Time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ion and preparation method and application thereof

Abstract

The application provides a time-resolved fluorescent immunochromatographic test strip for detecting heavy metal cadmium ions and a preparation method and application thereof. The test strip comprises a PVC bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption paper. The heavy metal cadmium antigen is coated on the T line on the nitrocellulose membrane, and the sheep anti-mouse antibody is coated on the C line. The europium time-resolved fluorescent microsphere-heavy metal cadmium monoclonal antibody immune complex is sprayed on the combination pad. The immune complex has the optical characteristics of the time-resolved fluorescent microsphere that can eliminate non-specific background fluorescence and the immune characteristics of the antibody. When the solution to be measured flows to the T line, the immune complex can be combined with the antigen. The T line presents red fluorescence under ultraviolet light. The rest is combined with the sheep anti-mouse antibody on the C line. The detection of the heavy metal cadmium ions can be realized through a time-resolved fluorescent reader. The method is sensitive, short in detection time, convenient to operate and carry, and can be applied to the rapid detection of cadmium ions in tea.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection technology of heavy metal ions, specifically relating to a time-resolved fluorescence immunochromatographic test strip for detecting cadmium ions, its preparation method and application, that is, using the time-resolved fluorescence immunochromatographic test strip to rapidly detect cadmium ions in food. Background Technology

[0002] Cadmium is a common heavy metal, usually existing in the form of cadmium ions. Affected by human activities such as industrial development, agricultural activities, and the combustion of fossil fuels, heavy metals released into the environment can further enter the biosphere cycle through the atmosphere, soil, and water. Since tea trees thrive in acidic soil, if the soil where tea trees are planted is contaminated with heavy metals, the acidic environment further facilitates the conversion of heavy metals into bioavailable forms, increasing the absorption of heavy metals by the tea tree roots and making it easier for heavy metals to migrate from the soil to the tea leaves. Cadmium ion pollution in tea not only leads to a decline in tea yield and quality but also endangers human health through direct contact or bioaccumulation in the food chain. Studies have shown that heavy metals can cause damage to organs such as the lungs, kidneys, liver, blood, gastrointestinal tract, and bones, thereby inducing hepatotoxicity, nephrotoxicity, carcinogenicity, teratogenicity, and reproductive toxicity. Tea contaminated with heavy metals not only weakens the competitiveness of tea-producing areas but also has a certain impact on the import and export market for tea. Traditional heavy metal detection techniques include atomic absorption spectrometry, atomic fluorescence spectrometry, and inductively coupled plasma atomic absorption spectrometry, which require complex and expensive large-scale instruments and experienced operators. In contrast, fluorescence immunoassay is simple to operate, highly specific, and has a short detection time, making it an important rapid detection method.

[0003] Time-resolved fluorescence immunoassay (TRFI) combines time-resolved fluorescence with immunological techniques to achieve quantitative labeling and fluorescence reading of antigens and antibodies. It leverages the advantages of time-resolved fluorescent materials, such as long fluorescence lifetime (typically in the μs to ms range), high quantum yield, narrow emission bandwidth, and large Stokes shift, while also possessing strong immunological specificity, high sensitivity, and rapid response. TRFI test strips are a commonly used rapid detection method, simple to operate, portable, and providing results within minutes, making them suitable for on-site testing in various settings such as farms, supermarkets, and homes. Currently, there are no reports on the use of TRFI test strips for the detection of cadmium ions in tea. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions, its preparation method, and its application. The test strip includes a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The nitrocellulose membrane has a detection line (T line) and a control line (C line). The T line is coated with heavy metal cadmium antigen, and the C line is coated with goat anti-mouse secondary antibody. The conjugate pad is coated with an europium time-resolved fluorescent microsphere-heavy metal cadmium monoclonal antibody immune complex. The europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex of this invention possesses both the optical properties of time-resolved fluorescent microspheres (eliminating non-specific background fluorescence) and the immunological properties of antibodies. Relying on the specific binding of antigen and antibody (i.e., binding to the antigen when the test solution flows to the T line, where it exhibits red fluorescence under ultraviolet light, and binding to the goat anti-mouse antibody on the C line), and based on the relationship between fluorescence intensity and heavy metal ion concentration, the detection of cadmium ions can be achieved (i.e., detection of cadmium ions can be achieved using a time-resolved fluorescence reader). This method is highly sensitive, has a short detection time, is easy to operate and carry, and can be applied to the rapid detection of cadmium ions in tea. It has significant reference value and implications for the research of rapid detection methods for heavy metals in food.

[0005] To achieve the above objectives, the solution of the present invention is:

[0006] A method for preparing a time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions includes the following steps:

[0007] (1) Preparation of europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex;

[0008] (2) Preparation of a binding pad coated with europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex;

[0009] (3) Coat the heavy metal cadmium antigen on the T line and coat the goat anti-mouse secondary antibody on the C line. After drying, attach them together with absorbent paper, sample pad and conjugate pad to assemble the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions.

[0010] As a preferred embodiment of the present invention, in step (1), the preparation method of the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex includes:

[0011] Europium time-resolved fluorescent microspheres were diluted with borate buffer and sonicated. Carbodiimide hydrochloride solution was added, and after activation, the supernatant was discarded by centrifugation. Goat anti-mouse secondary antibody was added, and after coupling, cadmium monoclonal antibody was added and coupled. After centrifugation, the supernatant was discarded. Blocking buffer was added to block unbound sites. After incubation and shaking, the reaction was carried out. After centrifugation, the supernatant was discarded, and the mixture was dissolved in borate buffer and stored under cold for later use.

[0012] In a preferred embodiment of the present invention, the concentration of the borate buffer is 0.01-0.05 mol / L and the pH is 6-9.

[0013] In a preferred embodiment of the present invention, the final concentration of the carbodiimide hydrochloride solution in the reaction solution is 0.2-0.4 μg / μL.

[0014] In a preferred embodiment of the present invention, the activation reaction time is 20-45 min and the activation reaction temperature is 30-40℃.

[0015] In a preferred embodiment of the present invention, the centrifugation speed is 12000-16000 rpm; the centrifugation time is 30-50 min.

[0016] As a preferred embodiment of the present invention, the preparation process of the heavy metal cadmium monoclonal antibody is as follows: the immunogen Cd-ITCBE-BSA is emulsified and injected into mice. After polyclonal antibody serum titer determination, cell fusion, and hybridoma cell screening, the hybridoma cells are injected into the peritoneal cavity of non-immunized mice to obtain mouse ascites. After dialysis, the heavy metal cadmium monoclonal antibody is obtained.

[0017] In a preferred embodiment of the present invention, the amount of heavy metal cadmium monoclonal antibody added is 12-80 μg / mL.

[0018] In a preferred embodiment of the present invention, the coupling time is 30-45 minutes and the coupling temperature is 2-8°C.

[0019] In a preferred embodiment of the present invention, the sealing time is 30-45 minutes and the sealing temperature is 30-40°C.

[0020] In a preferred embodiment of the present invention, the blocking solution is bovine serum albumin.

[0021] As a preferred embodiment of the present invention, the preparation method of the binding pad coated with europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex in step (2) includes:

[0022] Europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex were diluted with 4-hydroxyethylpiperazine ethanesulfonic acid buffer, then sprayed onto a glass fiber membrane and dried to obtain a binding pad coated with europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex.

[0023] As a preferred embodiment of the present invention, the concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid buffer is 0.01-0.05 mol / L, the pH is 6-8, and it contains 5% sucrose, 1% bovine serum albumin (BSA) and 0.1% Proclin-300.

[0024] In a preferred embodiment of the present invention, the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex is diluted 12-30 times with 4-hydroxyethylpiperazine ethanesulfonic acid buffer.

[0025] In a preferred embodiment of the present invention, the amount of coating is 4-8 μL / cm.

[0026] In a preferred embodiment of the present invention, the drying temperature is 30-50°C and the drying time is 2-6 hours.

[0027] In a preferred embodiment of the present invention, step (3) involves assembling the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions, including:

[0028] The heavy metal cadmium antigen was diluted with morpholine ethanesulfonate buffer, and the goat anti-mouse secondary antibody was diluted with morpholine ethanesulfonate buffer. Then, the two solutions were drawn onto the T and C lines of a nitrocellulose membrane, respectively. After drying the drawn nitrocellulose membrane, it was pasted onto a PVC base plate. Absorbent paper was pasted onto the top of the base plate, and the sample pad and conjugate pad were pasted from bottom to top at the bottom of the base plate. The overlap between each pasted component was 1-2 mm. The membrane was then cut into 3-4 mm wide test strips using a strip cutter and placed into the test strip holder to assemble the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions.

[0029] As a preferred embodiment of the present invention, the concentration of morpholine ethanesulfonic acid buffer is 0.01-0.05 mol / L, the pH is 6.5-8.0, and it contains 5% sucrose and 0.1% Proclin-300.

[0030] In a preferred embodiment of the present invention, the concentration of the heavy metal cadmium antigen after mixing and diluting with morpholine ethanesulfonic acid buffer, i.e., the T-line coating concentration, is 0.1-0.4 mg / mL, and the concentration of the goat anti-mouse secondary antibody after mixing and diluting with morpholine ethanesulfonic acid buffer, i.e., the C-line coating concentration, is 0.01-0.10 mg / mL.

[0031] In a preferred embodiment of the present invention, the amount of film applied is 0.4-0.8 μL / cm.

[0032] This invention provides a time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions, which is obtained by the above-described preparation method.

[0033] The present invention also provides an application of the above-mentioned time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions in food.

[0034] As a preferred embodiment of the present invention, the process of detecting cadmium ions in tea using a time-resolved fluorescence immunochromatographic test strip includes the following steps:

[0035] (a) Prepare cadmium ion standard solutions of different concentrations using heavy metal dilution solution, mix and react with tris(hydroxymethyl)aminomethane hydrochloride buffer, insert a time-resolved fluorescence reader after incubation, measure the fluorescence value, and construct a competitive inhibition standard curve.

[0036] (b) Pre-treat the tea leaves and add them to a time-resolved fluorescent immunochromatographic strip for detecting heavy metal cadmium ions. Calculate the concentration of cadmium ions based on the competitive inhibition standard curve.

[0037] As a preferred embodiment of the present invention, in step (a), the method for constructing the competitive inhibition standard curve is as follows: Cadmium ion standard solutions of different concentrations are prepared using hydrochloric acid, and then mixed with a mixed buffer of tris(hydroxymethyl)aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid solution. After incubation, a time-resolved fluorescence reader is inserted to detect the fluorescence signal. The obtained T-line fluorescence value is F. T The fluorescence value of the C line is F. C , with F T / F C The values ​​were used as the ordinate to fit and construct a competition suppression standard curve.

[0038] As a preferred embodiment of the present invention, the different concentrations of the cadmium ion standard solution are 0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, and 250 ng / mL.

[0039] In a preferred embodiment of the present invention, the concentration of hydrochloric acid is 0.1-0.5 mol / L, which contains 5% sodium chloride.

[0040] In a preferred embodiment of the present invention, in the mixed buffer solution of tris(hydroxymethyl)aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid solution, the concentration of tris(hydroxymethyl)aminomethane hydrochloride buffer is 0.2-0.8 mol / L, the pH is 7.0-9.0, and it contains 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300. The concentration of ethylenediaminetetraacetic acid solution is 1-10 mmol / L, and the volume ratio of tris(hydroxymethyl)aminomethane hydrochloride buffer to ethylenediaminetetraacetic acid solution is (5-3):1.

[0041] In a preferred embodiment of the present invention, the volume ratio of the cadmium ion standard solution to the mixed buffer solution is 1:5.

[0042] In a preferred embodiment of the present invention, in step (b), 1g of dried tea powder is mixed with acid solution for digestion, the supernatant is collected by centrifugation, diluted, and used as the tea sample to be tested. This tea sample is mixed with a mixed buffer of tris(hydroxymethyl)aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid solution at a volume ratio of 1:5, and added dropwise onto a time-resolved fluorescence immunochromatographic test strip for detecting cadmium ions. After incubation, a time-resolved fluorescence reader is inserted to detect the fluorescence signal. The obtained T-line fluorescence value is F. T The fluorescence value of the C line is F. C via F T / F C The content of cadmium ions in tea was calculated using the fluorescence signal value and the competitive inhibition standard curve of step (a).

[0043] In a preferred embodiment of the present invention, the acid solution is a mixture of concentrated nitric acid and 30% hydrogen peroxide, with a volume ratio of (5-3):1.

[0044] In a preferred embodiment of the present invention, the digestion time is 0.5-10h.

[0045] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0046] 1) The time-resolved fluorescent microspheres used in this invention have the characteristics of long fluorescence lifetime and high quantum yield. After setting a certain delay time, the short-lived background fluorescence cannot be detected and only the long-lived time-resolved fluorescence can be detected, thus effectively filtering out background interference. At the same time, after the surface of the time-resolved fluorescent microspheres is modified with carboxyl groups, it can be coupled with proteins to prepare conjugates, so that it has both the optical properties of fluorescent materials and the immune properties of immune proteins.

[0047] 2) The time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions constructed in this invention has a simple preparation method. By establishing the relationship between the concentration of the analyte and the fluorescence intensity, it can detect cadmium ions in food. Moreover, the detection speed is fast and the sensitivity is high. It is suitable for on-site detection in farmland, supermarkets, homes and other situations. Specifically, it can be applied to the detection of cadmium ions in tea, which has important practical significance and application value in tea quality control. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions according to the present invention.

[0049] Figure 2 The image shows the photoluminescence excitation and emission spectra of europium time-resolved fluorescent microspheres in Example 1 of this invention.

[0050] Figure 3These are transmission electron microscopy images of europium time-resolved fluorescent microspheres before (A) and after (B) coupling with heavy metal cadmium monoclonal antibody in Example 1 of the present invention.

[0051] Figure 4 This is a zeta potential diagram of europium time-resolved fluorescent microspheres-cadmium monoclonal antibody in Example 1 of the present invention.

[0052] Figure 5 This is a graph showing the change in fluorescence intensity of the time-resolved fluorescence immunochromatographic strip for detecting heavy metal cadmium ions under ultraviolet light, as described in Example 1 of the present invention, depending on the concentration of cadmium ions.

[0053] Figure 6 This is a competitive inhibition standard curve for detecting cadmium ions in Example 1 of the present invention. Detailed Implementation

[0054] This invention provides a time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions, its preparation method, and its application.

[0055] like Figure 1 As shown, the preparation method of the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions of the present invention includes the following steps:

[0056] (1) Preparation of europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex;

[0057] (2) Preparation of a binding pad coated with europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex;

[0058] (3) The heavy metal cadmium antigen is coated on the T line and the goat anti-mouse secondary antibody is coated on the C line. After drying, it is attached together with absorbent paper, sample pad and conjugate pad to assemble the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions.

[0059] Specifically, in step (1), the preparation method of the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex includes:

[0060] Dilute 20 μL europium time-resolved fluorescent microspheres with 200 μL borate buffer and sonicate to mix. Add 10 mg / mL carbodiimide hydrochloride solution and activate the reaction. Centrifuge and discard the supernatant. Add 1 mg / mL goat anti-mouse secondary antibody to make the final concentration 8-15 μg / mL. After conjugation, add heavy metal cadmium monoclonal antibody and conjugate again. Centrifuge and discard the supernatant. Add 200 μL of 1% (w / v) BSA blocking solution to block unbound sites. After incubation and shaking, centrifuge and discard the supernatant. Dissolve in 200 μL borate buffer and store for later use.

[0061] The concentration of the borate buffer solution can be 0.01-0.05 mol / L, preferably 0.05 mol / L; the pH can be 6-9, preferably 8.

[0062] The final concentration of the carbodiimide hydrochloride solution in the reaction solution is 0.2-0.4 μg / μL, preferably 0.3 μg / μL.

[0063] The activation reaction time can be 20-45 min, preferably 30 min; the activation reaction temperature can be 30-40℃, preferably 36℃.

[0064] The centrifugation speed can be 12,000-16,000 rpm, preferably 15,000 rpm; the centrifugation time can be 30-50 min, preferably 35 min.

[0065] The preparation process of heavy metal cadmium monoclonal antibody is as follows: Cd-ITCBE-BSA is emulsified and injected into mice. After polyclonal antibody serum titer determination, cell fusion, and hybridoma cell screening, the hybridoma cells are injected into the peritoneal cavity of unimmunized mice to obtain mouse ascites. After dialysis, heavy metal cadmium monoclonal antibody is obtained. The amount of heavy metal cadmium monoclonal antibody added can be 12-80 μg / mL, preferably 12-50 μg / mL, and more preferably 12.5 μg / mL.

[0066] The coupling time can be 30-45 min, preferably 30 min; the coupling temperature can be 2-8℃, preferably 4℃.

[0067] The sealing time can be 30-45 minutes, preferably 30 minutes; the sealing temperature can be 30-40℃, preferably 36℃.

[0068] In step (2), the preparation method of the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex coating conjugate includes: diluting the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex with 4-hydroxyethylpiperazine ethanesulfonic acid buffer by a certain factor, then spraying it onto a glass fiber membrane, and drying it to obtain the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex coating conjugate conjugate.

[0069] The concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid buffer can be 0.01-0.05 mol / L, preferably 0.05 mol / L; the pH is 6-8, preferably 8; and it contains 5% sucrose, 1% BSA and 0.1% Proclin-300.

[0070] The europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex can be diluted by 12-30 times with 4-hydroxyethylpiperazine ethanesulfonic acid buffer, preferably by 15 times.

[0071] The amount of coating can be 4-8 μL / cm, preferably 8 μL / cm.

[0072] The drying temperature can be 30-50℃, preferably 45℃; the drying time can be 2-6 hours, preferably 2 hours.

[0073] In step (3), the assembly process of the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions includes: mixing and diluting the heavy metal cadmium antigen with morpholine ethanesulfonic acid buffer, mixing and diluting the goat anti-mouse secondary antibody with morpholine ethanesulfonic acid buffer, and then tracing them onto the T and C lines of the nitrocellulose membrane, respectively. After drying the tracing nitrocellulose membrane at 45°C for 48-72 hours, it is pasted onto a PVC base plate. Absorbent paper is pasted onto the upper part of the base plate, and the sample pad and conjugate pad are pasted from bottom to top at the lower part of the base plate. The overlap between each pasted component is 1-2 mm. The strip is cut into test strips 3-4 mm wide using a strip cutter and placed into the test strip holder to assemble the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions.

[0074] The concentration of the morpholine ethanesulfonic acid buffer can be 0.01-0.05 mol / L, preferably 0.05 mol / L; the pH can be 6.5-8.0, preferably 7.4, and it contains 5% sucrose and 0.1% Proclin-300.

[0075] The concentration of the heavy metal cadmium antigen after mixing and diluting with morpholine ethanesulfonic acid buffer, i.e., the T-line coating concentration, can be 0.1-0.4 mg / mL, preferably 0.2 mg / mL; the concentration of the goat anti-mouse secondary antibody after mixing and diluting with morpholine ethanesulfonic acid buffer, i.e., the C-line coating concentration, can be 0.01-0.10 mg / mL, preferably 0.03 mg / mL.

[0076] The amount of film applied can be 0.4-0.8 μL / cm, preferably 0.6 μL / cm.

[0077] The present invention also provides a time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions obtained by the above preparation method.

[0078] Finally, this invention provides an application of the above-mentioned time-resolved fluorescent immunochromatographic test strip for detecting cadmium ions in food. Using europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex as a fluorescent probe, this probe is sprayed onto a conjugate pad and assembled with a PVC base plate, sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper to obtain the time-resolved fluorescent immunochromatographic test strip for detecting cadmium ions. This allows for rapid and highly sensitive detection of cadmium ions in tea samples.

[0079] The process of detecting cadmium ions in tea using time-resolved fluorescence immunochromatographic test strips of this invention includes the following steps:

[0080] (a) Prepare cadmium ion standard solutions of different concentrations using heavy metal dilution solution, mix and react with tris(hydroxymethyl)aminomethane hydrochloride buffer, insert a time-resolved fluorescence reader after incubation, measure the fluorescence value, and construct a competitive inhibition standard curve.

[0081] (b) Pre-treat the tea leaves and add them to a time-resolved fluorescent immunochromatographic strip for detecting heavy metal cadmium ions. Calculate the concentration of cadmium ions based on the competitive inhibition standard curve.

[0082] In step (a), the method for constructing the competitive inhibition standard curve of cadmium ions is as follows: Cadmium ion standard solutions of different concentrations (0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL) are prepared by dissolving a 10 mg / mL cadmium ion standard solution in hydrochloric acid. These solutions are then mixed with a mixed buffer of tris(hydroxymethyl)aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid solution, incubated at 37°C for 5-12 min, and the fluorescence signal is detected using a time-resolved fluorescence meter. The obtained T-line fluorescence value is F. T The fluorescence value of the C line is F. C , with F T / F C The values ​​were used as the ordinate to fit and construct a competition suppression standard curve.

[0083] Specifically, the concentration of hydrochloric acid can be 0.1-0.5 mol / L, preferably 0.2 mol / L, and contains 5% sodium chloride.

[0084] In the mixed buffer solution of tris(hydroxymethyl)aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid (EDTA) solution, the concentration of tris(hydroxymethyl)aminomethane hydrochloride buffer can be 0.2-0.8 mol / L, preferably 0.4 mol / L, and the pH can be 7.0-9.0, preferably 8.0. It contains 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300. The concentration of EDTA solution can be 1-10 mmol / L, preferably 1 mmol / L. The volume ratio of tris(hydroxymethyl)aminomethane hydrochloride buffer to EDTA solution can be (5-3):1, preferably 3:1.

[0085] The volume ratio of the cadmium ion standard solution to the mixed buffer solution of tris(hydroxymethyl)aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid solution was 1:5.

[0086] In step (b), 1g of dried tea powder is mixed with acid solution for digestion, centrifuged at 2000rpm and 25℃, and the supernatant is collected. This supernatant is diluted 50-100 times with 0.2mol / L hydrochloric acid (containing 5% sodium chloride) to obtain the tea sample for testing. This tea sample is mixed with a mixed buffer of tris(hydroxymethyl)aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid solution at a volume ratio of 1:5, and incubated at 37℃ for 5-12 minutes. 120-200μL is then added to a time-resolved fluorescence immunochromatographic strip for detecting cadmium ions. The strip is inserted into a time-resolved fluorescence meter to detect the fluorescence signal. The obtained T-line fluorescence value is F. T The fluorescence value of the C line is F. C via F T / F C The content of cadmium ions in tea was calculated using the fluorescence signal value and the competitive inhibition standard curve of step (a).

[0087] Specifically, the acid solution is a mixture of concentrated nitric acid and 30% hydrogen peroxide, with a volume ratio of (5-3):1, preferably 3:1; the digestion time can be 0.5-10h, preferably 2h.

[0088] The present invention will now be described in detail with reference to specific embodiments.

[0089] Example 1:

[0090] This embodiment of the process for detecting cadmium ions in tea using a time-resolved fluorescence immunochromatographic test strip includes the following steps:

[0091] (1) Preparation of europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex:

[0092] Dilute 20 μL of time-resolved fluorescent microspheres with 200 μL of 0.05 mol / L borate buffer (pH 8.0) and sonicate to mix. Add 10 mg / mL carbodiimide hydrochloride solution to bring the final concentration to 0.3 μg / μL. Activate at 36 °C for 30 min, centrifuge at 15000 rpm for 35 min and discard the supernatant. Add 1 mg / mL goat anti-mouse secondary antibody to bring the final concentration to 10 μg / mL. After conjugation, add cadmium monoclonal antibody to bring the final concentration to 12.5 μg / mL. Conjugate at 4 °C for 30 min, centrifuge at 15000 rpm for 35 min and discard the supernatant. Add 200 μL of 1% (w / v) BSA blocking solution to block unbound sites. Incubate at 36 °C with shaking for 30 min, centrifuge at 15000 rpm for 35 min and discard the supernatant. Dissolve in 200 μL of 0.05 mol / L borate buffer (pH 8.0) and store for later use.

[0093] (2) Preparation of a binding pad coated with europium time-resolved fluorescent microspheres and heavy metal cadmium monoclonal antibody immune complex:

[0094] Europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex were diluted 15-fold with 0.05 mol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer (pH 8.0, containing 5% sucrose, 1% BSA and 0.1% Proclin-300), and then sprayed onto a glass fiber membrane at a rate of 8 μL / cm. After drying at 45 °C for 2 h, the europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex-sprayed binding pad was obtained.

[0095] (3) Assemble a time-resolved fluorescence immunochromatographic strip for detecting heavy metal cadmium ions:

[0096] The heavy metal cadmium antigen was diluted with 0.05 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.2 mg / mL. Goat anti-mouse secondary antibody was diluted with 0.05 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.03 mg / mL. The diluted antibody was then drawn onto the T and C lines of a nitrocellulose membrane at a flow rate of 0.6 μL / cm. The drawn nitrocellulose membrane was dried at 45°C for 48 hours and then adhered to a PVC substrate. Absorbent paper was attached to the top of the substrate, and sample pads and conjugate pads were attached from bottom to top to the bottom, with a 2 mm overlap between each component. The substrate was then cut into 4 mm wide strips using a strip cutter and placed into the strip holder to assemble the time-resolved fluorescence immunochromatographic test strip for detecting cadmium ions.

[0097] (4) Fitting the competitive inhibition standard curve of cadmium ions:

[0098] Different concentrations of cadmium ion standard solutions (0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, and 250 ng / mL) were prepared by dissolving a 10 mg / mL cadmium ion standard solution in 0.2 mol / L hydrochloric acid (containing 5% sodium chloride). These solutions were then mixed with a 1:5 volume ratio of a 0.4 mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 8.0, containing 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300) and a 1 mmol / L ethylenediaminetetraacetic acid (EDTA) buffer. The mixture was incubated at 37°C for 5 min, and the fluorescence signal was detected using a time-resolved fluorescence meter. The obtained T-line fluorescence value was F.T The fluorescence value of the C line is F. C , with F T / F C The values ​​were used as the ordinate to fit and construct a competition suppression standard curve.

[0099] (5) Detection of cadmium ion content in tea:

[0100] 1g of dried tea powder was digested with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 3:1) for 2 hours. The mixture was centrifuged at 2000 rpm and 25℃, and the supernatant was collected. The supernatant was diluted 50 times with 0.2 mol / L hydrochloric acid (containing 5% sodium chloride) to obtain the tea sample for testing. 120 μL of this solution was added to a time-resolved fluorescence immunochromatographic strip for detecting cadmium ions. The strip was then inserted into a time-resolved fluorescence meter to detect the fluorescence signal. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C via F T / F C The content of cadmium ions in tea was calculated by comparing the fluorescence signal value with the competitive inhibition standard curve of step (4).

[0101] from Figure 2 As can be seen, the europium time-resolved fluorescent microspheres in this embodiment have a narrow emission bandwidth, exhibiting typical emission characteristics in the 400-800 nm range. 5 D0→ 7 F J The emission band characteristics (J=0, 1, 2, 3, 4) correspond to the emission peaks of europium at 580, 592, 616, 653, and 700 nm, respectively. Because... 5 D0→ 7 F0 and 5 D0→ 7 F3 is forbidden by the selective rules of forced dipole transitions, thus weaker emission peaks can be observed at the two transition locations (580 nm and 653 nm, respectively). There is also a weaker emission peak at 592 nm, which corresponds to... 5 D0→ 7 The F1 magnetic dipole transition typically plays a dominant role when europium occupies the inversion center site. The maximum emission peak of the europium time-resolved fluorescent microspheres is located at 616 nm, corresponding to... 5 D0→ 7 The electric dipole transition of F2, also known as the ultrasensitive transition, is the reason why Eu-TRFM emits red fluorescence under the corresponding excitation light.

[0102] from Figure 3 As can be seen, the europium time-resolved fluorescent microspheres in this embodiment have different morphologies before and after coupling with the heavy metal cadmium monoclonal antibody, and the surface becomes irregular after coupling.

[0103] from Figure 4As can be seen, in the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immunocomplex of this embodiment, the zeta potential of the conjugated complex changes significantly compared with that of the unconjugated heavy metal cadmium monoclonal antibody. This is because the europium time-resolved fluorescent microspheres are negatively charged after carboxyl modification. Under the mediation of carbodiimide hydrochloride, the amino-containing antibody and the carboxyl-containing microspheres are conjugated to form amide bonds, resulting in a change in their surface charge. This indicates that the presence of heavy metal cadmium monoclonal antibody affects the charge distribution on the surface of europium time-resolved fluorescent microspheres, thus proving the successful preparation of the europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immunocomplex.

[0104] from Figure 5 It can be seen that as the cadmium ion concentration increased from 0 ng / mL to 250 ng / mL, the fluorescence intensity of the time-resolved fluorescent immunochromatographic strip for detecting cadmium ions under ultraviolet light also changed. Visual observation showed that the C-line intensity gradually increased, while the T-line intensity gradually decreased, indicating that the presence of cadmium ions resulted in less europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex being captured by the T-line. Figure 6 It can be seen that the linear range of the constructed test strip is 2.52-56.34 ng / mL, and the limit of detection is 1.02 ng / mL, indicating that the method has high sensitivity.

[0105] Example 2:

[0106] This embodiment of the process for detecting cadmium ions in tea using a time-resolved fluorescence immunochromatographic test strip includes the following steps:

[0107] (1) Preparation of europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex:

[0108] Dilute 20 μL of time-resolved fluorescent microspheres with 200 μL of 0.04 mol / L borate buffer (pH 7.4), sonicate to mix, add 10 mg / mL carbodiimide hydrochloride solution to bring the final concentration to 0.4 μg / μL, react at 30 °C for 45 min, centrifuge at 12000 rpm for 50 min and discard the supernatant, add 1 mg / mL goat anti-mouse secondary antibody to bring the final concentration to 8 μg / mL, conjugate with heavy metal cadmium monoclonal antibody to bring the final concentration to 15 μg / mL, conjugate at 8 °C for 35 min, centrifuge at 12000 rpm for 50 min and discard the supernatant, add 200 μL of 1% (w / v) BSA blocking solution to block unbound sites, incubate at 30 °C with shaking for 45 min, centrifuge at 12000 rpm for 50 min and discard the supernatant, dissolve in 200 μL of 0.04 mol / L borate buffer (pH 7.4) and refrigerate for later use.

[0109] (2) Preparation of a binding pad coated with europium time-resolved fluorescent microspheres and heavy metal cadmium monoclonal antibody immune complex:

[0110] Europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex were diluted 15-fold with 0.04 mol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose, 1% BSA and 0.1% Proclin-300), and then sprayed onto a glass fiber membrane at a rate of 7 μL / cm. After drying at 30 °C for 6 h, the europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex-sprayed binding pad was obtained.

[0111] (3) Assemble a time-resolved fluorescence immunochromatographic strip for detecting heavy metal cadmium ions:

[0112] The cadmium heavy metal antigen was diluted with 0.04 mol / L morpholine ethanesulfonic acid buffer (pH 7.0, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.1 mg / mL. Goat anti-mouse secondary antibody was diluted with 0.04 mol / L morpholine ethanesulfonic acid buffer (pH 7.0, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.02 mg / mL. The diluted antibody was then drawn onto the T and C lines of a nitrocellulose membrane at a rate of 0.5 μL / cm. The drawn nitrocellulose membrane was dried at 45°C for 60 h and then adhered to a PVC substrate. Absorbent paper was attached to the top of the substrate, and sample pads and conjugate pads were attached from bottom to top to the bottom, with a 1.5 mm overlap between each component. The substrate was then cut into 3 mm wide strips using a strip cutter and placed into the strip holder to assemble the time-resolved fluorescence immunochromatographic test strip for detecting cadmium heavy metal ions.

[0113] (4) Fitting the competitive inhibition standard curve of cadmium ions:

[0114] Different concentrations of cadmium ion standard solutions (0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, and 250 ng / mL) were prepared by dissolving a 10 mg / mL cadmium ion standard solution in 0.1 mol / L hydrochloric acid (containing 5% sodium chloride). These solutions were then mixed with a 1:5 volume ratio of a 0.4 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 8.0, containing 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300) and a 1 mmol / L ethylenediaminetetraacetic acid solution. The mixture was incubated at 37°C for 8 min, and the fluorescence signal was detected using a time-resolved fluorescence meter. The obtained T-line fluorescence value was F.T The fluorescence value of the C line is F. C , with F T / F C The values ​​were used as the ordinate to fit and construct a competition suppression standard curve.

[0115] (5) Detection of cadmium ion content in tea:

[0116] 1g of dried tea powder was digested with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 5:1) for 8 hours. The mixture was centrifuged at 2000 rpm and 25℃, and the supernatant was collected. This supernatant was diluted 60 times with 0.1mol / L hydrochloric acid (containing 5% sodium chloride) and used as the tea sample for testing. 150μL of this solution was added to a time-resolved fluorescence immunochromatographic strip for detecting cadmium ions. The strip was then inserted into a time-resolved fluorescence meter to detect the fluorescence signal. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C via F T / F C The content of cadmium ions in tea was calculated by comparing the fluorescence signal value with the competitive inhibition standard curve of step (4).

[0117] Example 3:

[0118] This embodiment of the process for detecting cadmium ions in tea using a time-resolved fluorescence immunochromatographic test strip includes the following steps:

[0119] (1) Preparation of europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex:

[0120] Dilute 20 μL of time-resolved fluorescent microspheres with 200 μL of 0.03 mol / L borate buffer (pH 8.0) and sonicate to mix. Add 10 mg / mL carbodiimide hydrochloride solution to bring the final concentration to 0.2 μg / μL. React at 40 °C for 20 min, centrifuge at 14000 rpm for 40 min and discard the supernatant. Add 1 mg / mL goat anti-mouse secondary antibody to bring the final concentration to 12 μg / mL. Couple with heavy metal cadmium monoclonal antibody to bring the final concentration to 25 μg / mL. Couple at 2 °C for 40 min, centrifuge at 14000 rpm for 40 min and discard the supernatant. Add 200 μL of 1% (w / v) BSA blocking solution to block unbound sites. React at 40 °C with shaking for 30 min, centrifuge at 14000 rpm for 40 min and discard the supernatant. Dissolve in 200 μL of 0.03 mol / L borate buffer (pH 8.0) and refrigerate for later use.

[0121] (2) Preparation of a binding pad coated with europium time-resolved fluorescent microspheres and heavy metal cadmium monoclonal antibody immune complex:

[0122] Europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex were diluted 20-fold with 0.03 mol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer (pH 6.5, containing 5% sucrose, 1% BSA and 0.1% Proclin-300), and then sprayed onto a glass fiber membrane at a rate of 8 μL / cm. After drying at 45 °C for 4 h, the europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex-sprayed binding pad was obtained.

[0123] (3) Assemble a time-resolved fluorescence immunochromatographic strip for detecting heavy metal cadmium ions:

[0124] The heavy metal cadmium antigen was diluted with 0.05 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.3 mg / mL. Goat anti-mouse secondary antibody was also diluted with 0.05 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.05 mg / mL. The diluted antibody was then drawn onto the T and C lines of a nitrocellulose membrane at a rate of 0.5 μL / cm. The drawn nitrocellulose membrane was dried at 45°C for 48 hours and then adhered to a PVC substrate. Absorbent paper was attached to the top of the substrate, and sample pads and conjugate pads were attached from bottom to top to the bottom, with a 1.5 mm overlap between each component. The substrate was then cut into 4 mm wide strips using a strip cutter and placed into the strip holder to assemble the time-resolved fluorescence immunochromatographic test strip for detecting cadmium ions.

[0125] (4) Fitting the competitive inhibition standard curve of cadmium ions:

[0126] Different concentrations of cadmium ion standard solutions (0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, and 250 ng / mL) were prepared by dissolving a 10 mg / mL cadmium ion standard solution in 0.2 mol / L hydrochloric acid (containing 5% sodium chloride). These solutions were then mixed with a 1:5 volume ratio of a 0.4 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 8.0, containing 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300) and a 5 mmol / L ethylenediaminetetraacetic acid solution. The mixture was incubated at 37°C for 10 min, and the fluorescence signal was detected using a time-resolved fluorescence meter. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C , with F T / F CThe values ​​were used as the ordinate to fit and construct a competition suppression standard curve.

[0127] (5) Detection of cadmium ion content in tea:

[0128] 1g of dried tea powder was digested with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 4:1) for 8 hours. The mixture was centrifuged at 2000 rpm and 25℃, and the supernatant was collected. This supernatant was diluted 80 times with 0.2mol / L hydrochloric acid (containing 5% sodium chloride) and used as the tea sample for testing. 120μL of this solution was added to a time-resolved fluorescence immunochromatographic strip for detecting cadmium ions. The strip was then inserted into a time-resolved fluorescence meter to detect the fluorescence signal. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C via F T / F C The content of cadmium ions in tea was calculated by comparing the fluorescence signal value with the competitive inhibition standard curve of step (4).

[0129] Example 4:

[0130] This embodiment of the process for detecting cadmium ions in tea using a time-resolved fluorescence immunochromatographic test strip includes the following steps:

[0131] (1) Preparation of europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex:

[0132] Dilute 20 μL of time-resolved fluorescent microspheres with 200 μL of 0.05 mol / L borate buffer (pH 8.5), sonicate to mix, add 10 mg / mL carbodiimide hydrochloride solution to bring the final concentration to 0.4 μg / μL, react at 40 °C for 25 min, centrifuge at 16000 rpm for 30 min and discard the supernatant, add 1 mg / mL goat anti-mouse secondary antibody to bring the final concentration to 15 μg / mL, conjugate with heavy metal cadmium monoclonal antibody to bring the final concentration to 50 μg / mL, conjugate at 4 °C for 40 min, centrifuge at 16000 rpm for 30 min and discard the supernatant, add 200 μL of 1% (w / v) BSA blocking solution to block unbound sites, incubate at 36 °C with shaking for 40 min, centrifuge at 16000 rpm for 30 min and discard the supernatant, dissolve in 200 μL of 0.05 mol / L borate buffer (pH 8.5) and refrigerate for later use.

[0133] (2) Preparation of a binding pad coated with europium time-resolved fluorescent microspheres and heavy metal cadmium monoclonal antibody immune complex:

[0134] Europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex were diluted 25-fold with 0.05 mol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer (pH 8.0, containing 5% sucrose, 1% BSA and 0.1% Proclin-300), and then sprayed onto a glass fiber membrane at a rate of 5 μL / cm. After drying at 45 °C for 6 h, the europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex-sprayed binding pad was obtained.

[0135] (3) Assemble a time-resolved fluorescence immunochromatographic strip for detecting heavy metal cadmium ions:

[0136] The heavy metal cadmium antigen was diluted with 0.05 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.4 mg / mL. Goat anti-mouse secondary antibody was diluted with 0.05 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.08 mg / mL. The antigen was then drawn onto the T and C lines of a nitrocellulose membrane at a flow rate of 0.4 μL / cm. The drawn nitrocellulose membrane was dried at 45℃ for 66 h and then adhered to a PVC substrate. Absorbent paper was attached to the top of the substrate, and sample pads and conjugate pads were attached from bottom to top to the bottom, with a 2 mm overlap between each component. The substrate was then cut into 3 mm wide strips using a strip cutter and placed into the strip holder to assemble the time-resolved fluorescence immunochromatographic test strip for detecting cadmium ions.

[0137] (4) Fitting the competitive inhibition standard curve of cadmium ions:

[0138] Different concentrations of cadmium ion standard solutions (0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, and 250 ng / mL) were prepared by dissolving a 10 mg / mL cadmium ion standard solution in 0.1 mol / L hydrochloric acid (containing 5% sodium chloride). These solutions were then mixed with a 1:5 volume ratio of a 0.3 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.4, containing 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300) and an 8 mmol / L ethylenediaminetetraacetic acid solution. The mixture was incubated at 37°C for 12 min, and the fluorescence signal was detected using a time-resolved fluorescence meter. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C , with F T / F CThe values ​​were used as the ordinate to fit and construct a competition suppression standard curve.

[0139] (5) Detection of cadmium ion content in tea:

[0140] 1g of dried tea powder was digested with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 3:1) for 10h. The supernatant was collected by centrifugation at 2000rpm and 25℃ and diluted 70 times with 0.2mol / L hydrochloric acid (containing 5% sodium chloride) as the tea sample to be tested. 120μL of this solution was added to a time-resolved fluorescence immunochromatographic strip for detecting cadmium ions. The strip was then inserted into a time-resolved fluorescence meter to detect the fluorescence signal. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C via F T / F C The content of cadmium ions in tea was calculated by comparing the fluorescence signal value with the competitive inhibition standard curve of step (4).

[0141] Example 5:

[0142] This embodiment of the process for detecting cadmium ions in tea using a time-resolved fluorescence immunochromatographic test strip includes the following steps:

[0143] (1) Preparation of europium time-resolved fluorescent microsphere-cadmium monoclonal antibody immune complex:

[0144] Dilute 20 μL of time-resolved fluorescent microspheres with 200 μL of 0.02 mol / L borate buffer (pH 8.0), sonicate to mix, add 10 mg / mL carbodiimide hydrochloride solution to bring the final concentration to 0.3 μg / μL, react at 45 °C for 20 min, centrifuge at 16000 rpm for 30 min and discard the supernatant, add 1 mg / mL goat anti-mouse secondary antibody to bring the final concentration to 14 μg / mL, conjugate with heavy metal cadmium monoclonal antibody to bring the final concentration to 50 μg / mL, conjugate at 4 °C for 40 min, centrifuge at 16000 rpm for 30 min and discard the supernatant, add 200 μL of 1% (w / v) BSA blocking solution to block unbound sites, incubate at 33 °C with shaking for 40 min, centrifuge at 16000 rpm for 30 min and discard the supernatant, dissolve in 200 μL of 0.02 mol / L borate buffer (pH 8.0) and refrigerate for later use.

[0145] (2) Preparation of a binding pad coated with europium time-resolved fluorescent microspheres and heavy metal cadmium monoclonal antibody immune complex:

[0146] Europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex were diluted 20-fold with 0.02 mol / L 4-hydroxyethylpiperazine ethanesulfonic acid buffer (pH 6.0, containing 5% sucrose, 1% BSA and 0.1% Proclin-300), and then sprayed onto a glass fiber membrane at a rate of 8 μL / cm. After drying at 45 °C for 2 h, the europium time-resolved fluorescent microspheres-cadmium monoclonal antibody immune complex-sprayed binding pad was obtained.

[0147] (3) Assemble a time-resolved fluorescence immunochromatographic strip for detecting heavy metal cadmium ions:

[0148] The heavy metal cadmium antigen was diluted with 0.03 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.4 mg / mL. Goat anti-mouse secondary antibody was diluted with 0.03 mol / L morpholine ethanesulfonic acid buffer (pH 7.4, containing 5% sucrose and 0.1% Proclin-300) to a final concentration of 0.08 mg / mL. The antigen was then drawn onto the T and C lines of a nitrocellulose membrane at a flow rate of 0.4 μL / cm. The drawn nitrocellulose membrane was dried at 45℃ for 72 h and then adhered to a PVC substrate. Absorbent paper was attached to the top of the substrate, and sample pads and conjugate pads were attached from bottom to top to the bottom, with each component overlapping by 1 mm. The substrate was then cut into 4 mm wide strips using a strip cutter and placed into the strip holder to assemble the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions.

[0149] (4) Fitting the competitive inhibition standard curve of cadmium ions:

[0150] Different concentrations of cadmium ion standard solutions (0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, and 250 ng / mL) were prepared by dissolving a 10 mg / mL cadmium ion standard solution in 0.2 mol / L hydrochloric acid (containing 5% sodium chloride). These solutions were then mixed with a 1:5 volume ratio of a 0.8 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 8.0, containing 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300) and a 10 mmol / L ethylenediaminetetraacetic acid solution. The mixture was incubated at 37°C for 10 min, and the fluorescence signal was detected using a time-resolved fluorescence meter. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C , with F T / F CThe values ​​were used as the ordinate to fit and construct a competition suppression standard curve.

[0151] (5) Detection of cadmium ion content in tea:

[0152] 1g of dried tea powder was digested with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 3:1) for 4 hours. The mixture was centrifuged at 2000 rpm and 25℃, and the supernatant was collected. This supernatant was diluted 100 times with 0.2 mol / L hydrochloric acid (containing 5% sodium chloride) and used as the tea sample for testing. 200 μL of this solution was added to a time-resolved fluorescence immunochromatographic strip for detecting cadmium ions. The strip was then inserted into a time-resolved fluorescence meter to detect the fluorescence signal. The obtained T-line fluorescence value was F. T The fluorescence value of the C line is F. C via F T / F C The content of cadmium ions in tea was calculated by comparing the fluorescence signal value with the competitive inhibition standard curve of step (4).

[0153] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A preparation method of a time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions, characterized in that, It comprises the following steps: (1) Preparation of europium time-resolved fluorescent microspheres-heavy metal cadmium monoclonal antibody immune complex, the preparation method comprises: The europium time-resolved fluorescent microspheres are diluted with borate buffer solution, then ultrasonic mixed, carbonic acid imide hydrochloride solution is added, and activated reaction is carried out, then centrifuged and discarded supernatant, goat anti-mouse secondary antibody is added, coupled, then heavy metal cadmium monoclonal antibody is added, coupled, centrifuged and discarded supernatant, blocking solution is added to block unbound sites, constant temperature shaking reaction is carried out, then centrifuged and discarded supernatant, dissolved in borate buffer solution, and refrigerated for standby; the heavy metal cadmium monoclonal antibody is Cd-ITCBE-BSA antibody; (2) Preparation of europium time-resolved fluorescent microspheres-heavy metal cadmium monoclonal antibody immune complex spray coated binding pad, the preparation method comprises: The europium time-resolved fluorescent microspheres-heavy metal cadmium monoclonal antibody immune complex is diluted with 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, then sprayed on glass fiber membrane, dried to obtain europium time-resolved fluorescent microspheres-heavy metal cadmium monoclonal antibody immune complex spray coated binding pad; the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution is 0.01-0.05mol / L, and pH is 6-8, wherein 5% sucrose, 1% bovine serum albumin and 0.1% Proclin-300 are contained; (3) Heavy metal cadmium antigen is coated on T line, goat anti-mouse secondary antibody is coated on C line, dried, and then pasted with water absorption paper, sample pad and the binding pad to assemble to obtain a time-resolved fluorescent immunochromatography test strip for detecting heavy metal cadmium ions.

2. The preparation method of the time-resolved fluorescent immunochromatographic test strip for detecting heavy metal cadmium ions according to claim 1, characterized in that, In step (1), the concentration of the borate buffer solution is 0.01-0.05mol / L, and pH is 6-9; and / or, The final concentration of the carbonic acid imide hydrochloride solution in the reaction solution is 0.2-0.4μg / μL; and / or, The activation reaction time is 20-45min, and the activation reaction temperature is 30-40℃; and / or, The centrifugal speed is 12000-16000rpm, and the centrifugal time is 30-50min; and / or, The preparation process of the heavy metal cadmium monoclonal antibody is as follows: Cd-ITCBE-BSA is emulsified and injected into mice, then the antiserum titer is determined, cell fusion is carried out, hybridoma cells are screened, the hybridoma cells are injected into the abdominal cavity of non-immunized mice to obtain mouse ascites, and then the heavy metal cadmium monoclonal antibody is obtained after dialysis; and / or, The adding amount of the heavy metal cadmium monoclonal antibody is 12-80μg / mL; and / or, The coupling time is 30-45min, and the coupling temperature is 2-8℃; and / or, The blocking time is 30-45min, and the blocking temperature is 30-40℃; and / or, The blocking solution is bovine serum albumin.

3. The method for preparing a time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions according to claim 1, characterized in that, In step (2), the dilution multiple of the europium time-resolved fluorescent microspheres-heavy metal cadmium monoclonal antibody immune complex diluted with 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution is 12-30 times; and / or, The spraying amount is 4-8μL / cm; and / or, The drying temperature is 30-50℃, and the drying time is 2-6h.

4. The preparation method of the time-resolved fluorescent immunochromatographic test strip for detecting heavy metal cadmium ions according to claim 1, characterized in that, In step (3), the assembling process of the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions comprises: The heavy metal cadmium antigen is mixed and diluted with morpholine ethanesulfonic acid buffer solution, the goat anti-mouse secondary antibody is mixed and diluted with morpholine ethanesulfonic acid buffer solution, and then the two are respectively drawn on the T line and the C line of the nitrocellulose membrane. After the membrane is dried, it is pasted on a polyvinyl chloride base plate. The upper end of the base plate is pasted with a water absorption paper, and the lower end of the base plate is pasted with a sample pad and a binding pad from bottom to top. The overlapping distance between the pasted components is 1-2 mm. The components are cut into 3-4 mm wide test strips by using a cutting machine, and then the test strips are placed in a test strip card shell to obtain the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions; and / or The concentration of the morpholine ethanesulfonic acid buffer solution is 0.01-0.05 mol / L, and the pH is 6.5-8.0, and the buffer solution contains 5% sucrose and 0.1% Proclin-300; and / or The concentration of the heavy metal cadmium antigen mixed and diluted with the morpholine ethanesulfonic acid buffer solution is 0.1-0.4 mg / mL, and the concentration of the goat anti-mouse secondary antibody mixed and diluted with the morpholine ethanesulfonic acid buffer solution is 0.01-0.10 mg / mL; and / or The amount of the drawn membrane is 0.4-0.8 μL / cm.

5. A time-resolved fluorescent immunochromatographic test strip for detecting heavy metal cadmium ions, characterized in that, The time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions is obtained by the preparation method of any one of claims 1-4.

6. The time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions of claim 5 is used for detecting cadmium ions in food.

7. Use according to claim 6, characterized in that, The process of detecting cadmium ions in tea by using the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions comprises the following steps: (a) A heavy metal diluent is used to prepare cadmium ion standard solution with different concentrations, and the cadmium ion standard solution is mixed and reacted with a tris-hydroxymethyl aminomethane hydrochloride buffer solution. After incubation, the solution is inserted into a time-resolved fluorescence reader to measure the fluorescence value, and a competitive inhibition standard curve is constructed; (b) Tea is pretreated and added dropwise to the time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions, and the concentration of cadmium ions is calculated according to the competitive inhibition standard curve.

8. Use according to claim 7, characterized in that, In step (a), the method for constructing the competition inhibition standard curve is as follows: the cadmium ion standard solution is prepared into different concentrations of cadmium ion standard solution with hydrochloric acid, mixed with a mixed buffer of tris-hydroxymethyl aminomethane hydrochloride buffer and ethylenediaminetetraacetic acid solution, incubated, inserted into a time-resolved fluorescence reader, and the fluorescence signal is detected. The obtained T-line fluorescence value is F T , the C-line fluorescence value is F C , and the F T / F C value is taken as the ordinate to fit and construct the competition inhibition standard curve; and / or, The different concentrations of the cadmium ion standard solution are 0 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 62.5 ng / mL, 125 ng / mL, and 250 ng / mL; and / or The concentration of the hydrochloric acid is 0.1-0.5 mol / L, and the hydrochloric acid contains 5% sodium chloride; and / or In the mixed buffer solution of the tris-hydroxymethyl aminomethane hydrochloride buffer solution and the ethylenediaminetetraacetic acid solution, the concentration of the tris-hydroxymethyl aminomethane hydrochloride buffer solution is 0.2-0.8 mol / L, and the pH is 7.0-9.

0. The tris-hydroxymethyl aminomethane hydrochloride buffer solution contains 1% sucrose, 1% sodium chloride, 1% Tween-80, and 0.1% Proclin-300. The concentration of the ethylenediaminetetraacetic acid solution is 1-10 mmol / L. The volume ratio of the tris-hydroxymethyl aminomethane hydrochloride buffer solution to the ethylenediaminetetraacetic acid solution is (5-3):1; and / or The time-resolved fluorescence immunochromatographic test strip for detecting heavy metal cadmium ions is obtained by the preparation method of any one of claims 1-4. The volume ratio of the mixed buffer and the cadmium ion standard solution is 1:

5.

9. The use according to claim 7, characterized in that, In step (b), the dried tea leaf powder is mixed with acid solution for digestion, centrifuged to obtain supernatant, diluted to obtain a tea sample to be tested, mixed with a mixed buffer of tris-hydroxymethyl aminomethane hydrochloride buffer and ethylenediamine tetraacetic acid solution, incubated, inserted into a time-resolved fluorescence reader, and detected fluorescence signal, wherein the obtained T-line fluorescence value is F T , the C-line fluorescence value is F C , and the content of cadmium ion in the tea leaf is calculated by F T / F C value fluorescence signal value and the competitive inhibition standard curve of step (a); and / or, The acid solution is a mixture of concentrated nitric acid and 30% hydrogen peroxide, with a volume ratio of (5-3):

1.

10. The use according to claim 9, characterized in that, The digestion time is 0.5-10h.

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