A fluorescent lateral flow immunochromatographic assay method and preparation and application of test strip thereof

Abstract

The application discloses a fluorescent lateral flow immunoassay method and preparation and application of a test strip thereof, and the AuNCs-LFA test strip comprises a sample pad, a gold label pad, an NC membrane, an absorption pad and a PVC bottom plate; wherein the NC membrane comprises a signal probe AuNCs-Ab, an antigen ZEN-BSA and a goat anti-mouse lgG. The application provides a fluorescent LFA detection method based on metal nanocluster-labeled antibodies, which can be used for detecting ZEN and other small molecule compounds. Compared with a traditional colloidal gold LFA method, the method has improved speed and sensitivity for detecting small molecules.

Description

Technical Field

[0001] This invention belongs to the fields of food safety testing and nanobiosensing, specifically involving a fluorescent lateral flow immunochromatographic analysis method and the preparation and application of its test strips. Background Technology

[0002] Lateral chromatographic analysis (LFA), an in vitro diagnostic technique developed in the 1980s, boasts advantages such as simplicity, speed, and cost-effectiveness, and is now widely used in environmental monitoring, medical testing, food quality monitoring, and forensic investigations. However, traditional immunochromatographic methods still have significant limitations in detecting trace or ultra-trace analytes due to their low detection sensitivity, failing to meet the increasingly demanding testing requirements in various fields.

[0003] To improve the sensitivity and accuracy of LFA detection, various lateral flow immunochromatographic methods based on fluorescent nanomaterial labeling have been developed. Compared with traditional colloidal gold-based LFA methods (AuNPs-LFA), they exhibit significantly improved detection sensitivity.

[0004] Currently, the fluorescent labels used in fluorescence immunochromatography (LFA) mainly include fluorescent microspheres, upconversion nanoparticles, carbon dots, and quantum dots. Quantum dots, due to their advantages such as high luminescence intensity, broad excitation spectrum, and narrow emission spectrum, have been widely used in recent years to construct highly sensitive and multivariate immunochromatographic methods. However, quantum dots still pose risks related to toxic metal elements and complex preparation methods. Metal nanoclusters, as a new type of fluorescent material, possess the characteristics of low toxicity, high photostability, and ease of synthesis.

[0005] However, to date, very few studies have applied it to the development of fluorescence lateral flow immunochromatography (LFA) methods. Therefore, developing a highly sensitive fluorescence LFA method labeled with metal nanoclusters has significant application value. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a fluorescence-based immunochromatographic analysis AuNCs-LFA test strip.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: an immunochromatographic analysis (AuNCs-LFA) test strip based on fluorescent gold nanoclusters, comprising a sample pad, a gold label pad, an NC membrane, an absorbent pad, and a PVC base plate;

[0010] The NC membrane contains signal probe AuNCs-Ab, goat anti-mouse IgG, and antigen ZEN-BSA.

[0011] As a preferred embodiment of the AuNCs-LFA test paper of the present invention, a test line and a control line are provided on the NC membrane;

[0012] Among them, 0.4-0.6 mg / mL ZEN-BSA was sprayed on the test line, and 0.2-0.5 mg / mL goat anti-mouse IgG was sprayed on the control line, and dried at 37-40℃;

[0013] The distance between the test line and the control line is 4–8 mm.

[0014] In a preferred embodiment of the AuNCs-LFA test paper of the present invention, the preparation method of the signal probe AuNCs-Ab includes,

[0015] Add 20–100 μL of 0.02 mg / mL antibody to 25 μL of AuNCs solution, mix well, then add 5 μL of 10 mg / mL BSA solution and react for 5–30 min to obtain the fluorescent nanocluster-antibody label AuNCs-Ab.

[0016] In a preferred embodiment of the AuNCs-LFA test paper of the present invention, the AuNCs solution is prepared by a method comprising:

[0017] A solution of 6-aza-2-thiothymidine and a solution of chloroauric acid were mixed and reacted in the dark. After the reaction was completed, the mixture was purified to obtain a solution of AuNCs.

[0018] In a preferred embodiment of the AuNCs-LFA test paper of the present invention, the concentration of the 6-aza-2-thiothymidine solution is 80 mmol; the concentration of the chloroauric acid solution is 10 mg / mL; and the ratio of the 6-aza-2-thiothymidine solution to the chloroauric acid solution is 3 mL: 3 mL.

[0019] In a preferred embodiment of the AuNCs-LFA test paper of the present invention, the AuNCs have a diameter of 2-6 nm.

[0020] As a preferred embodiment of the AuNCs-LFA test paper of the present invention, wherein: the length of the overlap between the sample pad and the PVC adhesive backing is 1-2 mm, and the sample pad is placed on top of the PVC adhesive backing.

[0021] The overlap length between the sample pad, gold label pad, and NC membrane is 1-2 mm. The sample pad membrane is placed on top of the gold label pad, and the gold label pad is placed on top of the NC membrane.

[0022] The overlap between the NC membrane and the absorbent pad is 1-3 mm, and the absorbent pad is placed on top of the NC membrane.

[0023] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing AuNCs-LFA test strips, comprising:

[0024] The sample pad, gold label pad, NC membrane, and absorbent pad were sequentially attached to the PVC adhesive backing, each overlapping by 2 mm. Using a three-dimensional spraying platform, 0.05–0.1 mg / mL ZEN-BSA was sprayed on the T line of the NC membrane, and 0.1–0.2 mg / mL goat anti-mouse IgG was sprayed on the C line. The membrane was then dried at 30–37°C for 2 hours. The distance between the test line and the control line was 4–8 mm.

[0025] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of AuNCs-LFA test strip in the detection of zearalenone (ZEN).

[0026] As a preferred embodiment of the application described in this invention, the method includes: mixing the zearalenone sample to be tested with a running buffer to prepare a mixed solution;

[0027] Take 50-100 μL of the mixed solution and drop it onto the AuNCs-LFA test paper sample pad. After 3-6 minutes, heat it in a microwave oven on high for 1 minute and then interpret the test results.

[0028] The running buffer consists of 0.01M PBS, 0.5% Tween-20, pH 7.4.

[0029] Beneficial effects of this invention:

[0030] (1) Existing competitive LFA with colloidal gold as a probe has problems such as low colorimetric response efficiency and low sensitivity to weak positive samples. The preparation process of common fluorescent labeling materials is complicated or highly toxic, which limits the application of fluorescence-based LFA in practical detection. This paper proposes a side-flow chromatography (AuNCs-LFA) detection method based on fluorescent gold nanoclusters. Compared with the traditional colloidal gold LFA method, this method improves the visual discrimination sensitivity of small molecules.

[0031] (2) This invention provides a small-sized fluorescent probe that significantly shortens the time for LFA to detect small molecules. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0033] Figure 1 This is a schematic diagram illustrating the principle of detecting zearalenone in zearalenone using AuNCs-LFA according to the present invention.

[0034] Figure 2 The diagram shows the optimization of the AuNCs-LFA detection method of the present invention: (A) optimization of ZEN-BSA concentration; (B) optimization of the volume of the signal probe AuNCs-Ab; and (C) optimization of detection time.

[0035] Figure 3 The linear range diagram for detecting ZEN is shown, where (A) is the linear range of ZEN detected by AuNCs-LFA and (B) is the linear range of ZEN detected by AuNPs-LFA.

[0036] Figure 4 This is a specificity diagram of the AuNCs-LFA test strip of the present invention. Detailed Implementation

[0037] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0038] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0040] Example 1

[0041] (1) Preparation of ATT-AuNCs

[0042] The specific steps for preparing ATT-AuNCs are as follows:

[0043] 6-aza-2-thiothymine (ATT) was dissolved in NaOH (0.2M) solution; ATT (3 mL, 80 mM) solution was mixed with HAuCl4 (3 mL, 10 mg / mL) solution, and the mixture was stirred continuously at 25 °C in the dark for 1 hour to prepare ATT-AuNCs;

[0044] AuNC was then purified and synthesized via ultrafiltration (Millipore, 50 kDa).

[0045] (2) Preparation of signal probe AuNCs-Ab: 100 μL of antibody (0.02 mg / mL, ZEN monoclonal antibody, purchased from Wuxi Ditengmin Biotechnology Co., Ltd.) was added to AuNCs (25 μL), mixed evenly, and then 5 μL of BSA (10 mg / mL) solution was added. The reaction was carried out for 5 min to obtain fluorescent nanocluster-antibody label (AuNCs-Ab), which was stored at 4℃.

[0046] (3) Preparation of test strips

[0047] The side-flow chromatography device was prepared using five materials: a sample pad, a gold label pad, an NC membrane, and an absorbent pad, which were sequentially attached to a PVC backing, each overlapping by 2 mm.

[0048] Using a three-dimensional spraying platform, spray 0.5 mg / mL ZEN-BSA (1 mg / mL ZEN-BSA was diluted with PBS to 0.5 mg / mL) onto the test line (T line) of the NC membrane, spray 0.2 mg / mL goat anti-mouse IgG (purchased from Shanghai Jieyi Biotechnology Co., Ltd.) onto the control line (C line) of the NC membrane, and spray 8 μL AuNCs-Ab onto the gold label pad. Dry at 37°C for 4 hours. The distance between the T line and the C line is 5 mm.

[0049] Finally, cut the test strips into 4mm wide pieces and store them at 4℃.

[0050] Example 2

[0051] Optimization of the AuNCs-LFA detection method:

[0052] (1) Concentration of ZEN-BSA

[0053] Under the condition that other conditions remain unchanged (AuNCs-Ab volume 8μL, detection time 5min), only the ZEN-BSA concentration is changed (0.4mg / mL-0.6mg / mL, the purchased 1mg / mL ZEN-BSA is diluted with PBS to 0.4mg / mL-0.6mg / mL), and the signal strength is judged by the fluorescence intensity of the T line.

[0054] (2) AuNCs-Ab volume

[0055] The specific method is the same as step (1), only the volume of AuNCs-Ab is changed (4μL-10μL).

[0056] (3) Detection time

[0057] The specific method is the same as step (1), only the detection time is changed (2min-8min).

[0058] like Figure 2 AC, the three key parameters that were optimal were obtained through single-factor experiments: (1) the concentration of ZEN-BSA was 0.5 mg / mL; (2) the volume of AuNCs-Ab was 8 μL; and (3) the detection time was 5 min.

[0059] Example 3

[0060] Preparation of fluorescent metal nanocluster side-flow chromatography test paper:

[0061] 1. Preparation of test strips

[0062] The side-flow chromatography device was prepared using five materials: a sample pad, a gold label pad, an NC membrane, and an absorbent pad, which were sequentially attached to a PVC backing, each overlapping by 2 mm.

[0063] 0.5 mg / mL ZEN-BSA was sprayed onto the test line (T line) of the NC membrane using a three-dimensional spraying platform, 0.2 mg / mL goat anti-mouse IgG (purchased from Shanghai Jieyi Biotechnology Co., Ltd.) was sprayed onto the control line (C line) of the NC membrane, and 8 μL AuNCs-Ab was sprayed onto the gold label pad. The membrane was then dried at 37°C for 4 hours. The distance between the T line and the C line was 5 mm.

[0064] Finally, cut the test strips into 4mm wide pieces and store them at 4℃.

[0065] 2. Establishment of a detection method based on AuNCs-LFA test strips

[0066] Mix 80 μL of ZEN at different concentrations (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 ng / mL; preparation method: dissolve 10 mg ZEN standard in 1 mL of methanol, then dilute with ultrapure water to prepare ZEN solutions of 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 ng / mL) with 20 μL of Running Buffer and add to the sample pad. After 5 minutes, place the test strip in a microwave oven and heat for 1 minute. Then, use a portable reader to detect the results.

[0067] In the absence of ZEN, the AuNCs-Ab immunoprobe is captured by the antigen (ZEN-BSA) on the T line, forming a green fluorescent band.

[0068] In the presence of ZEN, ZEN competes with ZEN-BSA on the T-line for limited binding of AuNCs-Ab, at which point the green fluorescence signal on the T-line disappears. The schematic diagram for detecting zearalenone by AuNCs-LFA is shown below. Figure 1 .

[0069] Example 4

[0070] Detection of zearalenone in zearalenone:

[0071] (1) Determination of the sensitivity of zearalenone AuNCs-LFA test strip

[0072] Under the optimal experimental conditions determined in Example 2, different concentrations of zearalenone were detected to determine the sensitivity of zearalenone AuNCs-LFA.

[0073] The test method is the same as in Example 3, wherein:

[0074] Figure 3 The test conditions were as follows: ZEN-BSA concentration was 0.5 mg / mL; AuNCs-Ab volume was 8 μL; and detection time was 5 min.

[0075] Figure 3 The test conditions for B are: ZEN-BSA concentration of 2 mg / mL; AuNPs-Ab volume of 8 μL; and detection time of 10 min.

[0076] like Figure 3 As shown in AB, the signal intensity on the T line of the test strip gradually disappeared with increasing zearalenone concentration. The detection limit (LOD) for zearalenone detection by AuNCs-LFA was 0.0039 ng / mL. Figure 3 A), the LOD of zearalenone detected by AuNPs-LFA was 0.08 ng / mL. Figure 3 B). Compared with AuNPs-LFA, AuNCs-LFA has a 20-fold increase in detection sensitivity for ZEN and a 10-fold increase in visual detection sensitivity, making it suitable for detecting large batches of samples.

[0077] (2) Specificity of zearalenone AuNCs-LFA test strips

[0078] Several drugs with similar functions to zearalenone, such as aflatoxin M1, aflatoxin B1, vomitoxin, T-2 toxin, and ochratoxin, were used as potential interfering substances to detect the specificity of AuNCs-LFA. The procedure was as follows:

[0079] Mix 80 μL of zearalenone structural analog (20 ng / mL) with 20 μL of Runningbuffer buffer and add it to the sample pad. After 5 minutes, place the test strip in a microwave oven and heat for 1 minute. Then, use a portable reader to detect the result (detect at room temperature, no incubation required, excitation wavelength 365 nm, emission wavelength 510 nm, test method is the same as in Example 3).

[0080] like Figure 4 As shown, the established method exhibits high cross-reactivity with zearalenone structural analogs, while showing negative results for other mycotoxins. Therefore, the AuNCs-LFA established in this invention possesses high specificity for zearalenone.

[0081] (3) Using wheat simulated samples to test recovery rate

[0082] The recovery rate was tested using wheat simulants, and the steps were as follows:

[0083] Zearalenone was added to negative wheat samples to achieve final concentrations of 0, 0.5, 1, and 2 μg / kg, and then tested with AuNCs-LFA prepared in Example 3.

[0084] Table 1

[0085]

[0086] As shown in Table 1, the AuNCs-LFA test strip detection method exhibits excellent recovery rates (96.0%–102.0%) and a CV of less than 2.9%. Therefore, the AuNCs-LFA method provided by this invention can be used for the rapid detection of zearalenone in food.

[0087] In this invention, the antibody-based fluorescent lateral flow immunochromatographic assay strip detection method (AuNCs-LFA) exhibits a fluorescence signal pattern. In the absence of ZEN, the AuNCs-Ab immunoprobe is captured by the antigen (ZEN-BSA) on the T line, forming a green fluorescent band. In the presence of ZEN, ZEN competes with ZEN-BSA on the T line for the binding of a limited number of AuNCs-Abs, at which point the fluorescence signal on the T line disappears. Regardless of the presence or absence of ZEN, fluorescence on the C line is always present.

[0088] The high-sensitivity detection characteristic of this invention is reflected in the high sensitivity and high reactivity of the fluorescence signal of the nanoclusters. In traditional LFAs using AuNPs as signal probes, the signal intensity is relatively weak. However, in AuNCs-LFAs using AuNCs as probes, the signal intensity of the T-line is significantly enhanced, thereby achieving a significantly improved sensitivity.

[0089] Existing competitive fluorescent labeling methods using colloidal gold as a probe suffer from problems such as inefficient colorimetric response and low sensitivity to weakly positive samples. The preparation processes of common fluorescent labeling materials are complex or highly toxic, which limits the application of fluorescent LFA in practical detection. This paper proposes an LFA detection method based on fluorescent nanoclusters. Compared with the traditional colloidal gold LFA method, this method improves the sensitivity for detecting small molecules and also shortens the detection time.

[0090] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A fluorescent gold nanocluster-based immunochromatographic assay (AuNCs-LFA) test strip, characterized in that: include, Sample pads, gold label pads, NC film, absorbent pads, and PVC base plates; The NC membrane contains signal probe AuNCs-Ab, goat anti-mouse IgG, and zearalenone ZEN antigen ZEN-BSA. Test lines and control lines are set on the NC membrane; The test line was sprayed with 0.4–0.6 mg / mL ZEN-BSA, and the control line was sprayed with 0.2–0.5 mg / mL goat anti-mouse IgG, and dried at 37–40°C; the distance between the test line and the control line was 4–8 mm. The preparation method of the signal probe AuNCs-Ab includes adding 20~100μL of 0.02 mg / mL antibody to 25μL of AuNCs solution, mixing evenly, adding 5μL of 10 mg / mL BSA solution, reacting for 5~30 min to obtain fluorescent nanocluster-antibody label AuNCs-Ab; The AuNCs solution is prepared by mixing a 6-aza-2-thiothymidine solution and a chloroauric acid solution, reacting them in the dark, and purifying the mixture after the reaction to obtain the AuNCs solution. The concentration of the 6-aza-2-thiothymidine solution is 80 mmol, the concentration of the chloroauric acid solution is 10 mg / mL, and the ratio of the 6-aza-2-thiothymidine solution to the chloroauric acid solution is 3 mL:3 mL.

2. The immunochromatographic assay strip based on fluorescent gold nanoclusters as described in claim 1, characterized in that: The diameter of the AuNCs is 2~6nm.

3. The immunochromatographic assay strip based on fluorescent gold nanoclusters as described in claim 1, characterized in that: The overlap between the sample pad and the PVC backing is 1-2 mm, and the sample pad is placed on top of the PVC backing. The overlap length between the sample pad, gold label pad, and NC membrane is 1–2 mm. The sample pad membrane is placed on top of the gold label pad, and the gold label pad is placed on top of the NC membrane. The overlap between the NC membrane and the absorbent pad is 1–3 mm, with the absorbent pad placed on top of the NC membrane.

4. The method for preparing the immunochromatographic assay strip based on fluorescent gold nanoclusters according to any one of claims 1 to 3, characterized in that: include, The sample pad, gold label pad, NC membrane, and absorbent pad were sequentially attached to the PVC adhesive backing, each overlapping by 2 mm. Using a three-dimensional spraying platform, 0.05~0.1 mg / mL ZEN-BSA was sprayed on the T line of the NC membrane, and 0.1~0.2 mg / mL goat anti-mouse IgG was sprayed on the C line. The membrane was then dried at 30~37℃ for 2 hours. The distance between the test line and the control line was 4~8 mm.

5. The application of the immunochromatographic assay strip based on fluorescent gold nanoclusters as described in any one of claims 1 to 3 in the detection of zearalenone.

6. The application as described in claim 5, characterized in that: include, The zearalenone sample to be tested was mixed with the running buffer to prepare a mixed solution; Take 50~100μL of the mixed solution and drop it onto the sample pad of the immunochromatographic analysis test strip based on fluorescent gold nanoclusters. After 3~6 minutes, heat it in a microwave oven on high for 1 minute and then interpret the test results. The running buffer consists of 0.01 M PBS, 0.5% Tween-20, pH 7.4.

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