Bimodal immunochromatography test strip and application thereof

Abstract

The invention discloses a bimodal immunochromatography test strip and application thereof, and relates to the technical field of medical examination and food safety detection. The bimodal immunochromatography test strip takes AuNFs (at) TA / Zr as a probe, and a preparation method of the AuNFs (at) TA / Zr comprises the following steps: preheating deionized water, and adding a chloroauric acid aqueous solution to obtain a chloroauric acid solution; the preparation method comprises the following steps: dissolving tannic acid and a hydroxylamine hydrochloride aqueous solution in deionized water to obtain a mixed solution; reacting a chloroauric acid solution with the mixed solution to obtain an AuNFs (at) TA solution; zirconium chloride is added into the AuNFs (at) TA solution, and AuNFs (at) TA / Zr is obtained; the invention also provides application of the bimodal immunochromatography test strip in detection of pesticides or pathogenic bacteria. The immunochromatographic test strip has the beneficial effects that the immunochromatographic test strip with colorimetric and photo-thermal dual-mode signals is prepared by taking AuNFs (at) TA / Zr as a beacon carrier, and the immunochromatographic test strip has the characteristics of high stability and high detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical fields of medical testing and food safety detection, and particularly relates to a dual-modal immunochromatographic test strip and its application. Background Art

[0002] Immunochromatography is a rapid detection method based on the principle of specific binding between antigen and antibody. With its advantages of fast detection speed, strong specificity, simple operation and low cost, it has become the most mature on-site rapid detection technology at present and is widely used in the fields of clinical diagnosis, food safety detection, medical testing and environmental pollutant monitoring. Among them, the colloidal gold immunochromatographic test strip, as a classic application form, although it has the convenience of instant detection, its sensitivity is low and it is easily affected by interfering substances in complex sample matrices, which limits its application in trace analysis.

[0003] In view of the above limitations, in recent years, immunochromatography has reduced matrix interference by immunomagnetic separation and enrichment of target substances, combined with signal amplification strategies such as the biotin-streptavidin system and nanozymes to improve the sensitivity to the pg / mL level. At the same time, new labeling materials such as quantum dots and time-resolved fluorescence microspheres have been developed, and the traditional colloidal gold limitation has been broken through by using fluorescence enhancement or magnetic signal quantification modes, realizing the synergistic optimization of sensitivity and anti-interference, and promoting its application upgrade in trace biomarker detection and multi-target analysis.

[0004] For immunochromatographic detection technology, the stability and biocompatibility of nanoprobes are two important parameters. Stability determines the reliability of the detection method and the storage period of the detection product; biocompatibility affects the coupling of nanoprobes with recognition molecules, thus affecting the universality of the method. At present, the stability and biocompatibility of the nanoprobes used in immunochromatography in the prior art are average, and there are technical problems of poor universality and low detection sensitivity in the detection of pesticides or pathogenic bacteria, etc. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and provide a dual-modal immunochromatographic test strip and its application. Specifically, a dual-modal immunochromatographic test strip with metal polyphenol-coated flower-like gold nanoparticles (AuNFs@TA / Zr) and its application are designed. The present invention synthesizes a metal polyphenol-coated flower-like gold nanoparticles (AuNFs@TA / Zr) as a probe, which is applied to the fields of medical testing and rapid food safety detection, etc., and improves the stability and detection sensitivity of the test strip.

[0006] On the one hand, the present invention synthesizes a flower-like gold nanoparticle (AuNFs). AuNFs, with its unique dendritic multi-level structure, show performance advantages that are significantly superior to traditional colloidal gold, including a larger specific surface area (5-8 times that of spherical particles), enhanced localized surface plasmon resonance effect and peroxidase-like catalytic activity (efficiency increased by 3-5 times), which can significantly improve the signal transduction ability of the sensor probe. On the other hand, the present invention also uses metal (Zr) and polyphenol (tannic acid, referred to as TA) to coat AuNFs, thereby preparing a dual-modal (colorimetric and photothermal dual-modal signal) immunochromatographic test strip. The prepared AuNFs@TA / Zr has good dispersibility, good biocompatibility, good colorimetric performance, and superior photothermal performance, and can be used as a new colorimetric / photothermal signal label in a dual-modal immunochromatographic test strip. Compared with colloidal gold test strips, dual-modal immunochromatographic test strips prepared with metal polyphenol-coated flower-like gold nanoparticles (AuNFs@TA / Zr) as beacon carriers have the advantages of high sensitivity and short detection time.

[0007] The technical solution of the present invention is as follows: The first aspect of the present invention provides a dual-modal immunochromatographic test strip, wherein the dual-modal immunochromatographic test strip uses AuNFs@TA / Zr as a probe; The preparation method of AuNFs@TA / Zr comprises the following steps: S1. After preheating deionized water, add chloroauric acid aqueous solution and mix evenly to obtain chloroauric acid solution; S2, dissolving tannic acid and hydroxylamine hydrochloride aqueous solution in deionized water, and mixing thoroughly to obtain a mixed solution; S3, reacting the chloroauric acid solution with the mixed solution, cooling and centrifuging to obtain a precipitate after the reaction is completed; washing the precipitate and redissolving it in water to obtain an AuNFs@TA solution; S4. Add zirconium chloride to the AuNFs@TA solution, stir and mix, centrifuge and wash to obtain AuNFs@TA / Zr.

[0008] Preferably, in step S1, the temperature of the preheated deionized water is 65°C to 75°C, the mass concentration of the chloroauric acid aqueous solution is 0.5% to 1.5%, and the added volume ratio of deionized water to chloroauric acid solution is 95 to 103:1.

[0009] Preferably, in step S2, the addition ratio of tannic acid and hydroxylamine hydrochloride aqueous solution is 18 mg-22 mg:0.5 mL, and the concentration of hydroxylamine hydrochloride aqueous solution is 0.05 mol / L-0.15 mol / L.

[0010] Preferably, in step S3, the cooling condition is room temperature, the cooling time is 35 min to 45 min, the centrifugal speed is 3000 r / min to 12000 r / min, the centrifugal time is 10 min to 30 min, the number of washing times is 1 to 3 times, and the volume of water added for re-dissolution is 7 mL to 9 mL.

[0011] Preferably, in step S4, the addition ratio of zirconium chloride to chloroauric acid solution is 4 mg~6 mg:1 mL, the stirring speed is 1000 r / min~1400 r / min, the stirring time is 20 h~28 h, the centrifugal speed is 3000 r / min~12000 r / min, the centrifugal time is 10 min~30 min, and the number of washing times is 1 time~3 times.

[0012] Preferably, the AuNFs@TA / Zr is loaded on the dual-modal immunochromatographic test strip in the form of an AuNFs@TA / Zr labeled antibody complex, wherein the preparation method of the AuNFs@TA / Zr labeled antibody complex comprises the following steps: AuNFs@TA / Zr was dissolved in water to obtain AuNFs@TA / Zr solution. The antibody to be labeled is added to the AuNFs@TA / Zr solution, and a blocking agent is added after sufficient mixing. After the reaction, the solution is centrifuged to obtain a precipitate, and the obtained precipitate is redissolved to prepare an AuNFs@TA / Zr labeled antibody complex.

[0013] Preferably, in the method for preparing the AuNFs@TA / Zr labeled antibody complex, The final concentration of the antibody after adding the antibody to be labeled is 1 μg / mL to 100 μg / mL; After the blocking agent is added, the final mass concentration of the blocking agent is 0.1% to 1%, and the blocking agent is selected from any one of casein, bovine serum albumin, ovalbumin, polyethylene glycol, and skim milk; The centrifugal speed is 5000 r / min to 20000 r / min, and the centrifugal time is 10 min to 50 min; The precipitate after centrifugation was reconstituted with 0.01M-0.1M phosphate buffer, pH 6.0-9.0; The antibody to be labeled includes any one of monoclonal antibody, polyclonal antibody, nano antibody, and phage-expressed antibody.

[0014] Preferably, the dual-modal immunochromatographic test strip comprises: Base plate; A nitrocellulose membrane is arranged on the bottom plate, and the nitrocellulose membrane has detection lines and quality control lines arranged at intervals; A glass fiber pad is disposed at one end of the nitrocellulose membrane having a test line. One end of the glass fiber pad is disposed on the bottom plate, and the other end is disposed on the nitrocellulose membrane. The AuNFs@TA / Zr labeled antibody complex is disposed on the glass fiber pad; A blotting paper is disposed at one end of the nitrocellulose membrane having a quality control line. One end of the blotting paper is disposed on the bottom plate, and the other end is disposed on the nitrocellulose membrane; A sample pad, one end of which is disposed on the bottom plate and the other end is disposed on the glass fiber pad.

[0015] Preferably, the method for disposing the AuNFs@TA / Zr labeled antibody complex on the glass fiber pad includes: spraying the AuNFs@TA / Zr labeled antibody complex onto the glass fiber pad at a volume of 7 μL / cm to 9 μL / cm, and drying in vacuum for 1.5 h to 2.5 h.

[0016] Preferably, the test line is coated with an artificial conjugate antigen or an antibody of the analyte to be detected, and the quality control line is coated with a goat anti-mouse antibody.

[0017] The method for forming a test line and a quality control line on the nitrocellulose membrane includes the following steps: Using a PBS solution (phosphate buffer solution) with a concentration of 0.01 M to 0.5 M and a pH of 6.0 to 8.0 to adjust the artificial conjugate antigen or antibody of the analyte to be detected and the goat anti-mouse antibody to a concentration of 0.01 mg / mL to 10.0 mg / mL respectively; wherein, the antibody of the analyte to be detected includes monoclonal antibody, polyclonal antibody, nanobody, phage-expressed antibody; Spraying the artificial conjugate antigen or antibody of the analyte to be detected on the upper part of the nitrocellulose membrane as the test line, and spraying the goat anti-mouse antibody on the lower part of the nitrocellulose membrane as the quality control line; wherein, the spraying amount of the artificial conjugate antigen or antibody of the analyte to be detected and the goat anti-mouse antibody on the membrane is 0.25 μL / cm to 0.74 μL / cm; Drying the nitrocellulose membrane sprayed with the test line and the quality control line.

[0018] Preferably, the assembly of the test strip includes the following steps: (1) Overlapping and pasting the following materials on the bottom plate: filter paper, sample pad, glass fiber pad sprayed with AuNFs@TA / Zr labeled antibody complex, nitrocellulose membrane sprayed with an artificial conjugate antigen or antibody of the analyte to be detected as the test line and anti-mouse antibody / anti-rabbit antibody as the quality control line, and blotting paper, that is, assembling the dual-mode immunochromatographic test strip plate prepared by using AuNFs@TA / Zr as the beacon carrier of the present invention; (2)The assembled test strip plate is cut into the required width by a cutter, which is the dual-mode immunochromatographic test strip prepared by using AuNFs@TA / Zr as the beacon carrier in the present invention. This test strip can be used directly or can be used after being inserted into a plastic cartridge.

[0019] The second aspect of the present invention provides an application of the above-mentioned dual-mode immunochromatographic test strip in detecting pesticides or pathogenic bacteria for non-disease diagnosis purposes. The method for detecting pesticides or pathogenic bacteria by using the dual-mode immunochromatographic test strip includes the following steps: The processed sample to be tested is added to the dual-mode immunochromatographic test strip. The added sample volume is 50 μL - 200 μL, and the reaction time is 3 minutes - 30 minutes. Quantitative detection is achieved by reading the gray-scale data of the test strip and the imaging data of an infrared imager and calculating the concentration of the sample to be tested, or qualitative judgment of the sample to be tested is achieved by visually observing whether there are colored bands on the test line and the quality control line of the dual-mode immunochromatographic test strip.

[0020] The present invention has at least one of the following beneficial effects: The present invention synthesizes a flower-like gold nanoparticle (AuNFs), and uses metal and polyphenol to wrap the flower-like gold nanoparticle to form AuNFs@TA / Zr, and prepares a dual-mode immunochromatographic test strip with AuNFs@TA / Zr as the beacon carrier for detecting pathogenic bacteria or pesticides, etc. Compared with the traditional colloidal gold test strip, the dual-mode immunochromatographic test strip prepared in the present invention has colorimetric and photothermal dual-mode signals, and the signals are strong. The prepared dual-mode immunochromatographic test strip has high detection sensitivity and can realize rapid quantitative or qualitative detection of various analytes. Description of the Drawings

[0021] Figure 1 is a schematic structural diagram of the dual-mode immunochromatographic test strip. Among them, 1 is the sample pad, 2 is the glass fiber pad, 3 is the nitrocellulose membrane, 4 is the test line, 5 is the quality control line, 6 is the bottom plate, and 7 is the absorbent paper.

[0022] Figure 2 is the schematic detection principle diagram of the sandwich method AuNFs@TA / Zr immunochromatographic test strip. Among them, 8 is the analyte antibody on the test line, 9 is the anti-mouse antibody or anti-rabbit antibody on the quality control line, 10 is the macromolecular analyte, and 11 is the AuNFs@TA / Zr-antibody complex.

[0023] Figure 3 is the schematic detection principle diagram of the competitive method AuNFs@TA / Zr immunochromatographic test strip. Among them, 9 is the anti-mouse antibody or anti-rabbit antibody on the quality control line, 10 is the small molecule analyte, 11 is the AuNFs@TA / Zr-antibody complex, and 12 is the analyte artificial conjugate antigen on the test line.

[0024] Figure 4 The transmission electron microscopy images of AuNFs@TA / Zr are shown. Among them, a in the figure is the transmission electron microscopy image with a scale bar of 0.2 μm, b in the figure is the transmission electron microscopy image with a scale bar of 100 nm, c in the figure is the transmission electron microscopy image with a scale bar of 50 nm, and d in the figure is the transmission electron microscopy image with a scale bar of 10 nm. Figure 5 The colorimetric physical map of the dual-mode immunochromatographic test strip using AuNFs@TA / Zr as the beacon carrier for detecting Escherichia coli E. coli O157:H7 is shown.

[0025] Figure 6 The colorimetric physical map of the traditional colloidal gold test strip for detecting Escherichia coli O157:H7 is shown.

[0026] Figure 7 The photothermal physical map of the dual-mode immunochromatographic test strip using AuNFs@TA / Zr as the beacon carrier for detecting Escherichia coli O157:H7 is shown.

[0027] Figure 8 The colorimetric physical map of the dual-mode immunochromatographic test strip using AuNFs@TA / Zr as the beacon carrier for detecting clothianidin is shown.

[0028] Figure 9 The photothermal physical map of the dual-mode immunochromatographic test strip using AuNFs@TA / Zr as the beacon carrier for detecting clothianidin is shown.

[0029] Figure 10 The transmission electron microscopy images of metal polyphenol-coated non-flower-shaped gold nanoparticles are shown.

[0030] Figure 11 The physical map of the immunochromatographic test strip using metal polyphenol-coated non-flower-shaped gold nanoparticles as the beacon carrier for detecting Escherichia coli E. coli O157:H7 is shown.

[0031] Figure 12 The transmission electron microscopy images of AuNFs@polydopamine-Zr gold nanoparticles are shown. Figure 13 The physical map of the immunochromatographic test strip using AuNFs@polydopamine-Zr gold nanoparticles as the beacon carrier for detecting Escherichia coli E. coli O157:H7 is shown.

[0032] Figure 14 The transmission electron microscopy images of AuNPs@procyanidin-Zr gold nanoparticles are shown. Figure 15 The physical map of the immunochromatographic test strip using AuNPs@procyanidin-Zr gold nanoparticles as the beacon carrier for detecting Escherichia coliE. coli O157:H7 Physical diagram.

[0033] Figure 16 The transmission electron microscopy image of AuNPs@epicatechin-Zr nanogold particles is shown. Figure 17 The immunochromatographic test strip using AuNPs@epicatechin-Zr nanogold particles as the beacon carrier for detecting Escherichia coli E. coli O157:H7 Physical diagram.

[0034] Figure 18 The transmission electron microscopy image of AuNFs@TA flower-like nanogold particles is shown. Figure 19 The immunochromatographic test strip using AuNFs@TA flower-like nanogold particles as the beacon carrier for detecting Escherichia coli E. coli O157:H7 Physical diagram. Detailed implementation manners

[0035] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Example 1: This example provides a preparation method of AuNFs@TA / Zr, including the following steps: (1) Preparation of chloroauric acid solution Place 99 mL of deionized water in a microwave reactor and preheat it to 70 °C. Then, under continuous stirring, quickly add 1 mL of an aqueous solution of chloroauric acid (HAuCl4) with a mass concentration of 1% to the preheated deionized water, and mix evenly to obtain a chloroauric acid solution.

[0037] (2) Preparation of tannic acid-hydroxylamine hydrochloride-chloroauric acid mixed solution Dissolve 20 mg of tannic acid (relative molecular mass 1701.2 g / mol) and 0.5 mL of an aqueous solution of hydroxylamine hydrochloride with a concentration of 0.1 mol / L in deionized water, and mix well to form a homogeneous solution.

[0038] Quickly add the tannic acid-hydroxylamine hydrochloride mixed solution to the chloroauric acid solution obtained in step (1), and continue stirring to allow the components to react fully. Heat the mixed system at 70 °C for 1 min, and finally obtain a homogeneous mixed solution of tannic acid, hydroxylamine hydrochloride and chloroauric acid.

[0039] (3) Collection of AuNFs@TA solution Cool the homogeneous mixed solution at room temperature for 40 minutes, then centrifuge it at a speed of 12,000 r / min for 10 minutes to collect the precipitate. Wash the precipitate 3 times with deionized water and finally redissolve it in 8 mL of pure water to obtain the AuNFs@TA solution.

[0040] (4)Preparation of AuNFs@TA / Zr solution Add 5 mg of zirconium chloride (ZrCl4) to the AuNFs@TA solution obtained in step 3, and stir vigorously (1200 r / min) at room temperature overnight; then centrifuge to separate the precipitate, wash it and redissolve it in 1 mL of deionized water to obtain the AuNFs@TA / Zr solution, and store it at 4 °C.

[0041] Characterize the AuNFs@TA / Zr prepared in Example 1. Figure 4 The shown is the transmission electron microscopy image (TEM image) of AuNFs@TA / Zr. Figure 4 It can be seen that the AuNFs@TA / Zr prepared in Example 1 includes inner-layer gold nanoparticles (AuNFs) and a metal polyphenol network structure formed by tannic acid (TA) and zirconium (Zr) loaded on the outer surface of AuNFs. Among them, AuNFs have a flower-like structure and the characteristic of a large surface area; TA and Zr are evenly distributed on the surface of AuNFs, which is beneficial to improving biocompatibility.

[0042] Characterize the AuNFs@TA prepared in step (3) of Example 1. Figure 18 The shown is the transmission electron microscopy image (TEM image) of AuNFs@TA. Figure 4 and Figure 18 By comparison, it can be seen that the AuNFs@TA / Zr prepared in Example 1 has a dense cross-linked structure compared with AuNFs@TA, indicating that through the metal-polyphenol coordination of Zr 4+ and TA, a stable three-dimensional cross-linked network is formed, and the nanoparticles are arranged tightly and orderly. And the AuNFs in Example 1 show a more uniform flower-like morphology, and a more complete flower-like structure is shown in the TEM image of Example 1, indicating that the introduction of Zr enhances the structural stability of AuNFs.

[0043] Therefore, the introduction of Zr optimized the morphology, chemical state, and optical properties of AuNFs@TA / Zr through metal-polyphenol coordination, specifically manifested as follows: (1) Enhanced structural stability: The dense cross-linked network enhanced mechanical strength and probe uniformity; (2) Retention of functional sites: Reduced the shielding of TA on Au-S bonds and improved the efficiency of antibody-directed conjugation; (3) Enhanced signal amplification: Achieved highly sensitive detection through the LSPR effect and improved T-line aggregation. In contrast, AuNFs@TA lacked Zr cross-linking, resulting in significant defects in structure, function, and signal intensity, leading to a decrease in the sensitivity of the test strip.

[0044] Example 2: This example provides a method for preparing and detecting a sandwich immunoassay chromatographic test strip using AuNFs@TA / Zr as a beacon carrier for detecting Escherichia coli E. coli O157:H7 as shown in Figure 1 Figure, the immunoassay chromatographic test strip includes a bottom plate 6, and a filter paper, a sample pad 1, a glass fiber pad 2, a nitrocellulose membrane 3, and a blotting paper 7 that are sequentially lapped and pasted on the bottom plate 6. There are a test line 4 and a control line 5 on the nitrocellulose membrane 3.

[0045] I. Preparation process of the immunoassay chromatographic test strip 1. Preparation of the nitrocellulose membrane; Coating anti-Escherichia coli E. coli O157:H7 polyclonal antibody and anti-mouse antibody onto the nitrocellulose membrane: Dilute the anti-Escherichia coli E. coli O157:H7 polyclonal antibody with 0.01 M PBS (phosphate buffer) at pH 7.5 to a concentration of 0.5 mg / mL, and spray the resulting solution onto the membrane as the test line; dilute the anti-mouse antibody to a concentration of 0.5 mg / mL, and spray the resulting solution onto the membrane as the control line. The spraying volume of both lines is 0.74 μL / cm. The test line is spaced 10 mm from the top edge of the membrane, and the two lines are spaced 5 mm apart. Dry at 30 °C for 12 hours and store in a drying cabinet for later use.

[0046] 2. Preparation of the AuNFs@TA / Zr-antibody complex glass fiber pad: Take 50 μL of the AuNFs@TA / Zr solution prepared in Example 1 and sonicate for 3 minutes. Mix it with 450 μL of 0.02 M borate buffer at pH 7.0 by shaking. After mixing evenly, add 10 μg of Escherichia coli E. coli O157:H7The monoclonal antibody was fully mixed and stirred for 2 h. Then, 50 μL of bovine serum albumin with a final mass concentration of 0.5% was added, and the mixture was blocked at room temperature for 2 h. After that, it was centrifuged at 8000 r / min for 10 min. The precipitate was resuspended in 0.01 M phosphate buffer (PBS) with pH 7.4 to 1 / 10 of the original volume, and then sprayed onto the glass fiber pad at a volume of 8 μL / cm². It was dried in vacuo for 2 h and stored in a drying cabinet for later use.

[0047] 3. Assembly of the test strip: ⑴ The filter paper and sample pad had a specification of 1 cm × 30 cm; ⑵ The glass fiber pad sprayed with AuNFs@TA / Zr - antibody complex had a specification of 0.8 cm × 30 cm; ⑶ The nitrocellulose membrane sprayed with the test line and control line had a specification of 2.5 cm × 30 cm; ⑷ The absorbent paper had a specification of 1.2 cm × 30 cm; ⑸ The PVC bottom plate had a specification of 5.5 cm × 30 cm.

[0048] The above materials were sequentially pasted according to the positions of each component in the structural schematic diagram of the test strip. After assembly, the test strip was cut into strips with a size of 4 mm × 55 mm using a cutter, placed in a plastic cartridge, pressed tightly, then put into an aluminum foil bag. After adding a desiccant, it was sealed and stored. The shelf life at room temperature was 12 months.

[0049] II. Quantitative detection of Escherichia coli in the sample E. coli O157:H7 The above immunochromatographic test strip was used to detect Escherichia coli in the sample E. coli O157:H7 The method included the following steps: 1. Add 100 μL of the sample to the sample loading hole of the test strip and react for 15 min; 2. Insert the test strip into the detection window of the test strip reader. The intensity of the color development of the test line and control line will be displayed as a numerical value on the display. According to the standard curve already entered in the instrument, the content of Escherichia coli in the sample E. coli O157:H7 can be calculated to achieve the quantitative detection of Escherichia coli in the sample E. coli O157:H7 3. Establish a standard curve: Add standards to the negative matrix. The concentrations of Escherichia coli in the standard curve are: 0, 5×10

[0050] CFU / mL, 1×10 E. coli O157:H7 CFU / mL, 5×10 2 CFU / mL, 1×10 3 CFU / mL, 5×10 3 CFU / mL, 1×10 4 CFU / mL, 5×104 CFU / mL, 1×10 5 CFU / mL, 5×10 5 CFU / mL, 1×10 6 CFU / mL, 5×10 6 CFU / mL. The above samples were respectively loaded onto the sample pad, and the reaction time was 15 minutes.

[0051] As a control, the above samples were respectively loaded onto traditional colloidal gold test strips, and the reaction time was 15 min.

[0052] Figure 2 Shown is the schematic diagram of the detection principle of the sandwich immunoassay AuNFs@TA / Zr immunochromatographic test strip. Among them, 8 is the analyte antibody on the test line, 9 is the anti-mouse antibody or anti-rabbit antibody on the quality control line, 10 is the macromolecular analyte, and 11 is the AuNFs@TA / Zr-antibody complex.

[0053] Figure 5 Shown is the application of the immunochromatographic test strip prepared with AuNFs@TA / Zr as the beacon carrier in the present invention for detecting Escherichia coli E. coli O157:H7 of the physical result diagram; the results show that as the Escherichia coli E. coli O157:H7 increases, the colorimetric signal of the test line (T line) of the test strip gradually increases, and the lowest detectable limit visible to the naked eye is 5×10 4 CFU / mL.

[0054] Figure 7 Shown is the photothermal physical map of the immunochromatographic test strip prepared with AuNFs@TA / Zr as the beacon carrier in the present invention for detecting Escherichia coli at different concentrations (0 CFU / mL, 5×10 4 CFU / mL, 1×10 5 CFU / mL, 5×10 5 CFU / mL, 1×10 6 CFU / mL, 5×10 6 CFU / mL). The results show that as the Escherichia coli E. coli O157:H7 increases, the photothermal signal of the test line (T line) of the test strip gradually increases, indicating that the immunochromatographic test strip prepared with AuNFs@TA / Zr as the beacon carrier has good colorimetric and photothermal effects for detecting Escherichia coli E. coli O157:H7 . E. coli O157:H7 In addition, as a control,

[0055] showed the results of traditional colloidal gold test strips for detecting Escherichia coli at different concentrations Figure 6 O157:H7 E. coli , and the lowest detectable limit visible to the naked eye is 5×10 5 CFU / mL.Obvious signals appeared at 10⁶ CFU / mL, indicating that compared with the traditional colloidal gold test strip, the detection sensitivity of the test strip based on the AuNFs@TA / Zr beacon carrier was 10 times higher than that of the traditional colloidal gold test strip, verifying the significant performance advantages of this new material in the immunochromatographic detection system.

[0056] Example 3: This example provides a preparation and detection method of a competitive immunoassay chromatographic test strip for detecting clothianidin using AuNFs@TA / Zr as the beacon carrier, including the following steps: I. Preparation process of the immunoassay chromatographic test strip The competitive immunoassay chromatographic test strip for detecting clothianidin using AuNFs@TA / Zr prepared in Example 1 as the beacon carrier includes a PVC bottom plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane, and a blotting paper that are successively lapped and pasted on the bottom plate.

[0057] 1. Preparation of the nitrocellulose membrane; Coat clothianidin whole antigen and goat anti-mouse antibody on the nitrocellulose membrane: Dilute the concentration of clothianidin whole antigen to 1 mg / mL with 0.01 M PBS at pH 7.4, and the resulting solution is sprayed on the membrane as the test line; dilute the concentration of goat anti-mouse antibody to 0.5 mg / mL, and the resulting solution is sprayed on the membrane as the quality control line. The spraying volume of both lines is 0.74 μL / cm. The test line is spaced 10 mm from the top edge of the membrane, and the two lines are spaced 5 mm apart. Dry at 30 °C for 12 h and store in a drying cabinet for later use.

[0058] 2. Preparation of the AuNFs@TA / Zr-antibody complex glass fiber pad: Take 50 μL of AuNFs@TA / Zr (prepared in Example 1), ultrasonicate for 5 minutes, mix it with 450 μL of 0.02 M borate buffer at pH 8.0, after shaking and mixing evenly, add 2.5 μg of clothianidin monoclonal antibody, mix well, shake for 3 h, add 50 μL of bovine serum albumin with a final concentration of 10%, block at room temperature for 1 h, centrifuge at 8000 r / min for 20 min, and redissolve the precipitate in 0.01 M phosphate buffer (PBS) at pH 7.4 to 1 / 10 of the starting volume. Spray it onto the glass fiber pad at a volume of 8 μL / cm, dry it under vacuum for 2 h, and store in a drying cabinet for later use.

[0059] 3. Assemble the test strip: ⑴ The sample pad has a specification of 1 cm × 30 cm; ⑵ The glass fiber pad sprayed with the AuNFs@TA / Zr - antibody complex has a specification of 0.8 cm × 30 cm; ⑶ Nitrocellulose membrane sprayed with detection line and quality control line, with a specification of 2.5 cm × 30 cm; ⑷ Absorbent paper, with a specification of 1.2 cm × 30 cm; ⑸ PVC bottom plate, with a specification of 5.5 cm × 30 cm.

[0060] Paste the above materials in sequence according to the positions of each component in the structural schematic diagram of the test strip. After assembly, cut the test strip into 4 × 55 mm with a cutter, put it into a plastic cartridge, press it tightly and then put it into an aluminum foil bag. After adding a desiccant, seal it and store it. The shelf life at room temperature is 12 months.

[0061] II. Quantitative determination of clothianidin in samples The method for detecting clothianidin in samples with the above immunochromatographic test strip includes the following steps: 1. Add 100 μL of the sample to the sample addition hole of the test strip and react for 15 min; 2. Insert the test strip into the detection window of the test strip reader. The intensity of the color development of the detection line and the quality control line will be displayed as values on the display. According to the standard curve already entered in the instrument, the content of clothianidin in the sample can be calculated to achieve quantitative detection of positive samples.

[0062] 3. Prepare the standard curve of the test strip: Spike clothianidin in the negative matrix so that its concentrations in the negative matrix are: 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL. Add the above samples to the sample pad respectively and react for 15 min.

[0063] As a control, add the above samples to the traditional colloidal gold test strip respectively and react for 15 min.

[0064] Figure 3 It is the detection principle diagram of the competitive method AuNFs@TA / Zr immunochromatographic test strip. Among them, 9 is the anti-mouse antibody or anti-rabbit antibody on the quality control line, 10 is the small molecule analyte, 11 is the AuNFs@TA / Zr - antibody complex, and 12 is the artificial conjugate antigen of the analyte on the detection line.

[0065] Figure 8 、 Figure 9The figure shows the physical result diagram of the immunochromatographic test strip prepared with AuNFs@TA / Zr as the beacon carrier for the detection of clothianidin. Experiments show that as the concentration of clothianidin increases, both the colorimetric and photothermal signals of the test line on the test strip decrease in a concentration-dependent manner. Visible to the naked eye, an obvious inhibitory effect appears at 0.5 ng / mL, and the test line completely disappears at 50 ng / mL. This result confirms that the test strip based on AuNFs@TA / Zr can achieve semi-quantitative detection of clothianidin through dual-modal signals (colorimetric / photothermal). Its naked-eye detection sensitivity (0.5 ng / mL) and dynamic response range (0.5 ng / mL - 50 ng / mL) both meet the requirements of trace analysis, further verifying the practicability and reliability of this method.

[0066] Comparative Example 1 The difference from Example 1 is as follows: Prepare a non-flower-shaped gold nanoparticle coated with TA and Zr. The specific preparation method is as follows: 1. Synthesis of gold nanoparticle-tannic acid complex (AuNPs@TA) Dissolve 20 mg of tannic acid (TA) in 100 mL of phosphate buffer solution (PBS, pH 8.5), and stir magnetically to form a homogeneous solution. Under continuous stirring, add 3 mL of 1% aqueous solution of chloroauric acid (HAuCl4), and continue the reaction for 10 minutes. Centrifuge the reaction system at 8000 r / min for 10 minutes to collect the product, and then wash it three times with deionized water to remove unreacted substances. Finally, redisperse the purified AuNPs@TA complex in 8 mL of deionized water to obtain a stable nanoparticle dispersion.

[0067] 2. Synthesis of zirconium-coordinated modified gold nanocomposite (AuNPs@TA-Zr) Add 5 mg of zirconium tetrachloride (ZrCl4) to the above AuNPs@TA dispersion, and carry out a coordination reaction at room temperature (25 ± 2 °C) with continuous stirring at 1200 r / min for 12 h. After the reaction is completed, centrifuge the mixed solution at 8000 r / min for 10 min to collect the product, and wash it three times with deionized water to remove free zirconium ions. The obtained AuNPs@TA-Zr composite nanoparticles can be immediately used for subsequent functionalization applications, or stored in a 4 °C refrigerated environment to maintain stability.

[0068] Figure 10 The figure shows the transmission electron microscopy image of the metal polyphenol-coated non-flower-shaped gold nanoparticles. Clearly visible are the spherical nanoparticles.

[0069] The present invention uses metal polyphenol-coated non-flower-shaped gold nanoparticles (AuNPs@TA-Zr) as signal probes, and based on the sandwich-type immunochromatographic strip construction method described in Example 2, Escherichia coli E. coli O157:H7 is quantitatively detected. The detection results are as Figure 11 shown: when the target bacteria concentration gradient is (0 CFU / mL, 5×10 4 CFU / mL, 1×10 5 CFU / mL, 5×10 5 CFU / mL, 1×10 6 CFU / mL, 5×10 6 CFU / mL), the colorimetric signal intensity of the test line (T line) of the test strip is positively correlated with the bacteria concentration, and the lowest detection limit distinguishable by the naked eye is 1×10 5 CFU / mL. Through comparative experiments, it was found that the detection sensitivity of the test strip using the metal polyphenol-coated flower-shaped gold nanoparticles (AuNFs@TA / Zr) in Example 1 as the signal carrier was increased by 2 times compared with the non-flower-shaped structure system in Comparative Example 1. This difference is attributed to the following synergistic mechanisms: 1) The flower-shaped gold nanoparticles (AuNFs) have a higher specific surface area, significantly enhancing the loading efficiency of biomolecules such as antibodies and the interfacial reaction activity; 2) The metal polyphenol coating layer stabilizes the surface properties of the nanoparticles through Zr 4+ coordination, effectively inhibiting non-specific adsorption and improving biocompatibility; 3) The regulation of the nanoparticle morphology directly affects the probe-target binding efficiency, and the spatial topological properties of the flower-shaped structure are more conducive to the formation of highly sensitive immune complexes. The above results show that by synergistically optimizing the morphology design and surface coordination, the detection performance of the nanoprobe can be significantly enhanced.

[0070] Comparative Example 2 The difference from Example 1 is that a kind of gold nanoparticles coated with other polyphenols (such as polydopamine or procyanidin or epicatechin) replacing tannic acid and Zr is prepared. The specific preparation method is as follows: (1) Preparation of chloroauric acid solution Place 99 mL of deionized water in a microwave reactor and preheat it to 70 °C. Then, under continuous stirring, quickly add 1 mL of an aqueous solution of chloroauric acid (HAuCl4) with a mass concentration of 1% to the preheated deionized water, and mix evenly to obtain a chloroauric acid solution.

[0071] (2) Preparation of polydopamine or procyanidin or epicatechin-hydroxylamine hydrochloride-chloroauric acid mixed solution Dissolve 20 mg of polydopamine or procyanidin or epicatechin and 0.5 mL of an aqueous solution of hydroxylamine hydrochloride with a concentration of 0.1 mol / L in deionized water, and mix well to form a homogeneous solution.

[0072] Quickly add the mixed solution of the polydopamine or procyanidin or epicatechin-hydroxylamine hydrochloride to the chloroauric acid solution obtained in step (1), and continue stirring to allow the components to react fully. Heat the mixed system at 70 °C for 1 min, and finally obtain a homogeneous mixed solution of polydopamine or procyanidin or epicatechin, hydroxylamine hydrochloride, and chloroauric acid.

[0073] (3)Collection of AuNFs@TA solution Cool the homogeneous mixed solution at room temperature for 40 minutes, then centrifuge at a speed of 12,000 r / min for 10 minutes to collect the precipitate. Wash the precipitate 3 times with deionized water, and finally redissolve it in 8 mL of pure water to prepare the AuNFs@TA solution.

[0074] (4)Preparation of AuNFs@TA / Zr solution Add 5 mg of zirconium chloride (ZrCl4) to the AuNFs@TA solution obtained in step 3, and stir vigorously (1200 r / min) overnight at room temperature; then centrifuge to separate the precipitate, wash it, and redissolve it in 1 mL of deionized water to obtain the AuNFs@TA / Zr solution, and store it at 4 °C.

[0075] Use the prepared polydopamine or procyanidin or epicatechin and Zr-coated gold nanoparticles to prepare a sandwich immunoassay strip according to the method in Example 2 to detect Escherichia coli E. coli O157:H7 for detection.

[0076] Figure 12 Shown is the transmission electron micrograph of polydopamine-Zr-coated gold nanoparticles (AuNFs@polydopamine-Zr gold nanoparticles), presenting flower-shaped nanoparticles. The target bacteria concentration gradients are 0 CFU / mL, 5×10 2 CFU / mL, 1×10 3 CFU / mL, 5×10 3 CFU / mL, 1×10 4 CFU / mL, 5×10 4 CFU / mL, 1×10 5 CFU / mL, 5×10 5 CFU / mL, 1×10 6 CFU / mL, 5×10 6 CFU / mL.

[0077] The detection results of the test strip detection based on polydopamine and Zr-coated gold nanoparticles (AuNFs@polydopamine-Zr gold nanoparticles) as probes are shown in Figure 13The colorimetric signal intensity of the detection line (T line) is positively correlated with the bacterial concentration, but the signal intensity of the T and C lines has decreased, the color is not deep enough, and the lowest detection limit that can be identified by the naked eye is 1×10 5 CFU / mL, which is 2 times lower than the sensitivity of the dual-modal test strip based on AuNFs@TA / Zr of the present invention. Polydopamine-Zr coated gold nanoparticles were prepared according to the synthesis method of AuNFs@TA / Zr, and tannic acid was replaced with polydopamine. The other preparation conditions and steps were the same. Although the synthesized polydopamine-Zr coated gold nanoparticles showed a flower-like shape, the detection sensitivity was reduced.

[0078] Figure 14 Shown is a transmission electron micrograph of proanthocyanidin-Zr coated gold nanoparticles (AuNPs@proanthocyanidin-Zr gold nanoparticles), showing spherical nanoparticles.

[0079] The test results of the test strip based on proanthocyanidins and Zr-coated flower-shaped gold nanoparticles (AuNPs@proanthocyanidins-Zr gold nanoparticles) as probes are as follows Figure 15 As shown in the figure, the colorimetric signal intensity of the detection line (T line) is positively correlated with the bacterial concentration, but the signal intensity of the T and C lines has decreased, the color is wine red, and the lowest detection limit that can be identified by the naked eye is 5×10 5 CFU / mL. The sensitivity of the dual-modal test strip based on AuNFs@TA / Zr of the present invention is reduced by 10 times. According to the synthesis method of AuNFs@TA / Zr, proanthocyanidin-Zr coated nanogold particles were prepared, tannic acid was replaced by proanthocyanidin, and other preparation conditions and steps were consistent. The synthesized proanthocyanidin-Zr coated nanogold particles did not show a flower-like shape, the morphology of the nanoparticles changed, and the detection sensitivity decreased significantly.

[0080] Figure 16 Shown is a transmission electron micrograph of epicatechin-Zr coated gold nanoparticles (AuNPs@epicatechin-Zr gold nanoparticles), showing non-flower-shaped nanoparticles.

[0081] The test results of the test strip based on epicatechin and Zr-coated gold nanoparticles (AuNPs@epicatechin-Zr gold nanoparticles) as probes are as follows Figure 17 As shown in the figure, the test line and the quality control line failed to show color, indicating that the color was too light to be used for immunochromatographic test strips. Using the same preparation conditions and steps as AuNFs@TA / Zr, only replacing tannic acid with epicatechin, the morphology of the synthesized epicatechin-Zr coated nano-gold particles changed, not showing a flower shape, and the color was light gray, and it could not be colored on the test strip, resulting in it not being applicable to immunochromatography.

[0082] Comparative experiments showed that when polydopamine, procyanidin or epicatechin was used to replace tannic acid, due to the change in the type of polyphenols, the morphology, structure and color of the nanoparticles changed significantly, and the detection sensitivity decreased. This is because the high-density catechol groups and stereochemical structure of tannic acid have more advantages in metal coordination and network stability than other polyphenols such as polydopamine, procyanidin and epicatechin.

[0083] Comparative Example 3 The difference from Example 1 is as follows: Prepare a TA-encapsulated flower-like gold nanoparticle, and the specific preparation method is as follows: (1) Preparation of chloroauric acid solution Place 99 mL of deionized water in a microwave reactor and preheat it to 70 °C. Then, under continuous stirring, quickly add 1 mL of an aqueous solution of chloroauric acid (HAuCl4) with a mass concentration of 1% to the preheated deionized water, and mix evenly to obtain a chloroauric acid solution.

[0084] (2) Preparation of tannic acid-hydroxylamine hydrochloride-chloroauric acid mixed solution Dissolve 20 mg of tannic acid (relative molecular mass 1701.2 g / mol) and 0.5 mL of an aqueous solution of hydroxylamine hydrochloride with a concentration of 0.1 mol / L in deionized water, and mix well to form a homogeneous solution.

[0085] Quickly add the tannic acid-hydroxylamine hydrochloride mixed solution to the chloroauric acid solution obtained in step (1), and continue stirring to allow the components to react fully. Heat the mixed system at 70 °C for 1 min, and finally obtain a homogeneous mixed solution of tannic acid, hydroxylamine hydrochloride and chloroauric acid.

[0086] (3) Collection of AuNFs@TA solution Cool the homogeneous mixed solution at room temperature for 40 minutes, then centrifuge it at a centrifugal speed of 12,000 r / min for 10 minutes, and collect the precipitate. Wash the precipitate 3 times with deionized water, and finally redissolve it in 8 mL of pure water to prepare an AuNFs@TA solution.

[0087] Figure 18 The transmission electron micrograph of the TA-encapsulated flower-like gold nanoparticle is shown, presenting flower-like nanoparticles.

[0088] Use the prepared TA-encapsulated flower-like gold nanoparticle (AuNFs@TA flower-like gold nanoparticle) to prepare a sandwich immunoassay chromatographic strip according to the method in Example 2, and detect Escherichia coli E. coli O157:H7 for detection. The detection results are shown in Figure 19As shown, the colorimetric signal intensity of the test line (T line) of the test strip based on TA-coated flower-like gold nanoparticles (AuNFs@TA flower-like gold nanoparticles) as a probe is positively correlated with the bacterial concentration. However, the signal intensities of both the T and C lines have decreased, and the color is lighter. The lowest detection limit distinguishable by the naked eye is 1×10 5 CFU / mL. The sensitivity of the test strip based on AuNFs@TA / Zr in Example 1 has decreased by 2 times. The decrease in the sensitivity of the test strip based on TA-coated flower-like gold nanoparticles (AuNFs@TA flower-like gold nanoparticles) is mainly caused by the following two key reasons: (1) Decrease in probe labeling efficiency: The dense TA coating layer covers the active sites on the gold surface, resulting in a decrease in the antibody coupling density. At the same time, the non-specific adsorption of TA causes non-directional antibody coupling, reducing the antigen binding efficiency; (2) Insufficient signal amplification: The flower-like gold nanostructure formed by the coordination of TA alone is loose, the local surface plasmon resonance effect is weakened, and there is a lack of a dense polyphenol network cross-linked by Zr 4+ resulting in a decrease in the aggregation degree of gold particles in the T line and a significant weakening of the colorimetric signal.

[0089] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered within the protection scope of the present invention.

Claims

1. A dual-modal immunochromatographic test strip, characterized in that, The dual-modal immunochromatographic test strip uses AuNFs@TA / Zr as a probe; The preparation method of AuNFs@TA / Zr comprises the following steps: S1. After preheating deionized water, add chloroauric acid aqueous solution and mix evenly to obtain chloroauric acid solution; S2, dissolving tannic acid and hydroxylamine hydrochloride aqueous solution in deionized water, and mixing thoroughly to obtain a mixed solution; S3, reacting the chloroauric acid solution with the mixed solution, cooling and centrifuging to obtain a precipitate after the reaction is completed; washing the precipitate and redissolving it in water to obtain an AuNFs@TA solution; S4. Add zirconium chloride to the AuNFs@TA solution, stir and mix, centrifuge and wash to obtain AuNFs@TA / Zr.

2. The dual-modal immunochromatographic test strip according to claim 1, wherein In step S1, the temperature of the preheated deionized water is 65°C to 75°C, the mass concentration of the chloroauric acid aqueous solution is 0.5% to 1.5%, and the added volume ratio of deionized water to the chloroauric acid solution is 95 to 103:

1.

3. The dual-modal immunochromatographic test strip according to claim 1, characterized in that In step S2, the addition ratio of tannic acid and hydroxylamine hydrochloride aqueous solution is 18 mg-22 mg:0.5 mL, and the concentration of hydroxylamine hydrochloride aqueous solution is 0.05 mol / L-0.15 mol / L.

4. The dual-modal immunochromatographic test strip according to claim 1, wherein In step S3, the cooling condition is room temperature, the cooling time is 35 min~45 min, the centrifugal speed is 3000 r / min~12000 r / min, the centrifugal time is 10 min~30 min, the number of washing times is 1 time~3 times, and the volume of water added for re-dissolution is 7 mL-9 mL.

5. The dual-modal immunochromatographic test strip according to claim 1, characterized in that, In step S4, the addition ratio of zirconium chloride and chloroauric acid solution is 4 mg~6 mg:1 mL, the stirring speed is 1000 r / min~1400 r / min, the stirring time is 20 h~28 h, the centrifugal speed is 3000 r / min~12000 r / min, the centrifugal time is 10 min~30 min, and the number of washing times is 1 time~3 times.

6. The dual-modal immunochromatographic test strip according to claim 1, characterized in that, The AuNFs@TA / Zr is loaded on the dual-modal immunochromatographic test strip in the form of an AuNFs@TA / Zr labeled antibody complex, wherein the preparation method of the AuNFs@TA / Zr labeled antibody complex comprises the following steps: AuNFs@TA / Zr was dissolved in water to obtain AuNFs@TA / Zr solution. The antibody to be labeled is added to the AuNFs@TA / Zr solution, and a blocking agent is added after sufficient mixing. After the reaction, the solution is centrifuged to obtain a precipitate, and the obtained precipitate is redissolved to prepare an AuNFs@TA / Zr labeled antibody complex.

7. The dual-modal immunochromatographic test strip according to claim 6, wherein In the method for preparing the AuNFs@TA / Zr labeled antibody complex, The final concentration of the antibody after adding the antibody to be labeled is 1 μg / mL to 100 μg / mL; After the blocking agent is added, the final mass concentration of the blocking agent is 0.1% to 1%, and the blocking agent is selected from any one of casein, bovine serum albumin, ovalbumin, polyethylene glycol, and skim milk; The centrifugal speed is 5000 r / min to 20000 r / min, and the centrifugal time is 10 min to 50 min; The precipitate after centrifugation is resuspended with a phosphate buffer solution of 0.01 M to 0.1 M and pH 6.0 to 9.0; The antibody to be labeled includes any one of monoclonal antibodies, polyclonal antibodies, nanobodies, and phage-expressed antibodies.

8. The dual-modal immunochromatographic test strip according to claim 7, wherein The dual-modal immunochromatographic test strip includes: A bottom plate; A nitrocellulose membrane, disposed on the bottom plate, and having a detection line and a quality control line disposed at intervals thereon; A glass fiber pad, disposed at one end of the nitrocellulose membrane having the detection line, one end of the glass fiber pad is disposed on the bottom plate, and the other end is disposed on the nitrocellulose membrane, and the AuNFs@TA / Zr-labeled antibody complex is disposed on the glass fiber pad; Absorbent paper, disposed at one end of the nitrocellulose membrane having the quality control line, one end of the absorbent paper is disposed on the bottom plate, and the other end is disposed on the nitrocellulose membrane; A sample pad, one end of which is disposed on the bottom plate and the other end is disposed on the glass fiber pad.

9. The dual-modal immunochromatographic test strip according to claim 8, wherein The method for disposing the AuNFs@TA / Zr-labeled antibody complex on the glass fiber pad includes: spraying the AuNFs@TA / Zr-labeled antibody complex onto the glass fiber pad at a volume of 7 μL / cm to 9 μL / cm, and vacuum drying for 1.5 h to 2.5 h; The detection line is coated with an artificial conjugate antigen of the analyte or an antibody of the analyte, and the quality control line is coated with a goat anti-mouse antibody.

10. Use of a dual-modal immunochromatographic test strip according to any one of claims 1 to 9 in detecting pesticides or pathogenic bacteria for non-disease diagnosis purposes, characterized in that, The method for detecting pesticides or pathogenic bacteria using the dual-modal immunochromatographic test strip includes the following steps: The processed sample to be tested is added to the dual-modal immunochromatographic test strip, the added sample volume is 50 μL to 200 μL, the reaction time is 3 minutes to 30 minutes, and the concentration of the sample to be tested is calculated by reading the gray-scale data of the test strip and the imaging data of the infrared imager to achieve quantitative detection, or the qualitative judgment of the sample to be tested is achieved by visually observing whether there are color bands on the detection line and the quality control line of the dual-modal immunochromatographic test strip.

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