An aggregation-induced emission immunochromatographic test strip, a preparation method and application thereof

Abstract

The application discloses an aggregation-induced emission immunochromatographic test strip as well as a preparation method and application thereof, and belongs to the technical field of residual pesticide detection. The aggregation-induced emission immunochromatographic test strip utilizes the strong fluorescence characteristics of an AIE material and the autonomous movement ability of a nanomotor, increases the binding efficiency of an antigen and an antibody, reduces the detection limit of acetamiprid, improves the detection sensitivity, and can realize precise detection of low-concentration acetamiprid residues in fruits and vegetables. The aggregation-induced emission immunochromatographic test strip can reduce the influence of impurities in fruit and vegetable matrixes on the detection system based on the stability of the AIE material and the movement characteristics of the nanomotor, and can ensure stable detection performance in different fruit and vegetable samples. The aggregation-induced emission immunochromatographic test strip can greatly shorten the detection time by virtue of the active movement of the nanomotor to accelerate the detection reaction, does not need complex instruments and professional operation, and is suitable for rapid screening of acetamiprid residues in fruits and vegetables in field, market and other on-site scenes.

Description

Technical Field

[0001] This invention belongs to the field of pesticide residue detection technology, specifically relating to an aggregation-induced emission immunochromatographic test strip, its preparation method, and its application. Background Technology

[0002] Acetamiprid, chemically known as (E)-N¹-[(6-chloro-3-pyridyl)methyl]-N²-cyano-N¹-methylacetamidine, has the molecular formula C2. 10 H 11 ClN4 is a highly effective, broad-spectrum neonicotinoid insecticide. It works by acting on nicotinic acetylcholine receptors in the insect nervous system, blocking normal nerve conduction and causing paralysis and death in pests. It is widely used in crops such as vegetables, fruits, tea, and grains to control piercing-sucking pests such as aphids, planthoppers, and whiteflies.

[0003] However, with the widespread use of acetamiprid, its residues in agricultural products and environmental media (such as water and soil) are becoming increasingly prominent. Acetamiprid is chemically stable, difficult to degrade naturally, and can accumulate through the food chain, posing a potential threat to human health. Studies have shown that it may have neurotoxicity, reproductive toxicity, and endocrine-disrupting effects. In light of this, countries worldwide have established strict limits for acetamiprid residues in food and the environment.

[0004] Therefore, establishing efficient, sensitive, and rapid methods for detecting acetamiprid residues is of great significance for ensuring food safety, protecting the ecological environment, and promoting agricultural trade. Currently, routine methods for detecting acetamiprid mainly include chromatographic methods (such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS)) and immunoassay methods (such as enzyme-linked immunosorbent assay (ELISA). While these methods have advantages such as high reliability and low detection limits, they typically require expensive large-scale instruments, complex sample pretreatment processes, and specialized operators, resulting in significant limitations in practical applications.

[0005] Immunochromatographic strip (ICS) technology, with its advantages of speed, simplicity, and low cost, is considered one of the ideal platforms for rapid on-site detection. Traditional ICS typically uses colloidal gold as a signal tag, relying on visual observation of color changes for judgment. However, colloidal gold strips have relatively low sensitivity, weak signal intensity, and are difficult to quantify, posing a challenge when detecting low concentrations of residues.

[0006] To improve sensitivity, fluorescence immunochromatography (FIG) has emerged. This technique uses fluorescent materials such as quantum dots (QDs) and upconversion nanoparticles (UCNPs) as signal tags, achieving quantitative analysis by detecting fluorescence intensity, with significantly higher sensitivity than colloidal gold methods. However, these traditional fluorescent materials often undergo fluorescence quenching (ACQ) in the solid or aggregated state, which severely limits their signal loading and luminescence efficiency on the solid-phase carrier of test strips.

[0007] Aggregation-induced emission (AIE) materials are a novel class of luminescent materials with a luminescence mechanism opposite to that of traditional materials: they exhibit weak fluorescence in a dispersed state, but significantly enhanced fluorescence in an aggregated state. AIE materials effectively overcome the ACQ effect and possess outstanding advantages such as high fluorescence intensity, excellent photostability, and good biocompatibility. If applied to immunochromatographic test strips, they hold promise for constructing a fluorescence detection platform with ultra-high sensitivity and stability.

[0008] However, despite the promising prospects of AIE materials, a mature AIE fluorescent test strip solution for pesticide detection remains elusive. Furthermore, existing technologies still rely on Brownian motion for passive diffusion of signal probes within the chromatographic membrane, resulting in low efficiency. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides an aggregation-induced emission immunochromatographic test strip, its preparation method, and its applications. The aggregation-induced emission immunochromatographic test strip provided by this invention is a fluorescent test strip based on a self-driven AIE silica sphere probe. It utilizes the thrust generated by the decomposition of H2O2 to drive the probe's active movement, thereby improving detection performance. This invention integrates AIE fluorescence signal amplification, self-driven technology, and competitive immunochromatography technology to achieve the master-driven movement of the probe based on AIE hypersensitive luminescence. This provides an innovative tool for rapid on-site screening of acetamiprid in agricultural products, and can promote the transformation of food safety supervision from "laboratory dependence" to "on-site intelligentization."

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] One of the technical solutions of this invention is to provide a method for preparing an aggregation-induced emission immunochromatographic test strip, comprising the following steps:

[0012] (1) Polymethyl methacrylate, poly(maleic anhydride-alt-1-octadecene) and tetramethyl-4',4'',4''',4'''-(ethylene-1,1,2,2-tetramethyl)tetra([1,1'-biphenyl]-4-carboxylate) were dissolved in chloroform to obtain a mixture. The mixture was then added dropwise to an aqueous solution of sodium dodecyl sulfate and sonicated under ice bath conditions to obtain an emulsion. After the emulsion reaction, a microsphere suspension was obtained. After centrifugation, aggregation-induced emission microspheres were obtained.

[0013] (2) The aggregation-induced emission microspheres were added to anhydrous ethanol, sonicated, and then ammonia solution was added. The mixture was stirred evenly, and tetraethyl orthosilicate was added dropwise. The mixture was stirred and centrifuged to obtain aggregation-induced emission silicon spheres.

[0014] (3) The aggregation-induced light-emitting silicon spheres were added to anhydrous toluene, dispersed, and then 3-aminopropyltriethoxysilane was added. The mixture was refluxed and centrifuged to obtain aminated aggregation-induced light-emitting silicon spheres. The aminated aggregation-induced light-emitting silicon spheres were dispersed in N,N-dimethylformamide (DMF), and then succinic anhydride and catalyst were added. After stirring in the dark, the mixture was centrifuged to obtain carboxylated aggregation-induced light-emitting silicon spheres. The carboxylated aggregation-induced light-emitting silicon spheres were resuspended and coated on a carrier to prepare a monolayer film. Pt was sputtered onto the monolayer film, redispersed in water, and centrifuged to obtain self-driven aggregation-induced light-emitting silicon spheres.

[0015] (4) Add the self-driven aggregation-induced luminescent silicon spheres to MES buffer, then add N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir in the dark and centrifuge, resuspend the precipitate, add acetamiprid monoclonal antibody, shake in the dark and add bovine serum albumin solution, continue shaking and centrifuge to obtain the self-driven aggregation-induced luminescent silicon sphere probe;

[0016] (5) Prepare immunochromatographic test strips using the self-driven aggregation-induced light-emitting silicon sphere probe as a marker.

[0017] Preferably, in step (1), the ratio of polymethyl methacrylate, poly(maleic anhydride-alt-1-octadecene), tetramethyl-4',4'',4''',4'''-(ethylene-1,1,2,2-tetramethyl)tetra([1,1'-biphenyl]-4-carboxylate) to chloroform is 50 mg:25 mg:30 mg:1 mL; the volume ratio of the mixture to the sodium dodecyl sulfate aqueous solution is 1:5; the mass fraction of the sodium dodecyl sulfate aqueous solution is 0.25%; and the reaction conditions in step (1) are rotary evaporation at 37°C for 20 min.

[0018] Preferably, in step (2), the ratio of the aggregation-induced emission microspheres to the tetraethyl orthosilicate is 1 mg: 55 μL.

[0019] Preferably, in step (3), the ratio of the aggregate-induced light-emitting silicon spheres to the 3-aminopropyltriethoxysilane is 2 mg: 5 μL; the reflux reaction temperature is 80 °C and the time is 24 h.

[0020] Preferably, in step (3), the mass ratio of the aminated aggregation-induced light-emitting silicon spheres to the succinic anhydride is 4:5; the catalyst is triethylamine; and the stirring temperature in the dark is 25°C and the time is 12h.

[0021] Preferably, in step (3), the sputtering of Pt is performed using an ion sputtering instrument for a sputtering time of 40 seconds.

[0022] Preferably, in step (4), the mass ratio of the self-driven aggregation-induced light-emitting silicon spheres, the N-hydroxysuccinimide, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the acetamiprid monoclonal antibody is 4:2.4:3:0.2; the bovine serum albumin solution is prepared by adjusting the volume of 1g bovine serum albumin to 10mL; the light-protected shaking temperature is 25℃ for 4h; and the continued shaking temperature is 25℃ for 2h.

[0023] The second technical solution of the present invention provides an aggregation-induced emission immunochromatographic test strip prepared according to the above-mentioned preparation method of aggregation-induced emission immunochromatographic test strip.

[0024] The third technical solution of the present invention provides an application of the above-mentioned aggregation-induced emission immunochromatographic test strip in the detection of acetamiprid.

[0025] Preferably, hydrogen peroxide solution is used to dilute the sample during detection.

[0026] The beneficial technical effects of the present invention are as follows:

[0027] This invention utilizes the strong fluorescence properties of aggregation-induced emission (AIE) materials and the autonomous movement capability of nanomotors to increase the binding efficiency of antigens and antibodies, reduce the detection limit of acetamiprid, and improve detection sensitivity, enabling accurate detection of low concentrations of acetamiprid residues in fruits and vegetables.

[0028] Based on the stability of AIE materials and the motion characteristics of nanomotors, this invention can reduce the impact of impurities in fruit and vegetable matrices on the detection system, ensuring stable detection performance in different fruit and vegetable samples.

[0029] This invention utilizes the active motion of nanomotors to accelerate the detection reaction, which can greatly shorten the detection time. It requires no complicated instruments or professional operation and is suitable for rapid screening of acetamiprid residues in fruits and vegetables in field, market and other on-site scenarios. Attached Figure Description

[0030] Figure 1 This is a TEM image of the AIE microspheres in Example 1.

[0031] Figure 2 The zeta potentials of the AIE silicon spheres, aminated AIE silicon spheres, carboxylated AIE silicon spheres, and self-driven AIE silicon spheres prepared in steps (2) and (3) of Example 1.

[0032] Figure 3 The effect of different TEOS addition amounts on the thickness of the SiO2 layer (mSiO2) of the prepared AIE silicon spheres in step (2) of Example 1.

[0033] Figure 4 The effect of different TEOS addition amounts on the particle size of the self-driven AIE silicon balls obtained in step (2) of Example 1.

[0034] Figure 5 The mean square displacement (μm) of the self-driven AIE silicon spheres prepared in step (3) of Example 1 in hydrogen peroxide solutions of different concentrations 2 The relationship between () and time interval Δt (s).

[0035] Figure 6 The diffusion coefficient (μm) of the self-driven AIE silicon spheres prepared in step (3) of Example 1 in hydrogen peroxide solutions of different concentrations. 2 / s).

[0036] Figure 7 This is a schematic diagram of the structure of the AIE immunochromatographic test strip in Example 2.

[0037] Figure 8 The detection principle of the AIE immunochromatographic test strip prepared in Example 2 is explained.

[0038] Figure 9 This study examines the effect of different EDC addition amounts in step (4) of Example 1 on the detection performance of the prepared AIE immunochromatographic test strip.

[0039] Figure 10 This study examines the effect of different amounts of acetamiprid monoclonal antibody added in step (4) of Example 1 on the detection performance of the prepared AIE immunochromatographic test strip.

[0040] Figure 11 This study investigates the effect of different coating amounts of self-driven AIE silica ball probe solutions on the detection performance of the prepared AIE immunochromatographic test strips in Example 2.

[0041] Figure 12 This study investigates the effect of different coating amounts of acetamiprid and BSA conjugates on the detection performance of the prepared AIE immunochromatographic test strips in Example 2.

[0042] Figure 13The image shows the change in T-line fluorescence intensity over time when the AIE immunochromatographic test strip prepared in Example 2 detects a negative sample.

[0043] Figure 14 The results show the specificity of the AIE immunochromatographic test strip prepared in Example 2.

[0044] Figure 15 The results show the stability of the AIE immunochromatographic test strip prepared in Example 2. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0046] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0047] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] The reagents and materials used in the specific embodiments of the present invention are as follows:

[0051] Tetramethyl-4',4'',4''',4'''-(ethylene-1,1,2,2-tetramethyl)tetra([1,1'-biphenyl]-4-carboxylic acid ester) (TCBPE): Shanghai Mofang Chemical Technology Co., Ltd.;

[0052] Polymethyl methacrylate (PMMA): Shanghai Aladdin Biochemical Technology Co., Ltd., Product No.: B800058 (CAS 9011-14-7).

[0053] Poly(maleic anhydride-alt-1-octadecene) (PMAO): Sigma-Aldrich, catalog number: 418951 (CAS 25266-02-8).

[0054] Sodium dodecyl sulfate (SDS): Xilong Scientific Co., Ltd., Product No.: S8010 (CAS 151-21-3);

[0055] Chloroform (CHCl3): Sinopharm Chemical Reagent Co., Ltd., Product No.: 10006818 (CAS 67-66-3);

[0056] Sodium hydroxide (NaOH): Aladdin, product number: S111106 (0.01 M solution, CAS 1310-73-2).

[0057] Borate buffer (BB): Sigma-Aldrich, catalog number: B7660 (0.2 M, pH=8.0);

[0058] Ammonia water (NH4OH, 28 wt%): Sinopharm Chemical Reagent, Product No.: 10005908 (CAS 1336-21-6);

[0059] Anhydrous ethanol: Sinopharm Group, product number: 10009257 (CAS 64-17-5);

[0060] Ethyl orthosilicate (TEOS): Aladdin Biochemical Technology, Product No.: T104807 (CAS 78-10-4).

[0061] 3-Aminopropyltriethoxysilane (APTES), catalog number: 440140 (Sigma-Aldrich);

[0062] Anhydrous toluene, catalog number: 179418 (Sigma-Aldrich);

[0063] Succinic anhydride, catalog number: 239690 (Sigma-Aldrich);

[0064] Triethylamine, catalog number: T0886 (Sigma-Aldrich);

[0065] N,N-Dimethylformamide (DMF): Sigma-Aldrich;

[0066] Platinum (Pt) target (purity 99.99%), catalog number: PT-50-100 (Tianjin Aida Hengsheng);

[0067] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), catalog number: E106593 (Aladdin);

[0068] N-hydroxysuccinimide (NHS), catalog number: 130672 (Aladdin);

[0069] Acetamiprid monoclonal antibody (Shenzhen Kejie Industrial Development Co., Ltd.);

[0070] MES buffer (0.05 M, pH 6.5), catalog number: M2933 (Sigma-Aldrich);

[0071] Phosphate-buffered saline (PBS): Thermo Fisher Scientific (14190144);

[0072] Bovine serum albumin (BSA): Sigma-Aldrich (A7906);

[0073] NC membrane (Sartorius CN140): Sartorius, Germany, item number: CN140;

[0074] Sample pad / absorbent paper: Shanghai Jieyi Biotechnology, catalog number: JY-SP01;

[0075] PVC base plate: Shanghai Jieyi Biotechnology, item number: JY-PVC01.

[0076] Example 1

[0077] Fabrication of self-driven AIE silicon ball probes:

[0078] (1) Preparation of AIE microspheres

[0079] Dissolve 50 mg PMMA and 25 mg PMAO in 1 mL of chloroform solution, then add 30 mg TCBPE to form a PMMA / PMAO / TCBPE / chloroform solution. Add 240 μL of PMMA / PMAO / TCBPE / chloroform solution dropwise to 1.2 mL of SDS aqueous solution (0.25% w / w), immediately place in an ice bath, and sonicate at 120 W for 3 min to form a homogeneous emulsion.

[0080] The emulsion was rotary evaporated in a 37°C water bath for 20 minutes to obtain a microsphere suspension. The suspension was centrifuged at 12,000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was washed three times with 0.01 M NaOH solution. The final precipitate (AIE microspheres) was resuspended in 1.8 mL of borate buffer (0.2 M, pH 8.0) (concentration 10 mg / mL), transferred to a brown sample vial, and stored at 4°C protected from light.

[0081] (2) Preparation of AIE silicon spheres

[0082] Take 1 mL of AIE microsphere dispersion and wash it by centrifugation with anhydrous ethanol (the goal is to remove the borate buffer medium used to store the AIE microspheres). Add 60 mL of anhydrous ethanol and sonicate at 120 W for 30 min. Add 4.5 mL of ammonia (28 wt%) and 15 mL of ultrapure water, and stir magnetically at 25 °C (500 rpm) for 10 min. Slowly add 550 μL of TEOS and continue stirring for 6 hours. Centrifuge the solution at 12000 rpm for 20 min at 4 °C. Wash the precipitate three times each with anhydrous ethanol and ultrapure water. Finally, disperse the precipitate in 30 mL of anhydrous ethanol and store at 4 °C protected from light.

[0083] (3) Preparation of self-driven AIE silicon spheres

[0084] Take 10 mL of AIE silica sphere dispersion (concentration 4 mg / mL), wash with anhydrous toluene by centrifugation (to remove the storage medium of anhydrous ethanol for AIE silica spheres), disperse in 10 mL of anhydrous toluene, add 100 μL of APTES, reflux at 80 °C for 24 hours under nitrogen protection, centrifuge at 10000 rpm for 15 minutes, wash three times with ethanol by centrifugation to obtain aminated AIE silica spheres. Disperse the precipitate in 10 mL of DMF, add 50 mg of succinic anhydride and 50 μL of triethylamine, stir at 25 °C in the dark for 12 hours. Centrifuge at 12000 rpm for 20 minutes, wash the precipitate three times each with DMF and ultrapure water by centrifugation to obtain carboxylated AIE silica spheres.

[0085] The precipitate was dispersed in 2 mL of ethanol, and 40 μL was dropped onto a glass slide to form a uniform monolayer. The film was then dried at room temperature. Pt was sputtered using an ion sputtering apparatus for 40 seconds. The glass slide was then immersed in 5 mL of ultrapure water and sonicated for 60 seconds to remove the self-driven AIE silica spheres. After centrifugation at 8500 rpm for 10 minutes, the precipitate (self-driven AIE silica spheres) was dispersed in PBS (pH 7.4).

[0086] (4) Preparation of self-driven AIE silicon ball probes

[0087] Take 1 mL of self-driven AIE silica sphere dispersion (concentration 4 mg / mL), centrifuge and wash with MES buffer (the goal is to remove the PBS storage medium for the self-driven AIE silica spheres), add 2 mL of MES buffer, then add 2.4 mg NHS and 3 mg EDC, and stir at 25°C in the dark for 1 hour. Centrifuge the mixture at 12000 rpm for 10 minutes, discard the supernatant, and resuspend in PBS (pH 7.4). Add 0.2 mg acetamiprid monoclonal antibody, shake at 25°C in the dark for 4 hours, then add 100 μL of 10% (w / v, 1 g BSA to a final volume of 10 mL) BSA, shake at 25°C for 2 hours to obtain the self-driven AIE silica sphere probe. Wash three times with PBS solution containing 1% (w / v, 0.1 g BSA to a final volume of mL), resuspend in 1 mL of PBS solution containing 1% (w / v) BSA, and store at 4°C in the dark.

[0088] Take 1 μL of the AIE microsphere dispersion obtained in step (1), dilute it to 1 mL with ultrapure water, and drop an appropriate amount onto the surface of a 400-mesh carbon film copper mesh. After drying in a forced-air drying oven overnight, it is used for transmission electron microscopy (TEM) testing. The test results are shown in […]. Figure 1 .

[0089] The zeta potentials of the AIE silicon spheres, aminated AIE silicon spheres, carboxylated AIE silicon spheres, and self-driven AIE silicon spheres prepared in steps (2) and (3) were monitored, and the monitoring results are shown in […]. Figure 2 .

[0090] from Figure 2 As can be seen, the AIE silica spheres carry a negative charge, consistent with the characteristics of their surface hydroxyl groups (-OH); the aminated AIE silica spheres carry a positive charge, indicating that amino groups (-NH2) were successfully introduced, resulting in a positively charged surface; the carboxylated AIE silica spheres regain a negative charge, indicating that succinic anhydride successfully converted -NH2 to -COOH, restoring the surface to a negative charge; the self-driven AIE silica spheres still have a negative charge, indicating that Pt sputtering did not completely cover the carboxyl groups, and some -COOH was still retained, which can be used for subsequent antibody conjugation.

[0091] Referring to the method of Example 1, the amount of TEOS added in step (2) was adjusted to 450 μL, 65 μL, 700 μL, or 750 μL; the effect of different TEOS addition amounts on the SiO2 layer (mSiO2) thickness of the prepared AIE silicon spheres was observed, and the results are shown in […]. Figure 3 The effect of different TEOS addition amounts on the particle size of the prepared self-driven AIE silicon balls is shown in the figure. Figure 4 .

[0092] from Figure 3It can be seen that the amount of TEOS used is positively correlated with the thickness of the silica shell. Figure 4 As can be seen, the increase in the particle size of the self-driven AIE silicon spheres is a direct result of the increase in shell thickness, which is directly related to the amount of TEOS used. Therefore, the more TEOS added, the thicker the mSiO2 layer becomes, and the amount of TEOS used is a key synthesis parameter for controlling the size and structure of the self-driven AIE microspheres.

[0093] Hydrogen peroxide solutions with concentrations of 0.05 wt%, 0.10 wt%, 0.25 wt%, 0.50 wt%, 1.00 wt%, 2.50 wt%, and 5.00 wt% were prepared respectively. The mean square displacement (μm) of the self-driven AIE silicon spheres prepared in step (3) of Example 1 in hydrogen peroxide solutions of different concentrations was investigated. 2 The relationship between the time interval Δt (s) and the time interval is shown in the figure. Figure 5 ; and the diffusion coefficient (μm) of self-driven AIE silicon spheres in hydrogen peroxide solutions of different concentrations. 2 / s), results are shown Figure 6 .

[0094] about Figure 5 Mean square displacement (MSD) is a physical quantity that measures "average distance" a particle travels in a given time interval. A larger MSD value indicates that the particle is moving more vigorously and faster. With increasing hydrogen peroxide concentration, self-driven AIE silicon spheres become more active and faster within the same Δt. Regarding... Figure 6 The diffusion coefficient is a physical parameter that directly quantifies the mobility of particles. The higher the diffusion coefficient, the stronger the particle's migration ability and the faster its movement.

[0095] Figure 6 The study demonstrates a quantitative relationship between the mobility (diffusion coefficient) of the self-driven AIE silicon spheres and the H2O2 concentration. That is, the H2O2 concentration can control the movement of the self-driven AIE silicon spheres.

[0096] Example 2

[0097] Preparation of AIE immunochromatographic test strips:

[0098] Sample pad: The entire glass fiber membrane (CN140) was immersed in the treatment solution (12.1 g / L Tris, 10 g / L PVP, 5 g / L casein, 0.2 g / L NaN3, pH=8.0) for 3 min, and then dried overnight in a 37 ℃ forced-air drying oven. It was then stored in a desiccant for later use. The self-driven AIE silica ball probe solution obtained in Example 1 was diluted 100 times and sprayed onto the treated glass fiber membrane (spraying width of 1 mm) at a rate of 1.6 μL / cm. It was then dried in a 37 ℃ forced-air drying oven for 4 h and stored in a desiccant for later use. NC membrane coating: A solution containing a conjugate of 0.02 mg / mL acetamiprid and BSA (purchased from Beijing Bio-Long Immunotherapy Co., Ltd., catalog number ZD6136) and a 0.1 mg / mL capture antibody solution were sprayed onto a glass fiber membrane (spray width 1 mm) at a rate of 0.8 μL / cm to form a detection line (T line) and a control line (C line). The distance between the two lines was set to 6 mm. The membrane was dried in a 37 ℃ forced-air drying oven for 4 h and then stored in a desiccator for later use. Figure 7 As shown, the sample pad, NC membrane, and absorbent paper (absorbent pad) are pasted onto the PVC base plate in sequence, overlapping by 2 mm. The strips are then cut into 4 mm wide test strips using a cutter to obtain AIE immunochromatographic test strips. These strips are then assembled into plastic cartridges, sealed, and dried for storage.

[0099] The detection principle of the AIE immunochromatographic test strip prepared in Example 2 is described in [link to example]. Figure 8 .

[0100] Example 3

[0101] AIE Immunochromatographic Test Strip Detection Procedure:

[0102] Place the AIE immunochromatographic test strip in a 37°C incubator. Transfer 90 μL of the pre-treated test solution and mix it with 10 μL of 10.00 wt% hydrogen peroxide solution. Then, add the mixture to the sample well of the test strip and incubate for 15 min. Read the T and C line fluorescence signals of the test strip using a test strip fluorescence reader.

[0103] The dosage of EDC in step (4) of Example 1 was adjusted to 2 mg, 3 mg, 4 mg, 5 mg and 6 mg respectively. The detection effect of the prepared AIE immunochromatographic test strips was compared. The acetamiprid content in the test solution was 200 ng / mL. The fluorescence intensity of the T line is shown in the figure. Figure 9 .

[0104] Figure 9 The data shows that 3mg is the optimal dosage of EDC.

[0105] The dosage of acetamiprid monoclonal antibody in step (4) of Example 1 was adjusted to 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, and 0.3 mg, respectively. The detection performance of the prepared AIE immunochromatographic test strips was compared. The acetamiprid content in the test solution was 200 ng / mL. The fluorescence intensity of the T line is shown in the figure. Figure 10 .

[0106] Figure 10 The results showed that 0.2 mg of acetamiprid monoclonal antibody was the optimal dosage for monoclonal antibody use.

[0107] The spraying amount of the self-driven AIE silica ball probe solution in Example 2 was adjusted to 0.4 μL / cm, 0.8 μL / cm, 1.6 μL / cm, and 2.4 μL / cm. The detection performance of the prepared AIE immunochromatographic test strips was compared. The acetamiprid content in the test solution was 200 ng / mL. The fluorescence intensity and competitive inhibition rate of the T line are shown in [reference needed]. Figure 11 .

[0108] Figure 11 The optimal spraying amount of the self-driven AIE silicon ball probe solution is 1.6 μL / cm.

[0109] The concentrations of the acetamiprid-BSA conjugate solution from Example 2 were adjusted to 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, and 0.03 mg / mL. The detection performance of the prepared AIE immunochromatographic test strips was compared. The acetamiprid content in the test solution was 200 ng / mL. The fluorescence intensity and competitive inhibition rate of the T line are shown in [reference needed]. Figure 12 .

[0110] Figure 12 The optimal concentration of the acetamiprid-BSA conjugate solution was 0.02 mg / mL.

[0111] The detection time of the AIE immunochromatographic test strip prepared in Example 2 was tested using a negative sample. The change in fluorescence intensity of the T line of the AIE immunochromatographic test strip over time after adding the negative sample is shown in the figure. Figure 13 .

[0112] from Figure 13 As can be seen, the fluorescence intensity of the AIE immunochromatographic test strip is stable 10-15 minutes after detection.

[0113] The glass fiber membrane material in Example 2 was replaced with Pall Vivid 90 (PALL Corporation, USA) or YNHS (Shantou Yineng Membrane Industry Co., Ltd.). The non-specific adsorption of the prepared AIE immunochromatographic test strips was compared. The acetamiprid content in the test solution was 1 mg / mL. The adsorption results are shown in Table 1.

[0114] Table 1

[0115]

[0116] Table 1 shows that the optimal glass fiber membrane models are CN140 or Pall Vivid 90.

[0117] Example 4

[0118] Determination of actual samples:

[0119] Vegetable and fruit sample processing procedure: After homogenizing the vegetables and fruits, weigh 1.0 g of the homogenized sample, add 2 mL of methanol, and mix thoroughly by shaking for 2 minutes. Centrifuge at 5000 r / min for 5 minutes, collect the supernatant, and filter it through a 0.45 μm filter membrane to remove impurities. Dilute the filtrate 4 times with PBS buffer to obtain the processed test solution.

[0120] 1. Accuracy and precision assessment

[0121] A 200 ng / mL solution of acetamiprid was prepared in 11 different fruit and vegetable matrices, including cucumber, bok choy, leek, lettuce, cabbage, tomato, carrot, peach, pear, apple, and orange. The solution was tested using the test strips from Example 2 according to the immunochromatographic test strip detection procedure described above. The spiked recovery rate and coefficient of variation were calculated, and the results are shown in Table 2.

[0122] Table 2

[0123]

[0124] Table 2 shows that the recoveries of spiked samples were all >85%, and the coefficients of variation were all <9%.

[0125] 2. Specificity assessment

[0126] Using the test strips from Example 2, the fluorescence intensity of 20 ng / mL solutions of carbendazim, isocarbophos, pythil, triadimefon, fenpropathrin, aldicarb, chlorothalonil, carbofuran, parathion-methyl, and acetamiprid was determined according to the immunochromatographic test strip detection procedure described above. The fluorescence intensity of the T line is shown in the figure. Figure 14 .

[0127] Figure 14 The results show that the AIE immunochromatographic test strip prepared in this invention has good specificity for acetamiprid.

[0128] 3. Examination of storage conditions and duration

[0129] The test strips from Example 2 were stored in a 55°C oven for 20 days. During this period, the test strips were periodically removed, and the T-line and C-line fluorescence intensities of negative samples were tested according to the procedure. Evaluation was performed every other day for the first six days, and every two days thereafter. The ratios of T-line and C-line fluorescence intensities at different times are shown in the figure. Figure 15 .

[0130] Figure 15 The results show that the AIE immunochromatographic test strips prepared by this invention are not affected by temperature during storage and have a long storage time.

[0131] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an aggregation-induced emission immunochromatographic test strip, characterized in that, Includes the following steps: (1) Polymethyl methacrylate, poly(maleic anhydride-alt-1-octadecene) and tetramethyl-4',4'',4''',4'''-(ethylene-1,1,2,2-tetramethyl)tetra([1,1'-biphenyl]-4-carboxylate) were dissolved in chloroform to obtain a mixture. The mixture was then added dropwise to an aqueous solution of sodium dodecyl sulfate and sonicated under ice bath conditions to obtain an emulsion. After the emulsion reaction, a microsphere suspension was obtained. After centrifugation, aggregation-induced emission microspheres were obtained. (2) The aggregation-induced emission microspheres were added to anhydrous ethanol, sonicated, and then ammonia solution was added. The mixture was stirred evenly, and tetraethyl orthosilicate was added dropwise. The mixture was stirred and centrifuged to obtain aggregation-induced emission silicon spheres. (3) The aggregation-induced light-emitting silicon spheres were added to anhydrous toluene, dispersed, and then 3-aminopropyltriethoxysilane was added. The mixture was refluxed and centrifuged to obtain aminated aggregation-induced light-emitting silicon spheres. The aminated aggregation-induced light-emitting silicon spheres were dispersed in N,N-dimethylformamide, and then succinic anhydride and catalyst were added. After stirring in the dark, the mixture was centrifuged to obtain carboxylated aggregation-induced light-emitting silicon spheres. The carboxylated aggregation-induced light-emitting silicon spheres were resuspended and coated on a carrier to prepare a monolayer film. Pt was sputtered onto the monolayer film, redispersed in water, and centrifuged to obtain self-driven aggregation-induced light-emitting silicon spheres. (4) Add the self-driven aggregation-induced luminescent silicon spheres to MES buffer, then add N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, stir in the dark and centrifuge, resuspend the precipitate, add acetamiprid monoclonal antibody, shake in the dark and add bovine serum albumin solution, continue shaking and centrifuge to obtain the self-driven aggregation-induced luminescent silicon sphere probe; (5) Prepare immunochromatographic test strips using the self-driven aggregation-induced light-emitting silicon sphere probe as a marker.

2. The method for preparing the aggregation-induced emission immunochromatographic test strip according to claim 1, characterized in that, In step (1), the ratio of polymethyl methacrylate, poly(maleic anhydride-alt-1-octadecene), tetramethyl-4',4'',4''',4'''-(ethylene-1,1,2,2-tetramethyl)tetra([1,1'-biphenyl]-4-carboxylate) to chloroform is 50 mg:25 mg:30 mg:1 mL; the volume ratio of the mixture to the sodium dodecyl sulfate aqueous solution is 1:5; the mass fraction of the sodium dodecyl sulfate aqueous solution is 0.25%; and the reaction conditions are rotary evaporation at 37°C for 20 min.

3. The method for preparing the aggregation-induced emission immunochromatographic test strip according to claim 1, characterized in that, In step (2), the ratio of the aggregate-induced emission microspheres to the tetraethyl orthosilicate is 1 mg: 55 μL.

4. The method for preparing the aggregation-induced emission immunochromatographic test strip according to claim 1, characterized in that, In step (3), the ratio of the aggregate-induced light-emitting silicon spheres to the 3-aminopropyltriethoxysilane is 2 mg: 5 μL; the reflux reaction temperature is 80 °C and the time is 24 h.

5. The method for preparing the aggregation-induced emission immunochromatographic test strip according to claim 1, characterized in that, In step (3), the mass ratio of the aminated aggregation-induced light-emitting silicon spheres to the succinic anhydride is 4:5; the catalyst is triethylamine; the stirring temperature in the dark is 25°C and the time is 12h.

6. The method for preparing the aggregation-induced emission immunochromatographic test strip according to claim 1, characterized in that, In step (3), the sputtering of Pt is performed using an ion sputtering instrument for a sputtering time of 40 seconds.

7. The method for preparing the aggregation-induced emission immunochromatographic test strip according to claim 1, characterized in that, In step (4), the mass ratio of the self-driven aggregation-induced light-emitting silicon spheres, the N-hydroxysuccinimide, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the acetamiprid monoclonal antibody is 4:2.4:3:0.2; the bovine serum albumin solution is prepared by adjusting the volume of 1g bovine serum albumin to 10mL; the light-protected shaking temperature is 25℃ for 4h; and the shaking temperature for continued shaking is 25℃ for 2h.

8. An aggregation-induced emission immunochromatographic test strip prepared by the method of any one of claims 1 to 7.

9. The application of the aggregation-induced emission immunochromatographic test strip of claim 8 in the detection of acetamiprid.

10. The application according to claim 9, characterized in that, The sample to be tested is diluted with hydrogen peroxide solution during the test.

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