Fluorescent lateral flow immunochromatography analysis method and preparation and application of test strip thereof

Abstract

The invention discloses a fluorescent lateral flow immunochromatography analysis method and preparation and application of a test strip thereof. The AuNCs-LFA test strip comprises a sample pad, a gold label pad, an NC film, 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 invention provides a fluorescent LFA detection method based on a metal nano-cluster labeled antibody, which not only can be used for detecting ZEN, but also can be used for detecting other small molecule compounds. Compared with a traditional colloidal gold LFA method, the method has the advantages that the small molecule detection speed and sensitivity are improved.

Description

Technical Field

[0001] The invention belongs to the field of food safety detection and nano-biosensing, and specifically relates to a fluorescent lateral flow immunochromatography analysis method and the preparation and application of a test strip thereof. Background Art

[0002] Lateral flow analysis (LFA) is an in vitro diagnostic technology developed in the 1980s. It has the advantages of being simple, rapid and economical, and has been widely used in environmental monitoring, medical testing, food quality monitoring, forensic case determination, etc. However, the traditional immunochromatographic method has a low detection sensitivity and still has major defects in the detection of some trace or trace analyte residues, which makes it unable to meet the increasingly high detection requirements in various fields.

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

[0004] At present, the fluorescent markers used in LFA mainly include fluorescent microspheres, upconversion nanoparticles, carbon dots and quantum dots. Quantum dots have been widely used in recent years to construct highly sensitive and multivariate immunochromatography methods due to their advantages such as high luminescence intensity, wide excitation spectrum and narrow emission spectrum. However, quantum dots still have the risk of toxic metal elements and complex preparation. Metal nanoclusters, as an emerging type of fluorescent material, have the characteristics of low toxicity, high photostability and easy synthesis.

[0005] However, so far, very few studies have applied it to the development of fluorescent lateral flow immunochromatography methods. Therefore, the development of highly sensitive metal nanocluster-labeled fluorescent LFA methods has important application value. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

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

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a fluorescence-based immunochromatographic analysis AuNCs-LFA test paper.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: an immunochromatographic analysis (AuNCs-LFA) test paper based on fluorescent gold nanoclusters, comprising a sample pad, a gold label pad, an NC membrane, an absorption pad and a PVC bottom plate;

[0010] The NC membrane includes the 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, wherein: 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°C;

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

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

[0015] 20-100 μL of 0.02 mg / mL antibody was added to 25 μL of AuNCs solution, mixed evenly, and then 5 μL of 10 mg / mL BSA solution was added and reacted for 5-30 min to obtain the fluorescent nanocluster-antibody marker AuNCs-Ab.

[0016] As a preferred embodiment of the AuNCs-LFA test paper of the present invention, the preparation method of the AuNCs solution comprises:

[0017] The 6-aza-2-thiothymine solution and the chloroauric acid solution were mixed and reacted in the dark. After the reaction was completed, the AuNCs solution was purified to obtain the AuNCs solution.

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

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

[0020] As a preferred embodiment of the AuNCs-LFA test paper of the present invention, the overlapping portion between the sample pad and the PVC adhesive backing has a length of 1 to 2 mm, and the sample pad is placed above the PVC adhesive backing;

[0021] The overlapping length between the sample pad, gold label pad and NC membrane is 1 to 2 mm, the sample pad membrane is placed above the gold label pad, and the gold label pad is placed above the NC membrane;

[0022] The length of the overlapping portion between the NC film and the absorption pad is 1 to 3 mm, and the absorption pad is placed above the NC film.

[0023] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing AuNCs-LFA test paper, comprising:

[0024] Stick the sample pad, gold label pad, NC membrane and absorption pad on the PVC adhesive backing in sequence, with each overlapping by 2mm. Use a three-dimensional spray platform to spray 0.05-0.1mg / mL ZEN-BSA on the T line of the NC membrane and 0.1-0.2mg / mL goat anti-mouse IgG on the C line, and dry them at 30-37℃ for 2h. The distance between the test line and the control line is 4-8mm.

[0025] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of AuNCs-LFA test paper in detecting zearalenone (ZEN).

[0026] As a preferred embodiment of the application of the present invention, the method comprises: 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 at high heat for 1 min, and then read the test results.

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

[0029] Beneficial effects of the present invention:

[0030] (1) The existing competitive LFA using 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 fluorescent LFA in practical detection. This paper invented a lateral flow chromatography (AuNCs-LFA) detection method based on fluorescent gold nanoclusters. Compared with the traditional colloidal gold LFA method, the sensitivity of this method in detecting small molecules with naked eye is improved.

[0031] (2) The present invention provides a small-sized fluorescent probe, which significantly shortens the time of LFA detection of small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

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

[0034] Figure 2 Optimization diagram 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; (C) optimization of detection time.

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

[0036] Figure 4 This is a specific diagram of the AuNCs-LFA test paper of the present invention. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0038] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present 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 term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0040] Example 1

[0041] (1) Preparation of ATT-AuNCs

[0042] Preparation of ATT-AuNCs, the specific steps are as follows:

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

[0044] The synthesized AuNCs were then purified by 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 Di Tengmin Biotechnology Co., Ltd.) was added to AuNCs (25 μL), mixed evenly, and then 5 μL of BSA (10 mg / mL) solution was added and reacted for 5 min to obtain fluorescent nanocluster-antibody marker (AuNCs-Ab), which was stored at 4 °C.

[0046] (3) Preparation of test paper

[0047] Five materials were used to prepare the lateral flow chromatography device, namely, the sample pad, gold label pad, NC membrane and absorption pad were sequentially attached to the PVC adhesive backing, each overlapping by 2 mm.

[0048] 0.5 mg / mL ZEN-BSA (the purchased 1 mg / mL ZEN-BSA was diluted to 0.5 mg / mL with PBS) was sprayed on 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 on the control line (C line) of the NC membrane, and 8 ul AuNCs-Ab was sprayed on the gold label pad and dried at 37°C for 4 h. The distance between the T line and the C line was 5 mm;

[0049] Finally, the test strips were cut into 4 mm width and stored at 4 °C.

[0050] Example 2

[0051] Optimization of AuNCs-LFA detection method:

[0052] (1) Concentration of ZEN-BSA

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

[0054] (2) Volume of AuNCs-Ab

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

[0056] (3) Detection time

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

[0058] like Figure 2 AC, the three optimal key parameters 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; (3) the detection time was 5 min.

[0059] Example 3

[0060] Preparation of lateral flow chromatography test paper based on fluorescent metal nanoclusters:

[0061] 1. Preparation of test strips

[0062] Five materials were used to prepare the lateral flow chromatography device, namely, the sample pad, gold label pad, NC membrane and absorption pad were sequentially attached to the PVC adhesive backing, each overlapping by 2 mm.

[0063] 0.5 mg / mL ZEN-BSA was sprayed on 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 on the control line (C line) of the NC membrane, and 8 ul AuNCs-Ab was sprayed on the gold label pad and dried at 37 °C for 4 h. The distance between the T line and the C line was 5 mm;

[0064] Finally, the test strips were cut into 4 mm width and stored at 4 °C.

[0065] 2. Establishment of the detection method based on AuNCs-LFA test paper

[0066] 80 μL of different concentrations of ZEN (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 ng / mL, preparation method: dissolve 10 mg of ZEN standard in 1 mL of methanol, and then dilute with ultrapure water to 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 ng / mL ZEN solution) was mixed with 20 μL of Running buffer and added to the sample pad. After 5 minutes, the test strip was placed in a microwave oven and heated for 1 min, and the results were then detected using a portable reader.

[0067] In the absence of ZEN, the AuNCs-Ab immunoprobe was 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 binding to limited AuNCs-Ab, and the green fluorescence signal on the T line disappears. The schematic diagram of the detection of zearalenone by AuNCs-LFA is shown in Figure 1 .

[0069] Example 4

[0070] Detection of Zearalenone:

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

[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 A. The test conditions are: the concentration of ZEN-BSA is 0.5 mg / mL; the volume of AuNCs-Ab is 8 μL; and the detection time is 5 min.

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

[0076] like Figure 3 As shown in AB, with the increase of zearalenone concentration, the signal intensity on the T line of the test strip gradually disappeared. The detection line (LOD) of AuNCs-LFA for zearalenone 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 higher sensitivity for ZEN detection and a 10-fold higher sensitivity for naked eye detection, and can be used to detect large quantities of samples.

[0077] (2) Specificity of Zearalenone AuNCs-LFA Test Paper

[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 test the specificity of AuNCs-LFA. The steps were as follows:

[0079] 80 μL of zearalenone structural analog (20 ng / mL) was mixed with 20 μL of Runningbuffer buffer and added to the sample pad. After 5 minutes, the test strip was placed in a microwave oven and heated for 1 min. The results were then detected using a portable reader (detected at room temperature, without incubation, with an excitation wavelength of 365 nm and an emission wavelength of 510 nm. The test method was the same as in Example 3).

[0080] like Figure 4 As shown, the established method has a high cross-reaction with the structural analogs of zearalenone, while the detection results for other fungal toxins are negative. Therefore, the AuNCs-LFA established in the present invention has a high specificity for zearalenone.

[0081] (3) Recovery rate test using wheat simulated sample

[0082] The recovery rate was tested using wheat simulated samples. The steps are as follows:

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

[0084] Table 1

[0085]

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

[0087] In the present invention, the antibody-based fluorescent lateral flow immunochromatographic analysis test strip detection method (AuNCs-LFA) presents a fluorescent signal pattern. In the absence of ZEN, the AuNCs-Ab immune probe is captured by the antigen (ZEN-BSA) on the T line, forming a green fluorescent strip. In the presence of ZEN, ZEN competes with ZEN-BSA on the T line to bind to the limited AuNCs-Ab, and the fluorescent signal on the T line disappears. Regardless of whether ZEN is present, the fluorescence on the C line always exists.

[0088] The highly sensitive detection characteristics of the present invention are reflected in the high sensitivity and high responsiveness of the fluorescence signal of the nanocluster. In the traditional LFA using AuNPs as the signal probe, the signal intensity is weak. In the AuNCs-LFA using AuNCs as the probe, the signal intensity of the T line is significantly enhanced, thereby obtaining a significantly improved sensitivity.

[0089] The existing competitive LFA using colloidal gold as a probe has problems such as inefficient colorimetric response and low sensitivity to weakly positive samples. The preparation process of common fluorescent labeling materials is complicated or they are highly toxic, which limits the application of fluorescent LFA in actual detection. This article invents an LFA detection method based on fluorescent nanoclusters. Compared with the traditional colloidal gold LFA method, this method has improved sensitivity in detecting small molecules and 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 rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. An immunochromatographic analysis (AuNCs-LFA) test paper based on fluorescent gold nanoclusters, characterized in that: include, Sample pad, gold label pad, NC membrane, absorption pad and PVC base plate; The NC membrane includes the signal probe AuNCs-Ab, goat anti-mouse IgG and zearalenone (ZEN) antigen ZEN-BSA.

2. The AuNCs-LFA test paper according to claim 1, characterized in that: A test line and a control line are set on the NC membrane; 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°C; The distance between the test line and the control line is 4 to 8 mm.

3. The AuNCs-LFA test paper according to claim 1 or 2, characterized in that: The preparation method of the signal probe AuNCs-Ab comprises: 20-100 μL of 0.02 mg / mL antibody was added to 25 μL of AuNCs solution, mixed evenly, and then 5 μL of 10 mg / mL BSA solution was added and reacted for 5-30 min to obtain the fluorescent nanocluster-antibody marker AuNCs-Ab.

4. The AuNCs-LFA test paper according to claim 3, characterized in that: The AuNCs solution, the preparation method thereof comprises: The 6-aza-2-thiothymine solution and the chloroauric acid solution were mixed and reacted in the dark. After the reaction was completed, the AuNCs solution was purified to obtain the AuNCs solution.

5. The AuNCs-LFA test paper according to claim 4, characterized in that: The concentration of the 6-aza-2-thiothymine solution is 80 mmol; the concentration of the chloroauric acid solution is 10 mg / mL, and the ratio of the 6-aza-2-thiothymine solution to the chloroauric acid solution is 3 mL:3 mL.

6. The AuNCs-LFA test paper according to claim 4, characterized in that: The AuNCs have a diameter of 2 to 6 nm.

7. The AuNCs-LFA test paper according to claim 1, characterized in that: The overlapping portion between the sample pad and the PVC adhesive backing is 1 to 2 mm in length, and the sample pad is placed above the PVC adhesive backing; The overlapping length between the sample pad, gold label pad and NC membrane is 1 to 2 mm, the sample pad membrane is placed above the gold label pad, and the gold label pad is placed above the NC membrane; The length of the overlapping portion between the NC film and the absorption pad is 1 to 3 mm, and the absorption pad is placed above the NC film.

8. The method for preparing the AuNCs-LFA test paper according to any one of claims 1 to 7, characterized in that: include, Stick the sample pad, gold label pad, NC membrane and absorption pad on the PVC adhesive backing in sequence, with each overlapping by 2mm. Use a three-dimensional spray platform to spray 0.05-0.1mg / mL ZEN-BSA on the T line of the NC membrane and 0.1-0.2mg / mL goat anti-mouse IgG on the C line, and dry them at 30-37℃ for 2h. The distance between the test line and the control line is 4-8mm.

9. Use of the AuNCs-LFA test paper according to any one of claims 1 to 7 in detecting zearalenone.

10. The use according to claim 9, characterized in that: include, The zearalenone sample to be tested is mixed with a running buffer to prepare a mixed solution; 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 at high heat for 1 min, and then read the test results. The running buffer consisted of 0.01 M PBS, 0.5% Tween-20, pH 7.4.

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