is 0.45 ng / mL; in fluorescence mode, the visual detection limit is 0.25 ng / mL and the quantitative detection limit is 0.23 ng / mL.