Fluorescence quenching immunochromatography test strip for detecting lead ions as well as preparation and application of fluorescence quenching immunochromatography test strip

Abstract

The invention discloses a fluorescence quenching immunochromatography test strip for detecting lead ions as well as preparation and application thereof. The test strip sequentially comprises a PVC (polyvinyl chloride) bottom plate, a sample pad, an NC (nitrocellulose) membrane and a water absorption pad, the NC membrane comprises a detection area and a quality control area, the detection area is coated with a fluorescent donor probe-lead complete antigen mixture, and the quality control area is coated with a fluorescent donor probe-goat anti-mouse IgG antibody mixture; the fluorescence donor probe is a time-resolved fluorescent microsphere-BSA (Bovine Serum Albumin) compound; the sample pad is used for chromatography of a compound of an object to be detected and the fluorescent receptor probe, and the fluorescent receptor probe is a gold nanoflower-heavy metal lead monoclonal antibody compound; the lead complete antigen and the to-be-detected object compete to be combined with the fluorescent receptor probe; the gold nanoflowers are applied to the fluorescence quenching immunochromatography test strip as a fluorescence receptor, so that high-sensitivity and high-accuracy detection on heavy metal lead ions can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid food safety detection, and in particular to a fluorescence quenching immunochromatographic test strip for detecting lead ions, and preparation and application thereof. Background Art

[0002] Heavy metal lead pollution poses a serious threat to the ecological environment, food safety, and human health. Lead poisoning can cause damage to the nervous system, kidneys, and immune system. my country's national food safety standard GB 2762-2022, "Limits of Contaminants in Food," stipulates a lead limit of 0.2 mg / kg for cereals and their products, and 0.5 mg / kg for oatmeal, gluten, canned porridge, and rice and flour products with fillings. Currently, methods for detecting heavy metal ions of lead primarily include atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). However, these methods are limited by expensive equipment, complex operation, and long detection times, making them inadequate for rapid on-site screening of large numbers of samples. Therefore, strengthening lead detection in food and developing sensitive and rapid lead detection methods are crucial for ensuring food safety in my country.

[0003] Immunochromatographic technology has become a research hotspot in the field of rapid testing due to its advantages such as ease of use, low cost, and intuitive results. Traditional immunochromatographic test strips often use colloidal gold as a signal marker for colorimetric analysis, which suffers from issues such as insufficient sensitivity and susceptibility to matrix interference. Fluorescence analysis, on the other hand, offers higher signal intensity than colorimetric analysis, potentially improving test strip sensitivity. Immunochromatographic test strips for detecting small molecule targets such as heavy metal ions typically employ a competitive reaction model, quantifying the target by measuring the signal's "turn-off" behavior. However, when the analyte concentration is low, the resulting small changes in the detection signal are difficult to detect, resulting in low sensitivity. Recently, fluorescence quenching has been introduced into immunochromatographic technology as a novel signal enhancement strategy. This mechanism, triggered by specific antigen-antibody binding, provides a "turn-on" fluorescence signal output in the forward direction, improving test strip sensitivity and reducing background interference. The detection principle of fluorescence quenching immunochromatographic technology is based on the inner filter effect (IFE) between a fluorescent donor and a light-absorbing probe (quencher), converting the absorbance response into a change in fluorescence intensity, thereby achieving signal amplification. Among widely used fluorescent donors, time-resolved fluorescent microspheres (TRFM) offer advantages such as high brightness, excellent signal stability, and a large Stokes shift, making them ideal fluorescent materials. However, fluorescence quenching immunochromatographic test strips prepared using TRFM lack effective energy transfer, resulting in poor sensitivity and accuracy in lead ion detection.

[0004] Therefore, it is necessary to provide a fluorescence quenching immunochromatographic test strip that improves the detection sensitivity and accuracy of lead ions. Summary of the Invention

[0005] In view of this, the present application provides a fluorescence quenching immunochromatographic test strip for detecting lead ions and its preparation and application, which are used to solve the problem of how to improve the detection sensitivity and accuracy of lead ions.

[0006] In order to achieve the above technical objectives, this application adopts the following technical solutions: In the first aspect, the present application provides a fluorescence quenching immunochromatographic test strip for detecting lead ions, which comprises a PVC base plate, a sample pad, an NC membrane, and a water-absorbing pad in sequence; the NC membrane comprises a detection area and a quality control area, the detection area is coated with a fluorescent donor probe-lead complete antigen mixture, and the quality control area is coated with a fluorescent donor probe-goat anti-mouse IgG antibody mixture; the fluorescent donor probe is a time-resolved fluorescent microsphere-BSA complex; the sample pad is used to chromatograph the complex of the analyte and the fluorescent receptor probe, and the fluorescent receptor probe is a gold nanoflower-heavy metal lead monoclonal antibody complex; the lead complete antigen competes with the analyte for binding to the fluorescent receptor probe.

[0007] Preferably, the gold nanoflower is petal-shaped, has a diameter of 75-85 nm, and has a maximum absorption peak of 615 nm.

[0008] In a second aspect, the present application provides a method for preparing a fluorescence quenching immunochromatographic test strip for detecting lead ions, comprising the following steps: preparing a fluorescent acceptor probe solution and a fluorescent donor probe solution respectively; The NC membrane is pasted to the middle of the PVC base plate, and then the fluorescent donor probe solution is mixed with the complete lead antigen and sprayed on the detection area of ​​the NC membrane. The fluorescent donor probe solution is mixed with the goat anti-mouse IgG antibody and sprayed on the quality control area of ​​the NC membrane. Then it is dried, and the absorption pad and sample pad are pasted to the two ends of the PVC base plate respectively. After cutting into strips, a fluorescence quenching immunochromatographic test strip for detecting lead ions is obtained.

[0009] Preferably, the preparation method of the fluorescent receptor probe solution includes: adding potassium carbonate and lead monoclonal antibody to the gold nanoflower solution, mixing evenly and letting it stand, then adding BSA solution, mixing evenly and letting it stand, centrifuging, discarding the supernatant, and redissolving the resulting precipitate in a boric acid buffer containing PEG and a pH of 8-9 to obtain a fluorescent receptor probe solution.

[0010] Preferably, the preparation method of gold nanoflowers includes: Add trisodium citrate to the boiling chloroauric acid solution, heat and stir until the color is stable to obtain gold seed; Mix the gold seeds with the chloroauric acid solution, the trisodium citrate solution and the hydroquinone solution, and stir to react, to obtain the gold nanoflower.

[0011] Preferably, the preparation method of the fluorescent donor probe is as follows: taking the time-resolved fluorescent microspheres and BSA as raw materials, and performing coupling reaction through the activated ester method, to obtain the fluorescent donor probe.

[0012] Preferably, the concentration of the lead complete antigen is 0.04-0.05 mg / mL, and the spraying amount is 0.8-0.9 μL / cm.

[0013] Preferably, the concentration of the goat anti-mouse IgG antibody is 0.6-0.7 mg / mL, and the spraying amount is 0.8-0.9 μL / cm.

[0014] Preferably, the preparation method of the sample pad is as follows: soaking the glass fiber membrane in the blocking solution, and drying overnight, to obtain the sample pad.

[0015] In a third aspect, the application provides the application of the fluorescent quenching immunochromatography test strip for detecting lead ions in detecting heavy metal lead in food.

[0016] The application has the following beneficial effects: The test strip of the application has high sensitivity, and the molar extinction coefficient of the obtained gold nanoflower is higher than that of colloidal gold. The gold nanoflower is applied to the fluorescent quenching immunochromatography test strip as a fluorescent acceptor, so that high-sensitivity detection of heavy metal lead ions (the minimum detection limit is 0.04 ng / mL) can be realized. The fluorescent quenching immunochromatography test strip of the application has high sensitivity and good specificity, and has no obvious cross-reaction with heavy metal ions such as cadmium, cobalt, copper, mercury and arsenic. The fluorescent quenching immunochromatography test strip of the application is simple to operate and fast in detection, and can realize rapid screening of a large number of samples. Specifically, it can be applied to rapid quantitative determination of lead ion content in rice, and has important application value in grain quality control. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a structural schematic diagram of the fluorescent quenching immunochromatography test strip for detecting lead ions. Figure 2 It is a real object diagram of the gold seeds (A) and the gold nanoflower (B). Figure 3 It is a gold nanoflower identification result diagram. (A) is a transmission electron microscope diagram of the gold seeds; (B) is a transmission electron microscope diagram of the gold nanoflower; and (C) is an ultraviolet-visible absorption spectrum diagram of the gold seeds, the gold nanoflower and the gold nanoflower-antibody conjugate. Figure 4 It is a fluorescence scanning spectrum of the TRFM-BSA complex and an absorption spectrum superimposition diagram of the gold nanoflower-antibody conjugate. Figure 5 It is the standard working curve for the detection of lead ions by fluorescence quenching immunochromatographic test strips; Figure 6 This is a photo of a fluorescence quenching immunochromatographic test strip for detecting lead ions; Figure 7 This is the specific test result of the fluorescence quenching immunochromatographic test strip for detecting lead ions. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0019] like Figure 1 As shown, the present application provides a fluorescence quenching immunochromatographic test strip for detecting lead ions, which includes a PVC base plate, a sample pad, an NC membrane, and a water-absorbing pad in sequence; the NC membrane includes a detection area and a quality control area, the detection area is coated with a fluorescent donor probe-lead complete antigen mixture, and the quality control area is coated with a fluorescent donor probe-goat anti-mouse IgG antibody mixture; the fluorescent donor probe is a time-resolved fluorescent microsphere-BSA complex; the sample pad is used to chromatograph the complex of the analyte and the fluorescent receptor probe, and the fluorescent receptor probe is a gold nanoflower-heavy metal lead monoclonal antibody complex; the lead complete antigen competes with the analyte for binding to the fluorescent receptor probe.

[0020] In this application, gold nanoflowers with petal-like structures are labeled with heavy metal lead monoclonal antibodies as fluorescent receptor probes (gold nanoflower immune probes, AuNF-mAb), time-resolved fluorescent microspheres are used as fluorescent materials, time-resolved fluorescent microspheres modified with bovine serum albumin (BSA) are used as fluorescent donor probes, mixed with complete lead antigen and goat anti-mouse IgG antibody respectively, and sprayed on specific areas of the NC membrane as detection lines and quality control lines, using the complete lead antigen to compete with the analyte for binding to the heavy metal lead monoclonal antibody of the fluorescent receptor probe, as well as the inner filtration effect (IF) of gold nanoflowers and time-resolved fluorescent microspheres. E), assembled into a lateral flow chromatography test strip based on fluorescence quenching forward reading signal, the detection process: when there are no lead ions in the sample solution, the gold nanoflower immune probe binds to the lead antigen on the detection line, absorbs the emission light of the fluorescent microspheres and causes the fluorescence signal of the detection line to disappear or weaken, while the quality control line has fluorescence; when lead ions are present in the sample solution, the gold nanoflower immune probe preferentially binds to the lead ions. At this time, the number of gold nanoflower probes bound to the detection line decreases, resulting in an increase in the fluorescence signal of the test strip detection line, while the number of gold nanoflower probes bound to the quality control line increases, and the fluorescence signal of the quality control line decreases. The test strip provided by the present invention is simple to operate, highly sensitive, and easy to interpret results, and can be used for on-site rapid detection of heavy metal lead ions.

[0021] In some embodiments, the gold nanoflower is petal-shaped, has a diameter of 75-85 nm, and has a maximum absorption peak of 615 nm.

[0022] The gold nanoflowers of the present application are petal-shaped gold nanoparticles with a high extinction coefficient, large specific surface area, good colloidal stability, and significant local surface plasmon resonance (LSPR) effect. They have good extinction effect as fluorescence quenchers for time-resolved fluorescent microspheres. The absorption spectrum of the quencher overlaps with the excitation or emission spectrum of the fluorescence donor, and effective energy transfer can be obtained. The fluorescence quenching immunochromatographic test strips prepared with AuNF as the fluorescence acceptor and TRFM as the fluorescence donor are simple to operate, highly sensitive, and highly accurate, and can meet the existing grassroots needs for rapid screening of heavy metal lead.

[0023] The present application provides a method for preparing a fluorescence quenching immunochromatographic test strip for detecting lead ions, comprising the following steps: S1. Prepare fluorescent acceptor probe solution and fluorescent donor probe solution respectively; S2. Paste the NC membrane to the middle of the PVC base plate, then mix the fluorescent donor probe solution with the complete lead antigen and spray it on the detection area of ​​the NC membrane. Mix the fluorescent donor probe solution with the goat anti-mouse IgG antibody and spray it on the quality control area of ​​the NC membrane. Then dry it. Then, paste the absorption pad and sample pad to the two ends of the PVC base plate respectively. After cutting the strips, you will get the fluorescence quenching immunochromatographic test strip for detecting lead ions.

[0024] The fluorescence quenching immunochromatographic test strip provided by the present invention adopts a competitive method to quantitatively detect the content of heavy metal ion lead: the lead ions in the buffer solution first bind to the monoclonal antibody in the quencher gold nanoflower-labeled monoclonal antibody complex (fluorescent receptor probe), and are continuously chromatographed through the sample pad. The complex slowly moves on the NC membrane by relying on the chromatography effect, does not bind to the complete lead antigen in the detection line area, and the fluorescence donor in the detection line area is not quenched. The complex of the gold nanoflower-labeled antibody bound to the lead ion binds to the goat anti-mouse IgG antibody in the quality control line area. The fluorescence signal intensities of the detection line and the quality control line on the test strip can be automatically read by a fluorescence quantitative analyzer, and the linear range of detecting lead ions in the buffer solution is calculated to be 0.06-1.0 ng / mL, with good sensitivity and specificity.

[0025] In some embodiments, the preparation method of the fluorescent receptor probe solution includes: adding potassium carbonate and lead monoclonal antibody to the gold nanoflower solution, mixing evenly and letting it stand, and then adding BSA solution, mixing evenly and letting it stand, centrifuging, discarding the supernatant, and redissolving the resulting precipitate in a boric acid buffer containing PEG and a pH of 8-9 to obtain a fluorescent receptor probe solution.

[0026] In this embodiment, the heavy metal lead monoclonal antibody is physically adsorbed on the surface of the gold nanoflower to obtain a fluorescent receptor probe.

[0027] In some embodiments, the preparation method of the gold nanoflower comprises: K1. Adding trisodium citrate to a boiling chloroauric acid solution, and heating and stirring until the color is stable to obtain gold seeds; K2. Mixing and reacting the gold seeds with a chloroauric acid solution, a trisodium citrate solution, and a hydroquinone solution to obtain the gold nanoflower.

[0028] In this embodiment, the blue gold nanoflower is prepared by a seed-mediated growth method using spherical gold nanoparticles, sodium citrate, chloroauric acid, and hydroquinone as raw materials.

[0029] In some embodiments, the preparation method of the fluorescent donor probe is as follows: using time-resolved fluorescent microspheres and BSA as raw materials, and performing a coupling reaction by an activated ester method to obtain the fluorescent donor probe.

[0030] In some embodiments, the concentration of the lead complete antigen is 0.04-0.05 mg / mL, and the spraying amount is 0.8-0.9 μL / cm.

[0031] In some embodiments, the concentration of the goat anti-mouse IgG antibody is 0.6-0.7 mg / mL, and the spraying amount is 0.8-0.9 μL / cm.

[0032] In some embodiments, the preparation method of the sample pad is as follows: soaking a glass fiber membrane in a blocking solution, and drying overnight to obtain the sample pad.

[0033] The application provides an application of a fluorescent quenching immunochromatographic test strip for detecting lead ions in detecting heavy metal lead in food, and the specific application steps are as follows: mixing the fluorescent receptor probe with a sample to be detected, and then dropping the mixture on a sample pad, chromatographing the mixture to a water absorption pad layer through capillary action, allowing a lead complete antigen to compete with the sample to be detected to bind to the fluorescent receptor probe, and reading the fluorescence values on the detection line and the quality control line by using a fluorescence analyzer.

[0034] In some embodiments, the food is rice.

[0035] The present application is further described below through specific embodiments.

[0036] Raw material sources: Heavy metal lead monoclonal antibody: Wuxi Ditenmin Biotechnology Co., Ltd. Lead complete antigen: Wuxi Ditenmin Biotechnology Co., Ltd. Goat anti-mouse IgG antibody: Wuhan Yingke Biotechnology Co., Ltd. Time-resolved microspheres: Changsha Meiniu Biotechnology Co., Ltd. Sample pad: the glass fiber membrane was taken out after being fully immersed in the blocking solution, and dried at 37℃ overnight to obtain the sample pad. The blocking solution was prepared by adding 0.5% BSA, 0.5% PEG20000, 1% Tween 20 and 1% polyvinylpyrrolidone K-30 into 100 mL 0.01 M PBS (pH 7.4) solution.

[0037] Example 1 A fluorescence quenching immunochromatographic test strip for detecting lead ions comprises, in sequence, a PVC base plate, a sample pad, an NC membrane, and a water absorption pad. The NC membrane comprises a detection zone and a quality control zone. The detection zone is coated with a mixture of fluorescent donor probes and complete lead antigen, and the quality control zone is coated with a mixture of fluorescent donor probes and goat anti-mouse IgG antibody. The fluorescent donor probe is a time-resolved fluorescent microsphere-BSA complex. The sample pad is used to chromatograph the complex of the analyte and the fluorescent acceptor probe, and the fluorescent acceptor probe is a gold nanoflower-heavy metal lead monoclonal antibody complex. The complete lead antigen competes with the analyte to bind to the fluorescent acceptor probe.

[0038] The preparation method of the fluorescence quenching immunochromatographic test strip for detecting lead ions is as follows: S1. Gold nanoflowers were synthesized by seed-mediated growth method, and the steps were as follows: 2.7 mL of 1wt% trisodium citrate was quickly added into 100 mL of 0.01wt% chloroauric acid solution under boiling (100℃), and the solution was heated and stirred vigorously until the color changed to orange red. The heating and stirring state was maintained until the color of the solution no longer changed, and a seed gold solution with a diameter of 15 nm was obtained, i.e. the gold seed. Under room temperature (25℃) conditions, 1 mL of gold seed, 0.22 mL of 1wt% trisodium citrate solution and 10 mL of 0.03 M hydroquinone solution were added into 100 mL of 0.01wt% chloroauric acid solution under stirring, and stirred for 30 min to obtain a gold nanoflower solution. The prepared gold seed colloid was a grape wine red transparent liquid, as shown in Figure 2 (A); the gold nanoflower was an indigo blue transparent liquid without turbidity, as shown in Figure 2 (B); transmission electron microscopy observation showed that the gold seed particles had good dispersibility, and the diameter was 15 nm, as shown in Figure 3 (A); the gold nanoflower could be clearly seen as petal-like morphology under the scale of 100 nm, and had good dispersibility, with a diameter of about 80 nm, as shown in Figure 3 (B); ultraviolet-visible light spectrum scanning showed that the maximum absorption peak of the gold seed was 518 nm, the maximum absorption peak of the gold nanoflower was 615 nm, and the peak shape was relatively wide. After the gold nanoflower was coupled with the lead monoclonal antibody, the maximum absorption peak was 623 nm, which showed obvious red shift compared with the gold nanoflower, as shown in Figure 3(C) showed that the gold nanoflower and the conjugate with lead monoclonal antibody were successfully prepared; 1 mL of gold nanoflower (AuNF) solution was taken, 8 μL of 0.2 M K2CO3 solution and 6 μL of 1.0 mg / mL lead monoclonal antibody were added, the above solution was mixed uniformly, and then was left to stand at room temperature for 1 h, then 1% BSA solution was added, mixed uniformly, and then was left to stand at room temperature for 30 min, then was centrifuged at 22000 g for 15 min, the supernatant was discarded, and the precipitate was resuspended in 100 μL of 0.1 M boric acid buffer (pH 8.2) containing 0.2% PEG20000 for use, to obtain the gold nanoflower and monoclonal antibody complex (AuNF-mAb) as a fluorescent receptor probe; 0.1 mL of time-resolved fluorescent microspheres (1%) was taken, diluted with 0.9 mL of MES buffer solution with pH=6.0 and 25 mM, mixed uniformly, then centrifuged at 22000 g for 15 min, the supernatant was discarded, and the precipitate was resuspended in 0.4 mL of MES buffer solution, then 80 μL of 10 mg / mL EDC solution and 80 μL of 10 mg / mL NHS solution were added in sequence, incubated at room temperature for 30 min, centrifuged at 22000 g for 15 min, the supernatant was discarded, and the precipitate was resuspended in 0.4 mL of MES buffer solution, then 50 μL of 10 mg / mL BSA solution was added, incubated at room temperature for 2.5 h, then 20 μL of 10% BSA was added for blocking for 30 min, the complex solution was centrifuged, the supernatant was discarded, and the precipitate was resuspended in 200 μL of PBS buffer solution with pH=7.4 and 10 mM, to obtain the time-resolved fluorescent microsphere and bovine serum albumin complex (TRFM-BSA) as a fluorescent donor probe, the fluorescence scanning spectrum of the prepared TRFM-BSA complex and the superposition result of the absorption spectrum of the gold nanoflower-antibody conjugate are shown in Figure 4 The absorption spectrum of the gold nanoflower-antibody conjugate and the emission spectrum of the fluorescent donor completely overlap, which shows that the absorption spectrum of the quencher overlaps with the excitation or emission spectrum of the fluorescent donor, and effective energy transfer is obtained; S2. The prepared TRFM-BSA complex was diluted 200 times with 10 mM PBS buffer (pH 7.4) containing 1% BSA, and equally divided into two parts. One part was added with lead complete antigen at a final concentration of 0.04 mg / mL to form a mixture as coating solution I (fluorescent donor probe-lead complete antigen mixture) of the test line of the test strip. The other part was added with goat anti-mouse IgG antibody at a final concentration of 0.6 mg / mL to form a mixture as coating solution II (fluorescent donor probe-goat anti-mouse IgG antibody mixture) of the quality control line of the test strip. The NC membrane was pasted in the middle of the PVC base plate, and coating solution I (0.8 μL / cm) was sprayed on the NC membrane as the test line and coating solution II (0.8 μL / cm) was sprayed on the NC membrane as the quality control line by using a gold spraying membrane drawing instrument, with a distance of 10 mm between the two lines. The lengths of the overlapping regions of the sample pad, the NC membrane, and the water absorption pad were 2 mm. The PVC plate was dried in a 37°C oven for 2 h. The absorption pad and the sample pad were pasted at the two ends of the PVC base plate, respectively. Finally, the assembled test strip was cut into strips (3.9 mm) by a microcomputer automatic cutting machine to obtain the fluorescent quenching immunochromatography test strip for detecting lead ions, which was sealed and dried for storage.

[0039] Test and evaluation The fluorescent quenching immunochromatography test strip for detecting lead ions obtained in Example 1 was used for lead ion detection, and the process was as follows: The 1000 ng / mL lead ion standard solution was prepared into a mother liquor with 0.01 mol / L PBS buffer (pH=7.4) containing 10 μmol / L ethylenediaminetetraacetic acid. The mother liquor was diluted into a series of lead ion standard solution (0 ng / mL, 0.06 ng / mL, 0.09 ng / mL, 0.2 ng / mL, 0.3 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 2.0 ng / mL, 5.0 ng / mL) by using the PBS buffer. 100 μL of the solution and 10 μL of AuNF-mAb fluorescent receptor probe were simultaneously added into a micro-hole strip. The test strip was inserted into the micro-hole strip, and the chromatography reaction was carried out at 37°C for 8 min. The actual figure is shown in Figure 5 The fluorescence signal ratio of the test line and the quality control line of the test strip was read. The fluorescence signal ratio when no lead ion standard solution was added was F0, the fluorescence signal ratio when the lead ion standard solution was added was F, and the fluorescence signal ratio when the lead ion standard solution was saturated was Fmax. The value of (F-F0) / (Fmax-F0) was taken as the ordinate to fit the standard curve, and the result is shown in Figure 6 The regression equation of the standard curve was Y=0.787+0.546*lgX (R 2=0.991). The lowest detection limit (LOD) is defined as the average value of the fluorescence signal ratio F0 of 20 repeated measurements of the blank sample minus 3 times the standard deviation, which is brought into the standard curve equation to calculate the minimum detection limit. The test strip provided by the present application has a LOD of 0.04 ng / mL for lead ions, and a linear detection range of 0.06-1 ng / mL.

[0040] The fluorescence quenching immunochromatographic test strip obtained in Example 1 was evaluated for specificity test: the test strip of Example 1 was used to detect PBS buffer, 20 ng / mL lead ions, 20 ng / mL cadmium ions, 20 ng / mL cobalt ions, 20 ng / mL copper ions, 20 ng / mL mercury ions and 20 ng / mL arsenic ions, respectively, and the fluorescence signals during detection of different heavy metal ions were recorded, see Figure 7 . According to Figure 7 it can be known that when the test strip is used to detect the other 5 kinds of heavy metal ions except lead ions, the images of the test strip under ultraviolet light are obviously different from the detection results of lead ions. It is shown that the fluorescence quenching immunochromatographic test strip provided by the present application has high specificity for lead ion detection.

[0041] Actual detection of rice samples: 1 g of the powdered rice sample was mixed with 5 mL of 1 mol / L nitric acid solution, and after 3 min of vigorous shaking extraction, 4000 g centrifugation for 3 min, 100 μL of the supernatant was added to 250 μL of 0.5 mol / L sodium hydroxide solution, and then diluted 4 times with 0.01 mol / L PBS buffer (containing 10 μmol / L ethylenediaminetetraacetic acid) at pH=7.4 to obtain the rice sample to be detected, and then the test strip test was performed, and the test strip test time was 8 min. The detection results are shown in Table 1, and the detection accuracy of the fluorescence quenching immunochromatographic test strip of the present application was determined by the recovery test. As can be seen from Table 1, the recovery rate of the rice sample is between 96.4-104.9%, which shows that the test strip has good accuracy.

[0042] Table 1 Test strip detection results of rice samples with different concentrations of lead ions

[0043] As can be seen from the above results, the fluorescence quenching immunochromatographic test strip of the present application can effectively identify heavy metal ions lead, and can be applied to rapid detection of lead ions in rice, and has high sensitivity and accuracy.

[0044] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any changes or replacements easily thought of by those skilled in the art within the technical range disclosed by the present application should be covered within the protection scope of the present application.

Claims

1. A fluorescence quenching immunochromatographic test strip for detecting lead ions, characterized in that: It includes a PVC base plate, a sample pad, an NC membrane, and a water-absorbing pad in sequence; the NC membrane includes a detection area and a quality control area, the detection area is coated with a fluorescent donor probe-lead complete antigen mixture, and the quality control area is coated with a fluorescent donor probe-goat anti-mouse IgG antibody mixture; the fluorescent donor probe is a time-resolved fluorescent microsphere-BSA complex; the sample pad is used to chromatograph the complex of the analyte and the fluorescent receptor probe, and the fluorescent receptor probe is a gold nanoflower-heavy metal lead monoclonal antibody complex; the lead complete antigen competes with the analyte to bind to the fluorescent receptor probe.

2. The fluorescence quenching immunochromatographic test strip for detecting lead ions according to claim 1, wherein The gold nanoflower is in the shape of petals, the diameter of the gold nanoflower is 75-85 nm, and the maximum absorption peak of the gold nanoflower is 615 nm.

3. A method for preparing a fluorescence quenching immunochromatographic test strip for detecting lead ions according to any one of claims 1 to 2, characterized in that: The following steps are involved: preparing a fluorescent acceptor probe solution and a fluorescent donor probe solution respectively; The NC membrane is pasted to the middle of the PVC base plate, and then the fluorescent donor probe solution is mixed with the complete lead antigen and sprayed on the detection area of ​​the NC membrane. The fluorescent donor probe solution is mixed with the goat anti-mouse IgG antibody and sprayed on the quality control area of ​​the NC membrane, and then dried. The absorption pad and the sample pad are respectively pasted to the two ends of the PVC base plate. After cutting into strips, the fluorescence quenching immunochromatographic test strip for detecting lead ions is obtained.

4. The preparation method according to claim 3, characterized in that The preparation method of the fluorescent receptor probe solution includes: adding potassium carbonate and lead monoclonal antibody to the gold nanoflower solution, mixing evenly and letting it stand, then adding BSA solution, mixing evenly and letting it stand, centrifuging, discarding the supernatant, and redissolving the resulting precipitate in a boric acid buffer containing PEG and a pH of 8-9 to obtain the fluorescent receptor probe solution.

5. The preparation method according to claim 4, characterized in that The preparation method of the gold nanoflower comprises: Add trisodium citrate to the boiling chloroauric acid solution, heat and stir until the color is stable to obtain gold seed; The gold seed is stirred and mixed with chloroauric acid solution, trisodium citrate solution and hydroquinone solution to react, thereby obtaining the gold nanoflower.

6. The preparation method according to claim 3, characterized in that The preparation method of the fluorescent donor probe is as follows: using time-resolved fluorescent microspheres and BSA as raw materials, performing a coupling reaction through an activated ester method, and thus obtaining the fluorescent donor probe.

7. The preparation method according to claim 3, wherein The concentration of the lead complete antigen is 0.04-0.05 mg / mL, and the spray volume is 0.8-0.9 μL / cm.

8. The preparation method according to claim 3, wherein The concentration of the goat anti-mouse IgG antibody is 0.6-0.7 mg / mL, and the spray volume is 0.8-0.9 μL / cm.

9. The preparation method according to claim 3, characterized in that The sample pad is prepared by soaking a glass fiber membrane in a blocking solution and drying the membrane overnight to obtain the sample pad.

10. Use of the fluorescence quenching immunochromatographic test strip for detecting lead ions according to any one of claims 1 to 2 in detecting heavy metal lead in food.

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