A high-affinity self-assembled bichromatic metal-polyphenol network composite-based combined immunochromatographic test strip and application thereof

Abstract

The application belongs to the field of rapid detection of pesticide residues, and discloses a double-check immunochromatography test strip based on high-affinity self-assembled two-color metal-polyphenol network compound as well as a preparation method and application thereof. 2+ Fe is a metal ion, tannic acid and quercetin are polyphenol ligands, Fe-TA MPNCs and Fe-QUE MPNCs with different characteristic colors are prepared through a one-step self-assembly strategy, and are coupled with corresponding antibodies to form two-color detection probes. The MPNCs have good antibody coupling capacity and distinguishable color output. The two MPNCs with different colors are introduced into a double-check LFIA system, so that the synchronous visual detection of thiamethoxam and acetamiprid pesticide residues can be realized, and the rapid and intuitive interpretation of the detection result can be realized by establishing a 'one-to-one' colorimetric response relationship between the color signal and the target.

Description

Technical Field

[0001] This invention belongs to the field of rapid pesticide residue detection technology, and in particular relates to a combined detection immunochromatographic test strip based on a high-affinity self-assembled dual-color metal-polyphenol network complex and its application, aiming to achieve efficient and rapid on-site detection of thiamethoxam and acetamiprid pesticide residues. Background Technology

[0002] Thiamethoxam and acetamiprid are typical neonicotinoid pesticides, widely used in crop pest control due to their high efficiency and broad-spectrum insecticidal activity. However, pesticide residue problems caused by their residues are becoming increasingly prominent, posing a significant threat to consumer health, disrupting ecological balance, and impacting sustainable agricultural development. Therefore, developing accurate detection technologies for monitoring pesticide residues is of great importance. Currently, methods for detecting neonicotinoid pesticide residues mainly rely on large-scale instrumentation. While these methods offer good accuracy and reliable results, they suffer from drawbacks such as long processing times, complex operation, the need for specialized technicians, and expensive equipment, making them unsuitable for rapid on-site detection.

[0003] LFIA is a rapid detection technique based on the specific binding of a recognition element to a target on a paper-based substrate. It leverages the high specificity of immunological reactions and the high efficiency of chromatographic separation. Due to its ease of operation, rapid detection, and lack of complex instruments, it has gained widespread acceptance in fields such as food safety, clinical diagnostics, and environmental monitoring. However, traditional colorimetric LFIA using gold nanoparticles as signal markers is limited by poor color adjustment, cumbersome synthesis conditions, fixed material composition, and unclear nanobio-interactions with the recognition element, restricting its application in ultrasensitive detection and multi-pathway construction.

[0004] In recent years, self-assembly strategies have attracted attention due to their simplified synthesis processes and structural designability in the construction of nanomaterials. Among them, polyphenolic nanoparticles (MPNCs), typically formed by the coordination of metal ions and polyphenol molecules, are mainly used in functional coatings, biocarriers, and surface modification. However, the effective utilization of their inherent color properties and their compatibility with LFIA detection systems still lack systematic research. In particular, how to introduce MPNCs as stable and distinguishable colorimetric markers into multi-target LFIA systems and achieve clear differentiation and simultaneous detection among multiple targets remains an unsolved problem in current technologies.

[0005] Based on this, the present invention proposes a combined detection LFIA test strip based on high affinity self-assembled dual-color MPNCs, aiming to utilize the advantages of MPNCs such as easy color adjustment, simple synthesis conditions, diverse components, and enhanced nano-interface interaction to construct a "one-to-one" responsive combined detection LFIA for the simultaneous detection of thiamethoxam and acetamiprid. Summary of the Invention

[0006] The purpose of this invention is to provide a combined detection immunochromatographic test strip based on a high-affinity self-assembled dual-color metal-polyphenol network complex, its preparation method, and its application, for the simultaneous and rapid detection of thiamethoxam and acetamiprid. By constructing multi-color responsive MPNCs signal probes, the problem of single color signal and difficulty in achieving multi-color visualization detection in traditional colorimetric LFIA is solved.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a combined detection immunochromatographic assay (LFIA) test strip based on high affinity self-assembled dual-color metal-polyphenol network complexes (MPNCs), comprising a test strip body and a detection probe capable of binding to the analyte. The test strip body comprises a sample pad, a nitrocellulose membrane, absorbent paper, and a base plate connected in sequence. A first detection line (T1), a second detection line (T2), and a control line (C) are sequentially arranged along the chromatography direction on the nitrocellulose membrane. The first detection line (T1) is coated with thiamethoxam artificial conjugate antigen (THI-OVA), the second detection line (T2) is coated with acetamiprid artificial conjugate antigen (ACE-OVA), and the control line (C) is coated with goat anti-mouse secondary antibody. The detection probe comprises Fe... 2+ The first detection probe, Fe-TA MPNCs@mAb1, is obtained by conjugating purple Fe-TA MPNCs (formed by one-step self-assembly of tannic acid (TA)) with thiamethoxam antibody, and Fe... 2+ The second detection probe, Fe-QUE MPNCs@mAb2, was obtained by conjugating the brown-green Fe-QUEMPNCs formed by one-step self-assembly with quercetin (QUE) with acetamiprid antibody.

[0008] The test strips use two types of self-assembled MPNCs as colorimetric signal markers: (1) one type uses Fe 2+ Fe-TA MPNCs, which are formed by self-assembly with TA and are purple, are used to label thiamethoxam antibody (mAb1); (2) another type is Fe 2+ Fe-QUE MPNCs, which are brownish-green in color, are formed by self-assembly with QUE and are used to label acetamiprid antibody (mAb2). The two probes can competitively bind to the target pesticide molecule and are recognized and captured at the corresponding detection lines, thereby generating response signals of different colors.

[0009] Furthermore, the concentration of thiamethoxam artificial conjugate antigen sprayed on the first detection line is 0.1-0.6 mg / mL, the concentration of acetamiprid artificial conjugate antigen sprayed on the second detection line is 0.2-1.2 mg / mL, the concentration of goat anti-mouse secondary antibody sprayed on the quality control line is 0.8 mg / mL, the concentration of thiamethoxam antibody used in the first detection probe Fe-TA MPNCs@mAb1 is 1-6 μg, and the concentration of acetamiprid antibody used in the second detection probe Fe-QUE MPNCs@mAb2 is 1-6 μg.

[0010] Furthermore, the preparation method of the detection probe includes the following steps:

[0011] S1, adjust MPNCs to pH 7;

[0012] S2, Add the antibody to be labeled to the MPNCs obtained in step S1 and incubate;

[0013] S3, Add a blocking agent to the product obtained in step S2 to carry out a blocking reaction;

[0014] S4. The product obtained in step S3 is centrifuged to collect the precipitate, thus obtaining the detection probe.

[0015] Specifically, the construction methods of the first and second detection probes include: adjusting the pH of Fe-TA MPNCs or Fe-QUE MPNCs to 7, adding the corresponding antibody (Ab1 or Ab2) for incubation, then adding a blocking agent to block unbound sites, centrifuging to remove free antibody, and obtaining stable Fe-TA MPNCs@Ab1 and Fe-QUE MPNCs@Ab2 probes.

[0016] Furthermore, the synthesis method of the MPNCs includes the following steps: Fe 2+ The solution and polyphenol solution were added sequentially to phosphate buffer and reacted at room temperature for 30 minutes. The resulting MPNCs product was collected by centrifugation and washed at least twice with ultrapure water to remove unreacted components.

[0017] Both the first detection probe Fe-TA MPNCs@mAb1 and the second detection probe Fe-QUE MPNCs@mAb2 can be rapidly synthesized via a one-step self-assembly method. By simply replacing the polyphenol ligand, two products with different inherent colors can be obtained. This method has the advantages of mild synthesis conditions, simple operation, and easy color adjustment.

[0018] Furthermore, the MPNCs exhibit an irregular network structure with a particle size ranging from 100 to 400 nm; Fe 2+ The mass ratio of the solution to the polyphenol solution was 8:1; the pH of the phosphate buffer solution was 7.

[0019] Furthermore, the preparation method of the test strip body includes the following steps:

[0020] 1) Pretreatment of sample pads: Immerse the sample pads in 0.01 M phosphate buffer (pH 7.4) containing 1% BSA, 0.05% sodium azide and 0.5% Tween 20, and dry them for later use;

[0021] 2) Spraying the nitrocellulose membrane: Dilute the immunoreaction reagents (thiamethoxam artificial antigen, acetamiprid artificial antigen, and goat anti-mouse secondary antibody) in PBS buffer, and spray them onto the first detection line, second detection line, and control line of the nitrocellulose membrane, respectively, and place them at 37°C to dry overnight;

[0022] 3) Assemble the test strip: Attach the treated nitrocellulose membrane to the center of the base plate, attach the sample pad and absorbent paper to both ends, overlap by 1-2 mm and press firmly, cut the test strip to 3 mm wide, and store under dry conditions.

[0023] Furthermore, the preparation of the artificial conjugated antigen includes: covalently conjugating the hapten of thiamethoxam or acetamiprid with a protein carrier (OVA) using an activator (EDC / NHS), and purifying the resulting conjugate by dialysis and spraying it onto the T-line position.

[0024] The present invention also discloses the application of the above-mentioned immunochromatographic test strip based on a high-affinity self-assembled dual-color metal-polyphenol network complex in the detection of thiamethoxam and acetamiprid pesticide residues.

[0025] Specifically, the determination steps include: incubating the sample with the constructed probe, then dropping it onto the sample pad of the test strip. After chromatographic migration, the color changes of the first detection line (T1) and the second detection line (T2) are read by visual observation or an image analysis system to achieve rapid quantitative detection of the target pesticide residue.

[0026] Furthermore, the detection method is as follows:

[0027] The first detection probe Fe-TA MPNCs@mAb1 and the second detection probe Fe-QUE MPNCs@mAb2 were added to the mixed sample of thiamethoxam and acetamiprid and incubated. Then, they were dropped onto the sample pad of the test strip. After the chromatography reaction was completed, the color changes of the first detection line, the second detection line and the control line were observed for qualitative analysis.

[0028] or,

[0029] The first detection probe Fe-TA MPNCs@mAb1 and the second detection probe Fe-QUE MPNCs@mAb2 were added to the mixed sample of thiamethoxam and acetamiprid and incubated. Then, they were dropped onto the sample pad of the test strip. After the chromatographic reaction was completed, the test strip was photographed, and the gray values ​​of each line were analyzed using ImageJ software. A standard curve was plotted with the logarithm of the target concentration as the abscissa and the gray intensity as the ordinate for quantitative analysis.

[0030] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

[0031] (1) This study focuses for the first time on the inherent color properties of MPNCs and introduces them as multicolor signal markers into a combined detection LFIA platform, enabling the simultaneous detection of thiamethoxam and acetamiprid. The probe color can be controlled by adjusting the combination of metal ions and polyphenol ligands. Among them, the metal ion Fe... 2+ Due to their unfilled d orbitals, polyphenols exhibit unique electronic properties, enabling electronic transitions within complexes and generating specific optical responses. This determines the color attributes of MPNCs, allowing for tunable probe colors and different colorimetric outputs for different targets. This effectively overcomes the technical bottleneck of traditional gold nanoparticle (GNP) colorimetric LFIA, which suffers from single color and difficulty in distinguishing multiple targets. On the other hand, polyphenols, with their excellent coordination ability, tunability, and multifunctionality, can form stable coordination complexes with metal ions through their phenolic hydroxyl groups. By establishing a one-to-one colorimetric response relationship between the color signal and the target, rapid and intuitive interpretation of detection results is achieved, providing a novel, reliable, and easily applicable technical solution for rapid on-site detection of pesticide residues.

[0032] (2) MPNCs are constructed using a self-assembly strategy. The reaction conditions are mild, and the synthesis process does not require high temperature, high pressure, or complex modification. The steps are simple and reproducible, making it suitable for rapid preparation under ordinary laboratory conditions. This method is stable, the raw materials are widely available, and it is easy to scale up production, which is expected to reduce the overall cost of detection.

[0033] (3) MPNCs have a higher affinity for antibodies, enabling higher density and more stable antibody conjugation. The good surface chemical properties of MPNCs help improve probe construction efficiency, thereby enhancing the ability of immune recognition and colorimetric signal output.

[0034] (4) The dual-channel joint inspection system constructed in this invention has good specificity and sensitivity. The color response of the detection line is clear, the signal-to-noise ratio is high, the results can be read by the naked eye, and it is also suitable for image analysis, realizing qualitative or quantitative detection, and meeting the actual needs of rapid on-site screening and grassroots supervision.

[0035] (5) The technology established by this invention has good versatility and scalability. By changing the antibody and artificial antigen, it can be adapted to more types of pesticide, veterinary drug residues or other toxic small molecules. It has the potential to build a multi-stage rapid screening system. It has broad application prospects in agricultural product quality and safety testing, environmental pollution assessment and food processing monitoring, and can provide effective technical support for the food safety assurance system. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of (A) the preparation of multicolor MPNCs in this invention and (B) their application in multi-channel LFIA.

[0037] Figure 2 (A) Physical image, (B) Absorption spectrum, (C and D) Transmission electron microscopy images, and (E and F) Hydration particle size diagrams of Fe-TA MPNCs and Fe-QUE MPNCs.

[0038] Figure 3 The images show the zeta potentials of Fe-TA MPNCs before and after conjugation with thiamethoxam antibody (A and B), and the test strips before and after conjugation of Fe-QUE MPNCs with acetamiprid antibody (C and D), as well as the binding of the test strips to goat anti-mouse secondary antibody.

[0039] Figure 4 This study compares and evaluates the protein coupling performance of MPNCs probes and traditional GNPs probes, including: (A) schematic diagram of probe construction based on MPNCs and GNPs; (B) SDS-PAGE results of Fe-QUE MPNCs@mAb and GNPs@mAb for acetamiprid and (C) gray intensity analysis of the corresponding bands; (D) SDS-PAGE results of Fe-TA MPNCs@mAb and GNPs@mAb for thiamethoxam and (E) corresponding band intensity analysis.

[0040] Figure 5 To examine the (A) selective response of LFIA to different neonicotinoid pesticide combinations (thiamethoxam / acetamiprid concentrations i: 0 / 0; ii: 10 / 0; iii: 0 / 10; iv: 10 / 10 ng / mL) and (B) corresponding signal intensities.

[0041] Figure 6 (A) Physical image and (B and C) standard curves for multi-pathway LFIA for simultaneous detection of thiamethoxam and acetamiprid.

[0042] Figure 7 Heatmap showing the recovery rates of thiamethoxam and acetamiprid in food samples using multi-pathway LFIA. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0044] Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0045] Example 1: Fabrication of MPNC-based probes:

[0046] With Fe 2+ Taking the preparation of Fe-QUE MPNCs with quercetin (QUE) as an example, the specific synthesis steps are as follows ( Figure 1 A): Add 2 mL of phosphate buffer (0.02 M, pH 7) to a clean glass vial. While stirring vigorously, add 160 μL of Fe... 2+ The Fe-QUE MPNCs were prepared by reacting the mixture in 10 μL of solution (10 mg / mL) and 60 μL of QUE solution (5 mg / mL). After reacting at room temperature for 30 minutes, the mixture was centrifuged at 5500 rpm for 5 minutes to collect the assembled Fe-QUE MPNCs, and washed repeatedly with ultrapure water to remove residual reagents. Finally, the obtained Fe-QUE MPNCs were redispersed in ultrapure water for subsequent use. The preparation method of Fe-TA MPNCs is similar to that of Fe-QUE MPNCs, except that the corresponding polyphenol is replaced with tannic acid (TA), maintaining the molar ratio of metal ions to polyphenols at 8:1, and the final molar concentration of metal ions to polyphenols is the same as that of Fe. 2+ Same as QUE.

[0047] The prepared Fe-TA MPNCs and Fe-QUE MPNCs were purple and brownish-green, respectively. Figure 2 A), the positions of the characteristic peaks in the corresponding absorption spectra also differ ( Figure 2 B). Transmission electron microscopy images show ( Figure 2 (C and 2D), Fe-TA MPNCs and Fe-QUE MPNCs formed an irregular network structure with a rough surface and good dispersibility. The hydration particle sizes were measured to be 272.0 nm and 206.0 nm, respectively, with PDI values ​​of 0.147 and 0.136, respectively. Figure 2 E and 2F).

[0048] Thiamethoxam antibody (mAb1) and acetamiprid antibody (mAb2) were conjugated to Fe-TA MPNCs and Fe-QUE MPNCs, respectively. Taking Fe-QUE MPNCs@mAb2 as an example, 100 μL of mAb2 solution was added to 1 mL of Fe-QUE MPNCs solution (0.25 mg / mL), and incubated at room temperature for 1 hour. Then, 100 μL of blocking agent was added to the mixture and incubated for 0.5 hours to block excess conjugation sites on the Fe-QUE MPNCs surface. The mixture was centrifuged at 5500 rpm at 4°C for 5 minutes, the supernatant was discarded, and the mixture was redispersed in 100 μL of storage buffer. The preparation process for Fe-TA MPNCs@mAb1 was the same, using Fe-TAMPNCs as a signal marker and mAb1 as an immunorecognition element.

[0049] The successful preparation of Fe-TAMPNCs@mAb1 and Fe-QUEMPNCs@mAb2 was confirmed by changes in zeta potential and binding experiments with goat anti-mouse secondary antibody on test strips. Figure 3 After antibody conjugation, the zeta potential of the probes changed, and they could specifically bind to the C line of the test strip.

[0050] Example 2: Determination of probe coupling ability:

[0051] Selected MPNCs and traditional GNPs were conjugated with antibodies to prepare detection probes. Figure 4 A). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the bound antibody after probe surface denaturation treatment showed two clear bands at approximately 25 kDa (light chain) and 55 kDa (heavy chain), consistent with the control group (free antibody). Figure 4 B and 4D). Quantitative analysis of band intensity showed that the amount of antibody adsorbed on Fe-QUE MPNCs and Fe-TA MPNCs was higher than that on GNPs (B and 4D). Figure 4 (C and 4E) indicates that MPNCs have superior antibody adsorption capacity.

[0052] Example 3: Preparation of multi-channel LFIA test strips:

[0053] The multi-channel LFIA test strip consists of four parts: a sample pad, a nitrocellulose membrane with three parallel lines (two T lines and one C line), absorbent paper, and a base plate as a carrier support. Thiamethoxam artificial antigen (THI-OVA, 0.4 mg / mL), acetamiprid artificial antigen (ACE-OVA, 0.6 mg / mL), and goat anti-mouse secondary antibody (0.8 mg / mL) are sequentially sprayed onto the nitrocellulose membrane to form T1, T2, and C lines, respectively, and then dried in a 37°C oven. Before use, the sample pad is pretreated by soaking in phosphate buffer (0.01 M, pH 7.4) containing 1% BSA, 0.05% sodium azide, and 0.5% Tween 20, and then dried. Next, the nitrocellulose membrane is adhered to the center of the base plate, and the sample pad and absorbent paper are adhered to the sides, ensuring a 1-2 mm overlap between each component. After compression, cut into 3 mm wide test strips and store under dry conditions.

[0054] Example 4: Analytical performance of MPNCs-LFIA in detecting acetamiprid and thiamethoxam:

[0055] A typical testing procedure is as follows: 6 μL of Fe-TA MPNCs@mAb1 probe and 5 μL of Fe-QUE MPNCs@mAb2 probe are incubated with 100 μL of a mixed solution of thiamethoxam and acetamiprid (final concentrations of 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, and 50 ng / mL, respectively) for 5 minutes. Subsequently, the solution is dropped onto a sample pad for chromatographic reaction.

[0056] Observe the color of the T-line after 30 minutes. Next, photograph the test strip using a smartphone and analyze the grayscale intensity of the bands using ImageJ software. Plot a standard curve with the logarithm of the analyte concentration on the x-axis and grayscale intensity on the y-axis. For qualitative analysis, the COV (Cutoff Value) is defined as the lowest target analyte concentration at which the T-line disappears under naked-eye observation. For quantitative analysis, the LOD (Limit of Detection) is defined as the target analyte concentration corresponding to the grayscale intensity of a negative sample minus three standard deviations.

[0057] The detection employs a competitive strategy design. Figure 1 B): The detection antigens on the T1 and T2 lines compete with thiamethoxam or acetamiprid in the sample for binding to the detection probes. Increased concentrations of thiamethoxam or acetamiprid lead to a decrease in the number of probes captured by their respective T lines. Regarding signal output, the purple signal on the T1 line gradually weakens with increasing thiamethoxam concentration; the brown-green signal on the T2 line gradually weakens with increasing acetamiprid concentration. The goat anti-mouse secondary antibody on the C line specifically captures excess probes, ensuring valid results.

[0058] The dual detection capability of the LFIA (Fluid Level Immunoassay) system was verified by testing samples containing single and mixed pesticides. In pesticide-free samples, two different colored bands appeared at the two T-lines. For samples containing only one pesticide, the corresponding T-line band disappeared, while the T-line band for the other target analyte remained. In samples containing both pesticides, the colors of both T-lines disappeared. Figure 5 A). The results show that the LFIA can accurately distinguish between positive and negative samples without any cross-interference. Figure 5 B).

[0059] Analysis of mixed samples containing different concentrations of thiamethoxam and acetamiprid showed that as the concentrations of the two pesticides increased, the bands corresponding to the T1 and T2 lines gradually disappeared, and the COV for qualitative analysis of both was 5 ng / mL. -1 ( Figure 6 A). For quantitative analysis, the detection range of thiamethoxam using multi-pathway LFIA is 0.05-2 ng / mL. -1 The detection range for acetamiprid is 0.05-5 ng / mL. -1 The fitted linear regression equations are y = -3796 log(x) + 1528 (R²) 2 = 0.9840) and y = -3334log(x) + 2217 (R 2 = 0.9758), with corresponding LODs of 0.036 ng mL. -1 and 0.041 ng mL -1 ( Figure 6 B and 6C).

[0060] Example 5: Application in food samples:

[0061] The pretreatment methods for three food samples (honey, white radish, and melon) were as follows: First, the collected white radish and melon samples were thoroughly homogenized, while the honey sample was used directly. Then, a certain volume of thiamethoxam and acetamiprid standard solutions were spiked to 1 g of each sample at different concentrations. After standing for 2 hours, 5 mL of PBS buffer (0.01 M, pH 7.4) was added for extraction, followed by thorough shaking and vortex mixing. The mixture was centrifuged at 4000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter membrane. Finally, the filtrate was diluted 6-fold with PBS buffer (0.01 M, pH 7.4) to reduce matrix effects and used for subsequent multi-pathway LFIA analysis. The recovery rate was calculated using the following formula:

[0062]

[0063] Recovery results indicate that ( Figure 7The average recoveries of thiamethoxam ranged from 84.12% to 116.92%, while those of acetamiprid ranged from 86.81% to 112.41%, with coefficients of variation both below 15%, indicating that this method is suitable for the accurate detection of pesticide residues in food samples.

[0064] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A combined immunochromatographic test strip based on a high-affinity self-assembled dual-color metal-polyphenol network complex, comprising a test strip body and a detection probe capable of binding to the analyte, wherein the test strip body comprises a sample pad, a nitrocellulose membrane, absorbent paper, and a base plate connected in sequence, and a first detection line, a second detection line, and a control line are sequentially arranged on the nitrocellulose membrane along the chromatography direction, characterized in that: The first detection line is coated with thiamethoxam artificial conjugated antigen, the second detection line is coated with acetamiprid artificial conjugated antigen, and the quality control line is coated with goat anti-mouse secondary antibody; the detection probe comprises Fe 2+ The first detection probe, Fe-TA MPNCs@mAb1, was obtained by conjugating purple Fe-TA MPNCs (formed by one-step self-assembly with tannins) with thiamethoxam antibody, and Fe... 2+ The second detection probe, Fe-QUE MPNCs@mAb2, was obtained by conjugating the brown-green Fe-QUE MPNCs formed by one-step self-assembly with quercetin with acetamiprid antibody.

2. The test strip according to claim 1, characterized in that: The concentration of thiamethoxam artificial conjugate antigen sprayed on the first detection line is 0.1-0.6 mg / mL, the concentration of acetamiprid artificial conjugate antigen sprayed on the second detection line is 0.2-1.2 mg / mL, the concentration of goat anti-mouse secondary antibody sprayed on the quality control line is 0.8 mg / mL, the concentration of thiamethoxam antibody used in the first detection probe Fe-TAMPNCs@mAb1 is 1-6 μg, and the concentration of acetamiprid antibody used in the second detection probe Fe-QUEMPNCs@mAb2 is 1-6 μg.

3. The test strip according to claim 1, characterized in that: The method for preparing the detection probe includes the following steps: S1, adjust MPNCs to pH 7; S2, Add the antibody to be labeled to the MPNCs obtained in step S1 and incubate; S3, Add a blocking agent to the product obtained in step S2 to carry out a blocking reaction; S4. The product obtained in step S3 is centrifuged to collect the precipitate, thus obtaining the detection probe.

4. The test strip according to claim 3, characterized in that: The method for synthesizing the MPNCs includes the following steps: Fe 2+ The solution and polyphenol solution were added sequentially to phosphate buffer and reacted at room temperature for 30 minutes. The resulting MPNCs product was collected by centrifugation and washed at least twice with ultrapure water to remove unreacted components.

5. The test strip according to claim 4, characterized in that: The MPNCs exhibit an irregular network structure with a particle size ranging from 100 to 400 nm; Fe 2+ The mass ratio of the solution to the polyphenol solution was 8:1; the pH of the phosphate buffer solution was 7.

6. The test strip according to claim 1, characterized in that: The preparation method of the test strip body includes the following steps: 1) Pretreatment of sample pads: Immerse the sample pads in 0.01 M phosphate buffer containing 1% BSA, 0.05% sodium azide and 0.5% Tween 20, and dry them for later use; 2) Spraying the nitrocellulose membrane: Dilute the immunoreaction reagent in PBS buffer and spray it onto the first detection line, second detection line and control line of the nitrocellulose membrane, and dry it at 37°C overnight; 3) Assemble the test strip: Attach the treated nitrocellulose membrane to the center of the base plate, attach the sample pad and absorbent paper to both ends, overlap by 1-2 mm and press firmly, cut the test strip to 3 mm wide, and store under dry conditions.

7. The application of a combined immunochromatographic test strip based on a high-affinity self-assembled dual-color metal-polyphenol network complex as described in any one of claims 1-6 in the detection of thiamethoxam and acetamiprid pesticide residues.

8. The application according to claim 7, characterized in that, The detection method is as follows: The first detection probe Fe-TA MPNCs@mAb1 and the second detection probe Fe-QUE MPNCs@mAb2 were added to the mixed sample of thiamethoxam and acetamiprid and incubated. Then, they were dropped onto the sample pad of the test strip. After the chromatography reaction was completed, the color changes of the first detection line, the second detection line and the control line were observed for qualitative analysis. or, The first detection probe Fe-TA MPNCs@mAb1 and the second detection probe Fe-QUE MPNCs@mAb2 were added to the mixed sample of thiamethoxam and acetamiprid and incubated. Then, they were dropped onto the sample pad of the test strip. After the chromatographic reaction was completed, the test strip was photographed, and the gray values ​​of each line were analyzed using software. A standard curve was plotted with the logarithm of the target concentration as the abscissa and the gray intensity as the ordinate for quantitative analysis.

Images