Method for rapidly and quantitatively detecting cadmium element in cereals

Abstract

The invention discloses a method for rapidly and quantitatively detecting cadmium element in grain, which comprises the following specific steps: step 1, pretreating a grain sample to obtain supernate; and 2, dropwise adding the supernatant treated in the step 1 on a cadmium time-resolved fluorescence immunochromatography detection reagent card for detection, detecting a fluorescence signal by using a fluorescence analyzer, and calculating by comparing with a standard curve to obtain the cadmium concentration in the sample, wherein a sample pad is arranged at one end of the cadmium time-resolved fluorescence immunochromatography detection reagent card, and a marker pad, a chromatography pad and a water absorption pad are sequentially arranged on one side of the sample pad along the length direction of the chromatography detection reagent card; the marker pad is coated with cadmium antibody-fluorescent microspheres, the chromatography pad is provided with a line T and a line C, the line T is coated with cadmium antigen, and the line C is coated with secondary antibody-fluorescent microspheres; the cadmium antibody is a mouse monoclonal cadmium antibody, and the cadmium antigen is Cd < 2 + >-EDTA-BSA.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and in particular to a method for rapidly and quantitatively detecting cadmium elements in grains. BACKGROUND

[0002] Heavy metal cadmium (Cd) as a kind of environmental pollutants with special biological accumulation characteristics, in recent years, the pollution problem in grains is increasingly prominent. Cadmium is rapidly absorbed into the blood circulation after being absorbed through the digestive tract, and mainly accumulates in the kidney tissue. Because of its long biological half-life of 10-30 years, it can cause persistent damage to the human body. In addition to the kidney, cadmium also deposits in the lungs, bones, liver and other organs. Chronic cadmium exposure is closely related to many diseases, including kidney dysfunction, vitamin D metabolism abnormalities, osteoporosis, and may even increase the risk of lung cancer, and severe cases can cause typical "painful disease" symptoms.

[0003] The harm of heavy metal pollution to food crops has become a major global food safety problem. The use of cadmium-containing fertilizers and pesticides in agricultural activities, as well as the discharge of urban sewage, makes cadmium continue to enter the soil and groundwater system through osmosis. As a large agricultural country, China's heavy metal pollution situation is particularly severe, among which the problem of heavy metal pollution of rice is particularly prominent.

[0004] In the prior art, in order to accurately and efficiently detect heavy metal cadmium, traditional detection methods mainly include inductively coupled plasma mass spectrometry (ICP-MS), atomic fluorescence spectrometry (AFS), atomic absorption spectrometry (AAS), etc. However, these methods have various problems in actual use: on the one hand, in order to protect the precision instruments and exclude matrix interference, complex and time-consuming sample pretreatment processes (such as microwave digestion, concentrated acid cooking, etc.) are required; on the other hand, the high equipment purchase and maintenance costs require professional technical personnel to operate, which makes the traditional detection methods in the prior art more suitable for precise detection in laboratory environment, and it is difficult to meet the on-site rapid detection needs in the fields of food safety supervision and environmental monitoring. SUMMARY

[0005] In view of the above problems in the prior art, the purpose of the present application is to provide a method for rapidly and quantitatively detecting cadmium elements in grains, so as to solve the problems in the prior art that the detection method of cadmium in grains is expensive, has high maintenance cost, requires professional technical personnel to operate, and is difficult to meet the on-site rapid detection needs in the fields of food safety supervision and environmental monitoring.

[0006] In order to solve the above technical problems, the present application adopts the following technical solutions:

[0007] A method for rapidly and quantitatively detecting cadmium elements in grains, the specific steps are as follows:

[0008] Step 1: after the grain sample is crushed, the acid solution is added, vortexed and centrifuged, the supernatant is taken and the chelating agent solution and neutralizing agent are added to the supernatant, and the supernatant after chelation is collected; wherein the acid solution is a hydrochloric acid or nitric acid solution, the concentration of the acid solution is 0.19-3 mol / L; the chelating agent is EDTA-2Na, the concentration of EDTA-2Na in the chelating agent solution is 0.00001%-1%; the neutralizing agent is a Tris solution, the concentration is 0.125 mol / L-2 mol / L; 4 mL of acid solution is added per 1 g of crushed grain sample; the volume ratio of the supernatant, the chelating agent solution and the neutralizing agent is 5:1:5;

[0009] Step 2: the supernatant after step 1 is added dropwise to the cadmium time-resolved fluorescence immunochromatographic detection reagent card for detection, the fluorescence signal is detected by a fluorescence analyzer, and the cadmium concentration in the sample is calculated by comparing with the standard curve;

[0010] Wherein, a sample pad is arranged at one end of the cadmium time-resolved fluorescence immunochromatographic detection reagent card, a marker pad, a chromatographic pad and a water absorption pad are arranged on one side of the sample pad and along the length direction of the chromatographic detection reagent card; a cadmium antibody-fluorescent microsphere is coated on the marker pad, a T line and a C line are arranged on the chromatographic pad, the T line is coated with a cadmium antigen, and the C line is coated with a secondary antibody-fluorescent microsphere; the cadmium antibody is a mouse monoclonal cadmium antibody, the cadmium antigen is Cd 2+ -EDTA-BSA.

[0011] Preferably, in step 2, the cadmium antibody or the secondary antibody is connected to the fluorescent microsphere through a coupling reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-carboxyl thio succinimide.

[0012] Preferably, the coupling reaction is as follows:

[0013] S1: mix the fluorescent microsphere solution and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in water, then add N-carboxyl thio succinimide, react at 30-37°C for 10-15 min, and collect the solid product by centrifugation; in the reaction system, the addition amount of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 0.1-2 mg / mL, the addition amount of N-carboxyl thio succinimide is 0.1-2 mg / mL, the solid content in the fluorescent microsphere solution is 1-3 wt%, and 1 mL of water is mixed with 200 μL of the fluorescent microsphere solution;

[0014] S2: adding a phosphate buffer to the solid product to re-disperse and add one of the cadmium antibody or the secondary antibody, reacting at 30-37 DEG C for 1-1.5 hours, adding BSA to continue reacting for 30-40 minutes, collecting the solid substance after centrifugation, and adding a label storage solution to obtain a storage solution containing cadmium antibody fluorescent microspheres or secondary antibody fluorescent microspheres, respectively; wherein 4 mg of the solid product is added with 500 μL of the phosphate buffer, and the mass ratio of the solid product, one of the cadmium antibody or the secondary antibody, and BSA is 4:0.1:5, and 4 g of the solid substance is added with 500 μL of the label storage solution.

[0015] Preferably, the label storage solution is: 0.5 g of BSA, 0.2 g of Pc300, and 0.5 mL of Tween-20 are added into 100 mL of the phosphate buffer.

[0016] Preferably, the phosphate buffer has a pH of 7.8, a phosphate ion concentration of 50 mmol / L, a mass concentration of BSA of 0.2wt%-1.6wt%, and a mass concentration of Tween-20 of 0.2wt%-1.6wt%.

[0017] Preferably, the fluorescent microspheres have an emission light of 615 nm and an excitation spectrum of 360-410 nm, and the carboxyl content on the surface of the microspheres is 205-350 μmol / g.

[0018] Preferably, the secondary antibody is a goat anti-mouse or a rabbit anti-mouse.

[0019] Preferably, in step 2, the cadmium antigen-fluorescent microsphere coating concentration on the T line is 0.2-1.6 mg / mL.

[0020] Preferably, in step 2, the chromatography temperature is 20-25 DEG C, the chromatography time is 10-20 minutes, and the liquid volume added on the reagent card is 60-120 μL.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] 1.The present application improves the detection performance from the mechanism by combining the labeling system with specific immune response. On the one hand, europium time-resolved fluorescent microspheres are preferred as labels, which have a unique long fluorescence lifetime (millisecond level) that can eliminate background fluorescence interference by delayed detection, further reduce the influence of scattered light, and significantly improve the signal-to-noise ratio; on the other hand, the present application finds that different antigen-antibody combinations have a significant impact on the detection results. Therefore, the present application uses the principle of competitive immunochromatography to allow the specific binding of cadmium ions in the sample to fluorescently labeled antibodies, and then competes with the cadmium antigen on the test line (T line) for the binding site, and the two-antibody quality control mechanism of the quality control line (C line), which not only ensures the detection specificity, but also realizes accurate quantification through the fluorescence intensity ratio of T / C line, solving the problems of low sensitivity and easy interference of traditional colloidal gold method.

[0023] 2.The present application also studies the pretreatment method of grain samples to ensure the detection efficiency and accuracy from the mechanism. Dilute nitric acid is used as the extractant, and the acid environment is used to destroy the combined state structure of cadmium in the grain matrix, so that the cadmium ions are efficiently dissociated and free in the liquid phase. Compared with the traditional microwave digestion method, high temperature and high pressure are not required, and the change in the form of cadmium ions is avoided; EDTA-2Na chelating agent is added to maintain the recognizable form of cadmium ions by forming a stable five-membered ring complex with cadmium ions, providing a suitable substrate for subsequent immune response; and the pH value of the extract is adjusted to avoid the inhibition of antigen-antibody binding in acidic environment and prevent the precipitation of cadmium ions in strong alkaline conditions, ensuring that the extract is directly suitable for immunochromatographic detection. The entire pretreatment is completed within 20 minutes, meeting the demand for high efficiency of rapid detection.

[0024] 3.The present application further improves and optimizes the designed detection method to achieve a balance between detection stability and practicality. In the reagent preparation process, the optimal ratio of EDC / Sulfo-NHS activation system and the amount of antibody labeling are determined to ensure the stable coupling of fluorescent microspheres and antibodies and reduce non-specific binding; the sample pad is treated with BSA and Tween-20, BSA can block the non-specific adsorption sites on the surface of the sample pad, and Tween-20 can reduce the surface tension of the liquid and promote the uniform chromatography of the sample, thereby reducing matrix interference; the combination of room temperature chromatography and sample size ensures sufficient reaction of antigen-antibody and avoids signal drift caused by long chromatography time, and the effective period of 12 months at 4-8℃ is combined, so that the method described in the present application has long-term practical value in the field of on-site detection at the grassroots level. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The results of the different EDC / Sulfo-NHS usage experiments in Example 1 are shown in the figure.

[0026] Figure 2 The results of the different labeled monoclonal antibody usage experiments in Example 1 are shown in the figure.

[0027] Figure 3 Graph of results for different amounts of cadmium antibody labeling in Example 3.

[0028] Figure 4 Graph of results for different C-line raw material screening experiments in Example 4.

[0029] Figure 5 Graph of results for different coating and labeling amounts used in Example 5.

[0030] Figure 6 Graph of S / N results for different blocking agents and surfactant concentrations in Example 6.

[0031] Figure 7 Graph of fluorescence signal results for different blocking agents and surfactant concentrations in Example 6.

[0032] Figure 8 Graph of results for extractant optimization experiments for cadmium in grains in Example 9 and Example 10.

[0033] Figure 9 Graph of results for different cadmium extraction time experiments in grains in Example 11.

[0034] Figure 10 Graph of results for different concentrations of chelating agents DTPA and EDTA-2Na experiments in Example 12.

[0035] Figure 11 Graph of results for different concentrations of chelating agent EDTA-2Na experiments in Example 12.

[0036] Figure 12 Graph of results for different concentrations of neutralizing agents experiments in Example 13.

[0037] Figure 13 Graph of results for preparing standard curves for grain sample matrices with different cadmium concentrations in Example 14.

[0038] Figure 14 Standard curve for cadmium detection.

[0039] Figure 15 Graph of natural logarithm of k and reciprocal of temperature. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the present application belong to the scope of protection of the present application.

[0041] Unless otherwise indicated herein specifically, the numerical ranges listed herein are inclusive of the recited values and all sub-ranges encompassed therein, and are not limited to the specific values recited.

[0042] A method for rapidly and quantitatively detecting cadmium in grains

[0043] Step 1: After the grain sample is crushed, an acid solution is added, vortexed and centrifuged, the supernatant is taken and a chelating agent solution and a neutralizing agent are added to the supernatant, and the supernatant after chelation is collected; wherein the acid solution is a hydrochloric acid or nitric acid solution, the concentration of the acid solution is 0.19-3 mol / L; the chelating agent is EDTA-2Na, the concentration of EDTA-2Na in the chelating agent solution is 0.00001%-1%; the neutralizing agent is a Tris solution, the concentration is 0.125 mol / L-2 mol / L; 4 mL of acid solution is added per 1 g of crushed grain sample; the volume ratio of the supernatant, the chelating agent solution and the neutralizing agent is 5:1:5;

[0044] Step 2: The supernatant after step 1 is added dropwise to a cadmium time-resolved fluorescence immunochromatographic test reagent card for detection, a fluorescence analyzer is used to detect the fluorescence signal, and the cadmium concentration in the sample is calculated by comparing with a standard curve;

[0045] Wherein, a sample pad is arranged at one end of the cadmium time-resolved fluorescence immunochromatographic test reagent card, a marker pad, a chromatographic pad and a water absorption pad are arranged in sequence on one side of the sample pad and along the length direction of the chromatographic test reagent card; a cadmium antibody-fluorescent microsphere is coated on the marker pad, a T line and a C line are arranged on the chromatographic pad, the T line is coated with a cadmium antigen, and the C line is coated with a secondary antibody-fluorescent microsphere; the cadmium antibody is a mouse monoclonal cadmium antibody, the cadmium antigen is Cd 2+ -EDTA-BSA.

[0046] In the process of developing a method for rapidly and quantitatively detecting cadmium in grains, it is found that there are three core technical problems in traditional detection technology: first, large instrument detection (such as ICP-MS, AAS) relies on complex pretreatment (such as microwave digestion, concentrated acid cooking), which is time-consuming and requires professional personnel, and cannot meet the demand of on-site rapid detection; second, traditional immunodetection (such as colloidal gold test strip) is easily interfered by proteins, pigments and other substances in the grain matrix, the background fluorescence is strong, the sensitivity is low, and it is difficult to realize accurate quantification of trace cadmium (μg / kg level); third, in the existing dilute acid leaching pretreatment method, the concentration of the extractant, the selection of the chelating agent and the pH adjustment lack of synergistic optimization, either the cadmium ion dissociation is not sufficient, resulting in low extraction efficiency, or the extraction liquid environment destroys the subsequent immune reaction, resulting in poor detection accuracy.

[0047] To solve these problems, the present application starts from the perspective of the synergistic optimization of the three links of "marker system-immune response-pre-treatment": for the sensitivity problem, consider using time-resolved fluorescence technology to eliminate background interference, and improve the stability of the marker signal by screening lanthanide element fluorescent microspheres; for the contradiction between pre-treatment efficiency and compatibility, focus on the optimization of the ratio of dilute acid extractant, chelating agent and neutralizing agent, to ensure the efficient dissociation of cadmium ions while maintaining the liquid phase environment suitable for immune response; for the lack of detection specificity and quantitative ability, through antigen-antibody cross combination screening, to strengthen the specificity and linear range of immune response. In the optimization process, the present application accidentally found that when the screened europium fluorescent microspheres are matched with a specific activation system, not only the antibody labeling efficiency is maximized, but also the background fluorescence decay rate is reduced to a very low level through the wide displacement characteristics of the specific excitation light and emission light; at the same time, in the optimization of dilute acid extraction, the combination of dilute nitric acid extraction, chelating agent chelation, and neutralizing agent neutralization not only greatly shortens the extraction time of cadmium in grains (much faster than the traditional digestion method), but also stabilizes the pH of the extraction solution in the best interval for immune response, which exactly matches the environmental conditions required for immune response, ultimately making the detection limit and quantitative range of the detection method meet the trace detection requirements and have excellent linear correlation, and there is no cross reaction with common coexisting heavy metal ions, which not only solves the problems of fast detection adaptability, sensitivity and accuracy of traditional methods, but also realizes the efficient cooperation of the whole process of "pre-treatment-detection", and meets the practical needs of on-site trace cadmium screening.

[0048] In some embodiments of the present application, in step 2, the cadmium antibody or secondary antibody is connected to the fluorescent microspheres by a coupling reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-carboxyl thiosuccinimide. The coupling reaction is as follows:

[0049] S1: Mix the fluorescent microsphere solution and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in water, then add N-carboxyl thiosuccinimide, and react at 30-37°C for 10-15 min, centrifuge to collect the solid product; in the reaction system, the addition amount of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 0.1-2 mg / mL, the addition amount of N-carboxyl thiosuccinimide is 0.1-2 mg / mL, the solid content in the fluorescent microsphere solution is 1-3 wt%, and 1 mL of water is mixed with 200 μL (4 mg) of the fluorescent microsphere solution;

[0050] S2: adding a phosphate buffer to the solid product to re-dissolve and disperse, adding one of the cadmium antibody or the secondary antibody, reacting at 30-37℃ for 1-1.5h, adding BSA to continue reacting for 30-40min, collecting the solid substance after centrifugation, adding a label storage solution to obtain a storage solution containing cadmium antibody fluorescent microspheres or secondary antibody fluorescent microspheres; wherein 500μL of the phosphate buffer is added to every 4mg of the solid product, and the mass ratio of the solid product, one of the cadmium antibody or the secondary antibody, and the BSA is 4:0.1:5, and 500μL of the label storage solution is added to every 4g of the solid substance. This coupling reaction can be used to prepare a storage solution containing cadmium antibody fluorescent microspheres, cadmium antigen fluorescent microspheres, or secondary antibody fluorescent microspheres, respectively.

[0051] In some embodiments of the present application, the label storage solution is: 0.5g of BSA, 0.2g of Pc300, and 0.5ml of Tween-20 are added to 100mL of the phosphate buffer.

[0052] In some embodiments of the present application, the pH of the phosphate buffer is 7.8, the phosphate ion concentration is 50mmol / L, the mass concentration of the BSA is 0.2wt%-1.6wt%, and the mass concentration of the Tween-20 is 0.2wt%-1.6wt%.

[0053] In some embodiments of the present application, the fluorescent microspheres are europium time-resolved fluorescent microspheres, the emission light of the fluorescent microspheres is 615nm, the excitation spectrum is 360-410nm, and the carboxyl content on the surface of the microspheres is 205-350μmol / g. The europium time-resolved fluorescent microspheres are prepared by using Eu 3+ The polystyrene microspheres are filled with a chelating agent, and the surface is modified with -COOH to form uniform microspheres with high fluorescence yield.

[0054] In some embodiments of the present application, the secondary antibody is goat anti-mouse or rabbit anti-mouse.

[0055] In some embodiments of the present application, in step 2, the coating concentration of the cadmium antigen-fluorescent microspheres on the T line is 0.2-1.6mg / mL.

[0056] In some embodiments of the present application, in step 2, the chromatography temperature is 20-25℃, the chromatography time is 10-20min, and the volume of the liquid dropped on the reagent card is 60-120μL. II. DETAILED DESCRIPTION

[0058] 1. Screening and performance evaluation of europium time-resolved fluorescent microspheres

[0059] The main reagent materials and instruments used in the experiment are shown in Table 1:

[0060] Table 1

[0061] Material name CAS Specification Factory Eu time-resolved fluorescence microspheres / 100nm-300nm Chengdu Microspheres Biotechnology Co., Ltd. Goat anti-mouse / 7.9mg / mL Chengdu Microspheres Biotechnology Co., Ltd. Mouse IgG / 5mg / mL Chengdu Microspheres Biotechnology Co., Ltd. Nitrocellulose membrane / CN-140 Merck Chemical Technology Co., Ltd. EDC 1892-57-5 Analytically pure Merck Chemical Technology Co., Ltd. NHS 106627-54-7 Analytically pure Merck Chemical Technology Co., Ltd. Phosphate 10039-32-4 Analytically pure Beijing Solabio Technology Co., Ltd. Sodium hydroxide 1310-73-2 Analytically pure Beijing Solabio Technology Co., Ltd. Hydrochloric acid / 36％-38％ Chongqing Chuandong Chemical Industry Group Co., Ltd. Proclin300 124-07-2 Analytically pure Merck Chemical Technology Co., Ltd. Tween-20 9005-64-5 Analytically pure Merck Chemical Technology Co., Ltd. BSA 9048-46-8 ≥98％ Merck Chemical Technology Co., Ltd.

[0062] Table 2

[0063]

[0064]

[0065] Example 1:

[0066] The coupling method of time-resolved fluorescence microspheres and antibodies adopts the two-step method of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) plus N-carboxyl thiosuccinimide (Sulfo-NHS). The active intermediate formed by the reaction of the carboxyl group of the fluorescence microspheres with EDC is easy to hydrolyze, and the hydrolysis reaction decomposes the activated intermediate and re-forms the carboxyl microspheres. The addition of N-carboxyl thiosuccinimide (Sulfo-NHS) during the reaction process can make the intermediate form a stable Sulfo-NHS ester intermediate, and finally slowly react with R-NH2 on the antibody to form a stable amide bond. The coupling method using the two-step method has many advantages. Generally, carboxyl and primary amine groups exist on the antibody molecule at the same time, so it is necessary to remove the excess EDC before adding the protein to prevent the activation of the protein carboxyl group, which can cause the protein to couple itself. After the first step of activation in the two-step coupling method, the excess EDC in the solution can be removed by centrifugation, so that the antibody only reacts with the activated microspheres. At the same time, the activation reaction conditions and the amide bond formation conditions can be effectively separated, so that the activation and protein coupling can be carried out under the most effective reaction conditions.

[0067] Therefore, in this example, the use amount of the fixed mouse monoclonal antibody and the fluorescence microspheres is used, wherein the use amount of the mouse monoclonal antibody is in an excessive state, and different concentrations of EDC and Sulfo-NHS are used. The mouse secondary antibody is coated on the NC membrane, the fluorescence signal intensity captured by the secondary antibody is detected, and the change is analyzed.

[0068] Eu time-resolved fluorescence microspheres were purchased from the market, and 1 ml of 0.1% time-resolved fluorescence microspheres diluted with purified water was taken in 5 portions. The amount of EDC added was set to 2 mg / 1 mg / 0.5 mg / 0.25 mg / 0.1 mg. The same amount of Sulfo-NHS was added. Reaction was carried out at 37°C for 10 min, centrifuged to remove the supernatant, and 500 μL of phosphate buffer was added. After re-dissolving and dispersing, 0.5 mg of mouse monoclonal antibody was added to each group. Reaction was carried out at 37°C for 1 h. 20 mg of BSA was added for further reaction for 30 min. After reaction, the supernatant was discarded by centrifugation, and 500 μL of label storage solution was added. The label storage solution was prepared by adding 0.5 g of BSA, 0.2 g of Pc300, and 0.5 ml of Tween-20 to 100 ml of phosphate buffer.

[0069] The five different amounts of EDC-labeled fluorescence microspheres prepared above were diluted 200 times with PBS, and 100 μL was added to the mouse monoclonal antibody test strip to detect the fluorescence signal of the mouse monoclonal antibody microspheres captured on the line.

[0070] The concentration of the fixed fluorescence microspheres, EDC, and Sulfo-NHS was used in the second step of coupling the monoclonal antibody. Different amounts of monoclonal antibody were set in the second step. The mouse secondary antibody was coated on the NC membrane, and the fluorescence signal intensity captured by the secondary antibody was detected and analyzed. After maximum EDC activation, the amount of mouse monoclonal antibody added was 0.5 mg, 0.25 mg, 0.1 mg, 0.05 mg, and 0.025 mg. The five different amounts of monoclonal antibody-labeled fluorescence microspheres prepared above were diluted 20 times with PBS, and 100 μL was added to the mouse monoclonal antibody test strip to detect the fluorescence signal of the mouse monoclonal antibody microspheres captured on the line.

[0071] The results are shown in Figure 1 When the amount of EDC reaches 0.5 mg, the fluorescence signal increases slowly, and the activation amount reaches the maximum state, so 0.5 mg of EDC and 0.5 mg of Sulfo-NHS are selected as the best activation agent usage for subsequent examples.

[0072] At the same time, the results of the maximum mouse monoclonal antibody usage of the labeled fluorescence microspheres are shown in Figure 2 When the amount of monoclonal antibody is 0.25 mg, the fluorescence signal no longer increases significantly, so 0.25 mg of monoclonal antibody is selected as the maximum amount of monoclonal antibody for this labeled microsphere.

[0073] 2. Cadmium time-resolved fluorescence immunoassay card

[0074] (1) The main reagents and equipment used in the following examples are shown in Tables 3-4:

[0075] Table 3

[0076] Reagent name CAS Specification Factory Cadmium monoclonal antibody / 5.6mg / ml Chengdu Microspheres Biotechnology Co., Ltd. Cd 2+ - EDTA-BSA antigen / 4.05mg / ml Chengdu Microspheres Biotechnology Co., Ltd. Goat anti-mouse / 7.9mg / ml Hangzhou Qitai Biotechnology Co., Ltd. Rabbit anti-mouse / 5mg / ml Hangzhou Qitai Biotechnology Co., Ltd. Cd 2+ standard solution / 1000mg / Ml Shandong ZKRspectra Technology Co., Ltd. EDTA-2Na 6381-92-6 Analytically pure National Pharmaceutical Group Chemical Reagent Co., Ltd. Time-resolved fluorescence microspheres / 100nm-300nm Chengdu Microspheres Biotechnology Co., Ltd. Glass fiber pad / 8975 Tongsheng (Fuzhou) Paper Co., Ltd. Nitrocellulose membrane / CN140 Merck Chemical Technology Co., Ltd. Water absorption pad / 20cm*30cm*1.95mm Shanghai Jetning Biotechnology Co., Ltd. PVC plate / 65mm*30cm Shanghai Jetning Biotechnology Co., Ltd. Proclin300 124-07-2 Analytically pure Merck Chemical Technology Co., Ltd. Tween-20 9005-64-5 Analytically pure Merck Chemical Technology Co., Ltd. BSA 9048-46-8 ≥98％ Merck Chemical Technology Co., Ltd. Sodium chloride 7647-14-5 Analytically pure Beijing Solabio Technology Co., Ltd. Disodium hydrogen phosphate dodecahydrate 10039-32-4 Analytically pure Beijing Solabio Technology Co., Ltd. Sodium dihydrogen phosphate dihydrate 13472-35-0 Analytically pure Beijing Solabio Technology Co., Ltd. Potassium chloride 7447-40-7 Analytically pure Beijing Solabio Technology Co., Ltd. Tris (hydroxymethyl) aminomethane 77-86-1 Analytically pure Beijing Solabio Technology Co., Ltd.

[0077] Table 4

[0078] Experimental equipment Model Factory Test strip cutter Table type Hangzhou Glen Kun Technology Co., Ltd. pH meter FE20 Mettler Toledo Fluorescence analyzer JW-1706 Chengdu Microspheres Biotechnology Co., Ltd. High-speed refrigerated centrifuge CHT210R Hunan Xiangyi Laboratory Instrument Development Co., Ltd. Gold spraying and membrane drawing machine Table type Hangzhou Glen Kun Technology Co., Ltd. Air drying oven TST101A-1 Chengdu TST Instrument Co., Ltd.

[0079] (2) Solution preparation

[0080] Cadmium dilution solution: 60.57 g of Tris powder and 2.32 g of EDTA were weighed and placed in 800 mL of purified water, and the pH was adjusted to 7.0 with hydrochloric acid. Finally, purified water was used to make up to 1 L in a volumetric flask.

[0081] Phosphate buffer: 5.158 g of Na2HPO4·12H2O and 0.874 g of NaH2PO4·2H2O were accurately weighed and prepared in 1 L of purified water.

[0082] Fluorescent microsphere storage solution: 5.158 g of Na2HPO4·12H2O, 0.874 g of Na2HPO4·12H2O, 10 g of BSA, and 3002 g of PC were accurately weighed and prepared in 1 L of purified water.

[0083] Sample pad treatment solution: 6 g of Tris, 50 g of BSA, 3002 g of PC, and 5 g of Tween-20 were accurately weighed and dissolved in 800 mL of purified water, and the pH was adjusted to 8.0 with hydrochloric acid. Finally, make up to 1 L in a volumetric flask.

[0084] (3) Preparation of reagent card

[0085] The reagent card structure used in the present application is a conventional reagent card of the prior art, which includes a sample pad, a marker pad, a chromatography pad, and a water absorption pad. A sample pad is provided at one end of the reagent card, and a marker pad, a chromatography pad, and a water absorption pad are provided in sequence on one side of the sample pad and along the length direction of the chromatography detection reagent card. A cadmium antibody-fluorescent microsphere is coated on the marker pad, and a T line and a C line are provided on the chromatography pad, wherein the T line is coated with a cadmium antigen, and the C line is coated with a secondary antibody-fluorescent microsphere.

[0086] Embedding step: nitrocellulose membrane was pasted on a PVC plate, and Cd 2+ -EDTA-BSA and mouse secondary antibody were diluted to a certain concentration, and were drawn on the nitrocellulose membrane with a dot membrane gold spraying instrument, and were dried at 37°C for standby use.

[0087] Sample pad treatment step: glass fiber was cut into 30 cm x 20 mm specifications. The glass fiber was soaked in the treatment solution for 30 min, then taken out and dried in a forced air drying oven at 37°C for standby use.

[0088] Marker pad preparation: dilute the cadmium antibody fluorescent microspheres with the above storage solution to a certain concentration, uniformly spray on the glass fiber with the point film gold spraying instrument, and dry in the air drying oven at 37°C for standby.

[0089] Cut the absorbent paper into a size of 30 cm x 20 mm with a cutting knife. The prepared marker pad, sample pad, and absorbent paper are sequentially attached to the corresponding positions on the PVC plate to assemble a large plate, which is cut into a test strip with a width of 4 mm by a rolling cutter, and assembled into a reagent card for standby.

[0090] In the subsequent parameter optimization experiment, the reagent card prepared by the above method is used for optimization experiment, and any one of "different labeling amounts of cadmium antibody, different types of secondary antibody, different coating concentrations of cadmium antigen and cadmium antibody, different amounts of blocking agent and surfactant, and sample chromatography time, temperature and sample amount" is used as a single variable for optimization experiment. In the optimization experiment, the other fixed amount is set according to the corresponding parameter in the optimal scheme.

[0091] (4) Parameter optimization

[0092] Example 2: Optimization of the labeling amount of cadmium antibody

[0093] After determining the optimal cadmium antibody code, the labeling amount of cadmium antibody is set to a gradient amount, which is 2B, B, 1 / 2B, 1 / 4B, and 1 / 8B (B is the maximum labeled amount of monoclonal antibody). The five different labeling amounts of cadmium antibody fluorescent microspheres markers are combined with the cadmium antigen NC membrane to prepare a detection card. The fluorescence signal T / C of the positive control and the fluorescence signal T / C of the negative control are detected, and the ratio S / N is calculated, as shown in Table 1. Figure 3 The amount of fluorescent microsphere labeled antibody, the maximum labeling amount is not necessarily the best condition. When the fluorescent microsphere labeled antibody is used in the maximum labeling amount, the titer is the highest, but the sensitivity is not necessarily the best condition. Therefore, the optimal antibody optimized above is re-labeled with a gradient, and the best labeling amount is selected by considering the sensitivity and titer. On this basis, when the cadmium antibody used for labeling fluorescent microspheres is 0.05 mg, the ratio of the T / C of the negative control to the T / C of the positive control is the largest and is 5.4. When the amount is reduced by half, the ratio does not decrease significantly, but the T-line fluorescence intensity decreases significantly when detecting the negative control. In the competition method, in order to obtain a larger fluorescence range in the later debugging, thereby obtaining a larger linear range, the condition with a stronger T-line fluorescence of the negative control is preferred, and therefore 0.05 mg is selected as the most appropriate amount of fluorescent microsphere labeled cadmium antibody.

[0094] Example 3: Selection of quality control line raw materials

[0095] In order to screen the raw materials of the quality control line, the cadmium antibody is used as mouse monoclonal antibody, and the mouse secondary antibody is used as the quality control line of the test strip in this embodiment. According to this idea, four mouse secondary antibodies are collected from the market, including goat anti-mouse (2 sources) and rabbit anti-mouse (2 sources). The mouse secondary antibody is coated on the nitrocellulose membrane at the same concentration. According to the affinity of the mouse secondary antibody to the cadmium antibody, the relative standard deviation of the fluorescence of the C line and the accelerated stability, the optimal quality control line raw material is selected. Among them, the mouse secondary antibody refers to the secondary antibody of the anti-mouse primary antibody. It is a general term for universal secondary antibodies, not specific to the source species, and only specifies that its function is to bind all mouse primary antibodies. Goat anti-mouse and rabbit anti-mouse are both subtypes of it. Goat anti-mouse is a goat-derived secondary antibody for anti-mouse primary antibodies (Goat-derived Anti-Mouse Primary Antibody Secondary Antibody). The source species is a goat, that is, the secondary antibody is prepared from a goat, and the target object is a mouse primary antibody, that is, it is specifically designed to bind to an antibody prepared from a mouse. Rabbit anti-mouse is a rabbit-derived secondary antibody for anti-mouse primary antibodies (Rabbit-derived Anti-Mouse Primary Antibody Secondary Antibody). The source species is a rabbit, that is, the secondary antibody is prepared from a rabbit, and the target object is also a mouse primary antibody. Its function is the same as that of goat anti-mouse, only the source species is different.

[0096] The coating concentration of the mouse secondary antibody on the C line of the NC membrane is 0.5 mg / mL, the T line is not coated with any material, and the marker is labeled with a 5-fold dilution of cadmium antibody marker. After preparation, add cadmium dilution and record the detection fluorescence intensity of the C line. The reagent card is divided into 3 parts and placed in a 42°C oven for 4d, 8d and 12d respectively. After the baking is completed, add cadmium dilution and record the detection results. Record the fluorescence intensity of the C line, the change rate of the fluorescence of the C line, and the coefficient of variation of the fluorescence of the C line. This embodiment investigates the biological activity, uniformity and storage stability of several quality control line materials. The purpose of investigating the three indicators is to embed the C line raw material in the nitrocellulose membrane for a longer storage period, during which the change is minimal, and the detection value is minimally affected. High C fluorescence intensity will reduce the preparation cost of the detection card. The investigation of uniformity is also important. Different affinities of the C line will result in different uniformity of the C line. Selecting a C line raw material with better uniformity is more conducive to preparing detection cards with more stable batch-to-batch detection performance. The test results are shown in Table 1. Figure 4 As shown in Table 1, among the 4 groups of C line tests, rabbit anti-mouse 1 has obvious advantages, with a very low change rate in accelerated stability test, a small CV value, and a moderate fluorescence intensity. Therefore, rabbit anti-mouse 1 is selected as the best and most stable C line raw material.

[0097] Example 4: Optimization of the use of cadmium antigen and cadmium antibody-fluorescent microspheres

[0098] The coating concentration of cadmium antigen was set at 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, and 1.6 mg / mL, respectively. The cadmium antibody fluorescent microsphere marker pad was prepared according to 3.3.1.4 at different concentrations, set at 2-fold, 4-fold, 8-fold, and 16-fold dilution, respectively. Different coating and labeling concentrations were cross combined to detect the positive quality control 200 ng / mL and blank control prepared by cadmium diluent, and the detection sensitivity was used to screen the optimal cadmium antigen concentration and cadmium antibody marker concentration. Figure 5 As shown in the results, in the competition method, the smaller the S / N (positive control fluorescent signal / negative control fluorescent signal), the higher the sensitivity. When the cadmium antigen coating concentration was 0.8 mg / mL and the cadmium antibody fluorescent microsphere concentration was 8 or 16 times, the S / N reached the optimum. Considering that a strong fluorescent signal is conducive to a wide linear range, the cadmium antibody fluorescent microsphere concentration was selected as 8-fold dilution.

[0099] Example 5: Optimization of sample pad of test strip

[0100] The phosphate buffer has a pH of 7.8 and a phosphate ion concentration of 50 mmol / L. BSA and Tween-20 are the most commonly used blocking agents and surfactants in chromatography, and are widely available and stable in quality. In this study, the use of BSA and Tween-20 was set at 0.2%, 0.4%, 0.8%, and 1.6%, respectively. The optimal concentration of BSA and Tween-20 was selected by orthogonal test. The sample pad was treated with different concentrations of BSA and Tween-20, and the test reagent card was assembled with the treated sample pad. The cadmium standard solution and cadmium diluent with a concentration of 50 ng / mL were detected, the ratio S / N of the positive sample fluorescent signal T / C to the negative sample fluorescent signal T / C was calculated, and the average value of the background fluorescent signal intensity of the NC membrane was calculated. As shown in the results, Figure 6 and Figure 7 The smaller the S / N and background fluorescent signal, the better. The sample pad formula with better S / N was 0.8% BSA and 0.8%-1.6% Tween-20, and the sample pad formula with better fluorescent background was 0.2-0.8% BSA and 0.8%-1.6% Tween-20. Based on the above conditions, 0.8% BSA and 0.8% Tween-20 were selected as the best sample pad.

[0101] Example 6: Effect of sample chromatography time, temperature, and loading amount on detection results

[0102] The reagent card chromatography temperature was set at 2-8℃, room temperature and 37℃, respectively. The chromatography time was set at 5min, 10min, 15min, 20min. According to the minimum and maximum liquid volume of the reagent card chromatography, the sample volume was set at 60μL, 80μL, 120μL, and the positive and negative reference samples were detected under different conditions. The orthogonal test was also used to screen the optimal conditions in this experiment, and the results were analyzed by 3-factor 3-level 2-index range analysis. The range calculation method is as follows: (1) calculate the average value S of each index value under each factor and each level; (2) calculate the range R of each factor, which is the difference between the maximum and minimum values of the average values of the 3 levels under the factor.

[0103] The chromatography time (factor 1), chromatography temperature (factor 2), and sample volume (factor 3) were the three main factors, which were further divided into three small factors. The chromatography time had three levels (chromatography time factor 1 was 10min, chromatography time factor 2 was 15min, and chromatography time factor 3 was 20min), the chromatography temperature had three levels (chromatography temperature factor 1 was 2-8℃, chromatography temperature factor 2 was room temperature, and chromatography temperature factor 3 was 37℃), and the sample volume had three levels (sample volume factor 1 was 60μL, sample volume factor 2 was 80μL, and sample volume factor 3 was 120μL). The orthogonal test table was designed as shown in Table 5.

[0104] Table 5

[0105] Number Factor 1 Factor 2 Factor 3 1 Chromatography time factor 1 Chromatography temperature factor 1 Loading amount factor 1 2 Chromatography time factor 1 Chromatography temperature factor 2 Loading amount factor 3 3 Chromatography time factor 1 Chromatography temperature factor 3 Loading amount factor 2 4 Chromatography time factor 2 Chromatography temperature factor 1 Loading amount factor 3 5 Chromatography time factor 2 Chromatography temperature factor 2 Loading amount factor 2 6 Chromatography time factor 2 Chromatography temperature factor 3 Loading amount factor 1 7 Chromatography time factor 3 Chromatography temperature factor 1 Loading amount factor 2 8 Chromatography time factor 3 Chromatography temperature factor 2 Loading amount factor 1 9 Chromatography time factor 3 Chromatography temperature factor 3 Loading amount factor 3

[0106] According to the test results of the factor levels in Table 5, the change rate S of S / N and chromatography T / C was analyzed, S=(T1-T2) / T1×100% (wherein, T1 is the fluorescence intensity before change, and T2 is the fluorescence intensity after 5 minutes), and the statistical table was recorded as shown in Table 6.

[0107] Table 6

[0108] No. S / N S 1 0.11 23.0 2 0.14 2.7 3 0.13 9.7 4 0.12 12.4 5 0.14 6.6 6 0.14 7.7 7 0.16 7.3 8 0.16 3.9 9 0.16 1.3

[0109] The 3-factor 3-level 2-index range analysis was performed on the above, and the results were shown in Table 7.

[0110] Table 7

[0111] Factor Chromatography time Chromatography temperature Loading amount Level 1 mean (S / N) 0.128 0.129 0.136 Level 2 mean (S / N) 0.130 0.146 0.140 Level 3 mean (S / N) 0.160 0.143 0.142 Range (S / N) 0.032 0.016 0.006 Level 1 mean (S%) 11.8 14.2 11.5 Level 2 mean (S%) 8.9 4.4 7.9 Level 3 mean (S%) 4.2 6.2 5.5 Range (S%) 7.6 9.8 6.1

[0112] Two indicators of sensitivity (S / N) and change rate (S) are of great significance. A small S / N value represents a higher sensitivity in detection. In the case of the smallest S change rate, the optimal detection conditions can be recommended during the use of the reagent card, and the time period has more detection stability under the conditions. Combined with the two indicators, the chromatography time of 10 min, the chromatography temperature of room temperature, and the loading amount of 120 μL are selected as the optimal detection conditions. This condition takes into account both the change rate and the sensitivity indicators. As can be seen from the range analysis table, the three factors affecting the sensitivity are ranked in order of influence size: chromatography time > chromatography temperature > loading amount; the three factors affecting the change rate are ranked in order of influence size: chromatography temperature > chromatography time > loading amount. The purpose of this example is to explore a relatively stable detection environment for the detection card, in which the detection can ensure accuracy. Chromatography time, chromatography temperature and loading amount will affect each other. The entire chromatography process is carried out in a dynamic process, and the release of the marker pad microspheres is clean at the end of the chromatography period. The release is relative. If the chromatography time is too long, the test time of the detection card will be longer. At the same time, the chromatography process is greatly affected by temperature. On the one hand, temperature affects the antigen-antibody reaction. Suitable temperature (such as body temperature 37℃) is more conducive to the antigen-antibody reaction. On the other hand, temperature affects the chromatography speed of the chromatography liquid. The higher the temperature, the faster the chromatography liquid moves. If the chromatography speed is too fast, the antigen-antibody reaction time will be shortened, which is not conducive to the immune reaction. Therefore, orthogonal test must be used to verify multiple factors.

[0113] 3. Optimization of the pretreatment method for cadmium extraction in cereals

[0114] The reagents and equipment used in the following examples are shown in Tables 8 and 9.

[0115] Table 8

[0116] Reagent name CAS / No. Specification Manufacturer Hydrochloric acid 7647-01-0 Chongqing Chuandong Chemical Industry (Group) Co., Ltd. Nitric acid 7697-37-2 Chongqing Chuandong Chemical Industry (Group) Co., Ltd. Diethylene triamine pentaacetic acid 67-43-6 Analytically pure Tengzhun Biological Disodium ethylenediaminetetraacetate 6381-92-6 Analytically pure National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai) Tris 77-86-1 Analytically pure Beijing Solabio Technology Co., Ltd. Cadmium negative rice / / Chongqing Metrology and Quality Inspection Research Institute Cadmium negative corn / / Chongqing Metrology and Quality Inspection Research Institute Cadmium negative wheat / / Chongqing Metrology and Quality Inspection Research Institute Cadmium negative sorghum / / Chongqing Metrology and Quality Inspection Research Institute Positive rice sample / Cadmium content 0.41 mg / kg Chongqing Metrology and Quality Inspection Research Institute

[0117] Table 9

[0118] Experimental equipment Model Manufacturer Fluorescence detector JW-1706 Chengdu Micro-Rui Biological Technology Co., Ltd. Centrifuge TD4 Kate Experimental Instrument Co., Ltd. Multi-tube vortex oscillator MIX-200 TUOHE Company

[0119] In the following examples, different concentrations of nitric acid and hydrochloric acid were used to extract negative rice spiked samples (cadmium spiked concentration of 0.2 mg / kg), positive rice samples (cadmium content of 0.41 mg / kg), and negative rice samples. The change in the ratio of fluorescence signal T / C was observed, and the smaller the ratio of positive T / C to negative T / C (i.e. S / N) was, the better the condition was. Because different concentrations of acid correspond to different concentrations of neutralizing liquid, different concentrations of acid-base neutralizing liquid were prepared before the experiment, and then positive and negative quality controls were prepared from the neutralizing liquid to investigate the S / N change at that time.

[0120] Example 7:

[0121] Accurately take 1 g of crushed cereal sample in a 10 mL centrifuge tube, add 4 mL of acid solution in the centrifuge tube, put it into a multi-tube vortex oscillator for vortex oscillation for a certain time, then put it into a centrifuge at 4000 rpm / min for 5 min. Take 100 μL of supernatant, add 20 μL of chelating agent (Note: the concentration of chelating agent refers to mass g / purified water volume mL), then add 100 μL of neutralizing solution for neutralization, and finally add 120 μL of neutralized supernatant to the cadmium time-resolved fluorescence immunochromatographic detection card for detection.

[0122] Example 8:

[0123] In Example 7, 1.5 mol / L, 0.75 mol / L, 0.38 mol / L, 0.19 mol / L. The concentration of neutralizing agent Tris corresponds to the concentration of acid, respectively 2 mol / L, 1 mol / L, 0.5 mol / L, 0.25 mol / L, 0.125 mol / L.

[0124] Example 9:

[0125] Based on Example 7, the difference is that hydrochloric acid solution is used, and the concentration of hydrochloric acid is 3.0 mol / L, 1.5 mol / L, 0.75 mol / L, 0.38 mol / L, 0.19 mol / L. The concentration of neutralizing agent Tris corresponds to the concentration of acid, respectively 2 mol / L, 1 mol / L, 0.5 mol / L, 0.25 mol / L, 0.125 mol / L.

[0126] The detection results of Example 8 and Example 9 are shown in Figure 8 The concentration of acid solution has a significant effect on S / N value. Whether hydrochloric acid or nitric acid, when the molar concentration of acid is 0.25 mol / L-0.5 mol / L, the S / N detection value is the smallest. The overall extraction efficiency of nitric acid is higher than that of hydrochloric acid. Considering the reduction of acid dosage, the subsequent example selects dilute nitric acid with a concentration of 0.25 mol / L as the best extraction agent for cadmium in cereals.

[0127] Example 10:

[0128] According to the operation steps of Example 7, the concentration of nitric acid solution is 0.25 mol / L, and the concentration of chelating agent (EDTA-2Na) is 0.002% (Note: mass g / purified water volume mL). The oscillation extraction time is set to 2 min, 5 min, 10 min, 15 min, and 20 min, respectively. Negative rice samples (cadmium spike concentration is 0.2 mg / kg), positive rice samples (cadmium content is 0.41 mg / kg), and negative rice samples are extracted, respectively. Observe the change of fluorescence signal T / C ratio, where the smaller the S / N, the better the condition. The results are as followsFigure 9 As shown, after 5 minutes of oscillation, the S / N ratio changes very little. The extraction efficiency is close to the maximum efficiency after 5 minutes and meets the requirements for rapid detection. Therefore, 5 minutes was selected as the oscillation extraction time for cadmium in cereal powder.

[0129] Example 11:

[0130] Based on Example 7, the nitric acid solution concentration was 0.25 mol / L, the shaking extraction time was 5 min, and two common chelating agents, EDTA-2Na and DTPA, were selected. Different dosages were set, and the extracted samples were rice samples with added cadmium (cadmium added concentration of 0.2 mg / kg) and negative rice samples. Extraction experiments were first conducted with EDTA-2Na and DTPA concentrations of 1%, 0.1%, 0.01%, 0.001%, 0.0001%, and 0.00001% (Note: mass g / purified water volume mL). Then, isochronous dilution tests were performed based on the chelating agent concentration near the optimal conditions found. The S / N ratio was calculated for each experimental scheme.

[0131] The results are as follows Figures 10-11 As shown, excessive DTPA has a negative impact on sensitivity; the optimal concentration of DTPA does not significantly improve sensitivity, and its chelation effect is much worse than that of EDTA-2Na. Figure 10 Therefore, an EDTA-2Na mass fraction of 0.0001% was chosen for further optimization experiments. The concentrations of EDTA-2Na were set to 0.0005%, 0.0002%, 0.0001%, 0.00005%, and 0.00002%, and the optimal S / N ratio was selected. Figure 11 As shown in the figure, the S / N ratio was lowest when the mass fraction of EDTA-2Na was 0.0001%, therefore this mass fraction was chosen as the optimal amount of chelating agent. In immunoassay methods for heavy metal detection, heavy metals must bind to a chelating agent to become haptens. Haptens generally bind to proteins to form fully immunogenic antigens. The choice of chelating agent is partly to accommodate the immunogenicity of cadmium monoclonal antibodies, and partly related to the efficiency of the chelating agent in chelating cadmium. Excessive chelating agent can actually reduce detection sensitivity. The reason for this might be that excessive chelating agent can form a three-dimensional structure different from the "cadmium-chelating agent-protein" site of the immunogen. This three-dimensional structure cannot be recognized by the cadmium antibody, thus competing with the cadmium monoclonal antibody and reducing detection sensitivity.

[0132] Example 12:

[0133] On the basis of Example 7, the concentration of nitric acid solution was 0.25 mol / L, the oscillation extraction time was 5 min, the chelating agent was EDTA-2Na, and the concentration was 0.0001% (the concentration here refers to the mass g / purified water volume mL). Different concentrations of Tris were selected as neutralizing reagents, and the supernatant of positive sample and the supernatant of negative sample were taken as four groups. After adding the chelating agent, Tris was added, and the concentration was set to 1.4 mol / L, 0.7 mol / L, 0.35 mol / L, and 0.18 mol / L. The extracted samples were rice spiked samples (the cadmium spiking concentration was 0.2 mg / kg) and negative rice samples. The S / N under each experimental scheme was calculated to determine the optimal concentration of the neutralizing agent. The results are shown in Figure 12 Table 2. The use of Tris at a concentration of 0.35 mol / L to 0.7 mol / L has good sensitivity. The concentration of 0.35 mol / L of Tris is selected as the optimal concentration of the neutralizing agent. In the case of insufficient amount of neutralizing agent Tris, the overall liquid environment is acidic, which will seriously affect the detection S / N, and will also greatly reduce the fluorescence values of C line and T line. The main reason is that in an acidic environment, the antigen-antibody binding reaction is weakened, and in an acidic environment, the effect of the chelating agent is greatly reduced, so it is necessary to adjust to an alkaline environment for detection. However, cadmium ions will produce insoluble precipitates under strong alkaline conditions, affecting the concentration of cadmium ions in the solution and thus affecting the chelation effect.

[0134] Example 13

[0135] According to the method of Example 7, the concentration of nitric acid solution was 0.25 mol / L, the oscillation extraction time was 5 min, the chelating agent was EDTA-2Na, and the concentration was 0.0001% (the concentration here refers to the mass g / purified water volume mL), and the content of neutralizing agent Tris was 0.35 mol / L. Different negative grain samples (including rice, corn, sorghum, and wheat) were added with different concentrations of cadmium standard, and the optimized pretreatment method was used for extraction and detection to draw standard curves of different matrices. Four different grain matrices were added with cadmium at concentrations of 3000 μg / kg, 1200 μg / kg, 600 μg / kg, 300 μg / kg, 120 μg / kg, 60 μg / kg, 30 μg / kg, and 12 μg / kg. The logarithm of the concentration was taken as the X-axis, and the logarithm of the detection value T / C was taken as the Y-axis to draw the standard curve. As shown in Figure 13As shown, the standard curves of the four substrates of sorghum, corn, rice and wheat are uniform, and the correlation R2 of the standard curves after adding each substrate remains between 0.9877-0.9963. The example evaluates the substrate interference of the four common cereals, and the purpose is to test the interference of different substrates in the sample detected by the detection card on the standard curve. When preparing the reagent card, several cereal samples can be tested by the same standard curve, which is convenient for testers to use.

[0136] 4. Optimal parameters of extraction and reagent card optimization

[0137] The optimal extraction conditions of the cereal sample are as follows: 0.25 mol / L dilute nitric acid is used as the extraction agent, room temperature oscillation extraction is performed for 5 min, then 0.0001% EDTA-2Na is added for chelation, and finally 0.35 mol / L Tris buffer is used for neutralization treatment. The pretreatment process can be completed within 20 min. The treated extract can be directly used for time-resolved fluorescence immunochromatographic detection, which provides a reliable pretreatment scheme for the on-site rapid screening of cereal cadmium pollution.

[0138] The cadmium-specific antibody is coupled with the time-resolved fluorescence microspheres and fixed on the glass fiber pad, the cadmium-EDTA-BSA complex antigen is coated on the nitrocellulose (NC) membrane detection line, the rabbit anti-mouse IgG is used as the quality control line, and the sample pad, the water absorption pad and the PVC back plate are assembled into a complete detection device. Through system optimization, the key process parameters are determined as follows: the optimal labeling amount of the fluorescence microsphere-labeled cadmium antibody is 0.05 mg / mL; the T-line cadmium antigen coating concentration is 0.8 mg / mL, the C-line rabbit anti-mouse IgG coating concentration is 0.5 mg / mL, and the cadmium antibody-labeled fluorescence microsphere concentration is 8 times; the best sample pad treatment liquid is 0.8% BSA and 0.8% Tween-20; the detection condition is room temperature, the sample amount is 120 μL, and the chromatographic reaction time is 10 min.

[0139] 5. Efficiency verification of the reagent card and application thereof

[0140] The reagent materials used in the following experiments are shown in Table 10:

[0141] Table 10

[0142]

[0143] (1) Detection limit determination and verification of the reagent card

[0144] 1) Detection limit determination of the reagent card

[0145] The detection limit of cadmium in grains was determined according to the detection limit determination method (blank standard deviation method) in GB 5009.295-2023 "General rules for verification of chemical analysis methods". Ten cadmium-negative rice samples, ten cadmium-negative wheat samples and ten cadmium-negative corn samples were taken, and the T / C of 30 negative samples was detected, and the average value and standard deviation were calculated. The rice negative sample was added to a series of different concentrations of cadmium standard substance added sample, and the T / C was detected. The T / C of the estimated detection limit was equal to the average value of the blank sample minus 3 times the blank standard deviation, and the T / C corresponding to the series of different concentrations of cadmium standard substance was detected to determine the minimum detection limit concentration of the detection card. The results are shown in Table 11:

[0146] Table 11

[0147]

[0148] Table 12 Gradient addition concentration sample detection result table

[0149] Concentration T / C 3000 0.027 1200 0.038 600 0.050 300 0.082 120 0.123 60 0.179 30 0.290 12 0.381 6 0.450

[0150] The estimated detection limit concentration is close to 6 μg / kg, and the T / C is 0.454. This T / C is used as the threshold for judging the positivity of the reagent kit. In order to achieve a detection limit detection rate of 95%, the estimated true detection limit concentration is 10 μg / kg, and the T / C of 20 rice samples added with this concentration is verified, as shown in Table 13.

[0151] Table 13

[0152] Sample number Sample addition concentration (μg / kg) T / C greater than threshold sample number T / C less than threshold sample number Limit of detection detection rate 20 10 0 20 100％

[0153] 2) The reagent card detection limit verification of the application

[0154] The detection rate of the target analyte at the detection level limit is not less than 95%, and 20 detection samples of the detection limit concentration are added to the blank rice samples, and the T / C of the detection samples is detected. The T / C value of 95% is lower than the T / C of the detection limit concentration, which is considered to be qualified.

[0155] (2) The detection range determination of the reagent card of the application

[0156] 1) Standard curve preparation

[0157] In the negative rice sample, the cadmium standard was added in gradient (concentration n), extracted according to the extraction steps, and the T / C of each cadmium concentration gradient was detected by cadmium time-resolved fluorescence immunochromatographic detection reagent card. The logarithm of concentration is y axis, that is, y1 = log (n1), y2 = log (n2), y3 = log (n3) ……; The logarithm of the detected T / C is the x axis, that is, x1 = log (t1 / c1), x2 = log (t2 / c2), x3 = log (t3 / c3) ……; Scatter plot is drawn in excel, and the relationship formula between fluorescence signal (T / C) and cadmium concentration is found according to the regression formula: y = ax + b. According to the observation, the best R 2 linear range is selected.

[0158] 2) Standard curve range determination

[0159] According to GB 5009.295-2023 "General rules for verification of chemical analysis methods", the highest concentration and the lowest concentration in the linear range and the concentration of interest should be verified for correctness and precision, and the correctness and precision should meet the corresponding requirements. According to the standard requirements of GB 2762-2022 "National food safety standard limit of contaminants in food", the limit standard of cadmium in grains is 0.2 mg / kg, so this concentration is tested as the concentration of interest, and 6 parallel tests are performed. The relative deviation calculation formula is:

[0160] Relative deviation = (detection value - standard concentration) ÷ standard concentration x 100%.

[0161] 3) Determination results

[0162] According to the experimental data in Table 12, scatter plot (such as Figure 14 ) is drawn. When the detection range is 12-3000 μg / kg, the linear correlation coefficient R2 = 0.9933. The standard curve formula can be obtained from the following scatter plot:

[0163] Cadmium concentration (μg / kg): c = 10 (-1.9632×ogT / c+1)

[0164] According to the estimation method of the limit of quantification in GB 5009.295-2023 "General rules for verification of chemical analysis methods", the limit of quantification is equal to 3 times the detection limit, and the limit of quantification is set to 30 μg / kg. According to the requirements of correctness in this document, when the concentration content is 100 μg / kg-1000 mg / kg, the relative deviation requirement is -20% to +10%, so the upper limit concentration is set to 2600 μg / kg. And verify the accuracy and precision of 30 μg / kg, 200 μg / kg, 2600 μg / kg, as shown in Table 14.

[0165] Table 14

[0166]

[0167]

[0168] (3) The repeatability test of the reagent card according to the present application

[0169] 1) Precision test within the same batch

[0170] The upper limit, lower limit and concentration of interest within the quantitative detection range of each card in the same batch were detected respectively, and the relative standard deviation of the detection results was calculated. The batch precision evaluation of the cadmium time-resolved fluorescence immunochromatographic detection card was evaluated using the data determined in the previous part 2) of the standard curve range. The standard deviation (S) calculation formula is as follows:

[0171]

[0172] The relative standard deviation (RSD) calculation formula is as follows:

[0173]

[0174] 2) Precision test between different batches

[0175] Three batches of prepared detection reagent cards were used to detect high, low and concentration of interest of cadmium prepared in grain matrix, and each batch of card detected each concentration for 6 times, and the relative standard deviation of each batch was calculated.

[0176] 3) Verification results

[0177] The batch repeatability verification results are shown in Table 15:

[0178] Table 15

[0179]

[0180]

[0181] The repeatability relative deviation of the upper limit of the quantitative range is 19%, the repeatability relative deviation of the lower limit is 10%, which meets the repeatability requirements of GB5009.295-2023 "General rules for verification of chemical analysis methods", and the repeatability relative deviation of the concentration of interest is 12%, which is greater than the standard requirement of 10%.

[0182] (4) Cross reaction determination of the reagent card according to the present application

[0183] Prepare a chromium standard solution and a lead standard solution at 2.5 mg / mL, and a cadmium standard solution at 0.025 μg / mL. Detect the two heavy metal solutions with the cadmium fluorescent quantitative detection card, and record the detection results. The cadmium fluorescent quantitative detection card detects the 2.5 mg / mL lead and chromium standard, and the detection results show that both are less than 20 μg / kg. There is no cross-reaction at this concentration.

[0184] (5) The reagent card validity period determination of the application

[0185] Based on the Arrhenius equation recommended by EP25-A Stability Evaluation of In Vitro Diagnostic Reagents published by the United States CLSI, the validity period of the cadmium fluorescent quantitative detection card is predicted. The detection result drift of the cadmium fluorescent detection card needs to be tested under different high temperature conditions. According to the change of the series of detection values under different temperatures, the degradation reaction constant at each temperature is calculated, and then the storage temperature reaction constant is calculated according to different temperatures and reaction constants. The detection validity period of the reagent is deduced by the reaction constant of the storage temperature. The prepared cadmium fluorescent detection reagent card is placed at 25℃, 37℃, 42℃ and 56℃, respectively. After 4d, 14d, 24d and 34d of accelerated baking, it is taken out and stored in a 2-8℃ refrigerator. The standard substance of cadmium is detected at the same time, and the Cd concentration of interest (0.2 μg / kg) is detected. The standard is set as follows: the detection result cannot exceed 30% of the true concentration. Record the detection results as shown in Table 16.

[0186] Table 16

[0187]

[0188] First, calculate the slope (k) of the detection value change with time at each baking temperature in Table 15. The calculation method is to draw a scatter plot of the detection time (d) and the natural logarithm of the relative value of the detection value in excel, and obtain the slope ki at each temperature. Record it in Table 17. And according to the natural logarithm of ki and the reciprocal of temperature (converted into Kelvin), draw a scatter plot again, and obtain the corresponding relationship between reaction constant and temperature, as shown in Table 18. Figure 15

[0189] Table 17

[0190] Temperature (Kelvin) k Temperature reciprocal ln(k) 298.15 0.0034 0.003354 -5.68398 310.15 0.0058 0.003224 -5.1499 315.15 0.0077 0.003173 -4.86653 329.15 0.0201 0.003038 -3.90704

[0191] The corresponding relationship between the reaction constant and the temperature is as follows:

[0192] k = e^(-5.633.1 x 1 / Kelvin + 13.109)

[0193] ​Thus the reaction constant k at 4℃ (277k) is 0.000735 and at 8℃ (281k) is 0.000981.

[0194] According to Arrhenius derivation formula:

[0195]

[0196] Where t stab is the shelf life (d), k norm is the reaction constant, C i is the real-time value, C0is the original detection value, and according to the set qualification standard, the maximum acceptance is 30% deviation from the true value, so C i / C0is 0.7. Substituting the above calculation formula can deduce that the shelf life at 4-8℃ is 346d-485d. It is estimated that the shelf life of cadmium fluorescence detection reagent card at 4-8℃ is 1 year.

[0197] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the technical solutions. Those of ordinary skill in the art should understand that modifications or equivalent replacements to the technical solutions of the present application without departing from the purpose and scope of the technical solutions should be covered in the scope of the claims of the present application.

Claims

1. A method for rapid quantitative detection of cadmium element in grains, characterized in that, The specific steps are as follows: Step 1: After the grain sample is crushed, it is added to an acid solution, vortexed and centrifuged, and then the supernatant is taken and a chelating agent solution and a neutralizing agent are added to the supernatant, and the supernatant after chelation is collected; wherein the acid solution is a hydrochloric acid or nitric acid solution, and the concentration of the acid solution is 0.19-3 mol / L; the chelating agent is EDTA-2Na, and the concentration of EDTA-2Na in the chelating agent solution is 0.00001%-1%; the neutralizing agent is a Tris solution, and the concentration is 0.125 mol / L-2 mol / L; 4 mL of acid solution is added per 1 g of crushed grain sample; the volume ratio of the supernatant, the chelating agent solution and the neutralizing agent is 5:1:5; Step 2: The supernatant after step 1 is added dropwise to a cadmium time-resolved fluorescence immunochromatographic test reagent card for detection, a fluorescence analyzer is used to detect the fluorescence signal, and the cadmium concentration in the sample is calculated by comparing with a standard curve; Wherein, at one end of the cadmium time-resolved fluorescent immunochromatographic test reagent card, a sample pad is arranged, on one side of the sample pad and along the length direction of the chromatographic test reagent card, a marker pad, a chromatographic pad and a water absorption pad are arranged in sequence; on the marker pad, a cadmium antibody-fluorescent microsphere is coated, on the chromatographic pad, a T line and a C line are arranged, in the T line, a cadmium antigen is coated, and in the C line, a secondary antibody-fluorescent microsphere is coated; the cadmium antibody is a mouse monoclonal cadmium antibody, the cadmium antigen is Cd 2+ -EDTA-BSA.

2. The method of claim 1, wherein, In step 2, the cadmium antibody or secondary antibody is coupled with the fluorescent microspheres through a coupling reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-carboxyl thio succinimide.

3. The method of claim 2, wherein, The coupling reaction is as follows: S1: The fluorescent microsphere solution and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide are mixed in water, followed by the addition of N-carboxyl thio succinimide, and the reaction is carried out at 30-37°C for 10-15 min, and the solid product is collected by centrifugation; in the reaction system, the amount of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide added is 0.1-2 mg / mL, the amount of N-carboxyl thio succinimide added is 0.1-2 mg / mL, the solid content in the fluorescent microsphere solution is 1-3 wt%, and 1 mL of water is mixed with 200 μL of the fluorescent microsphere solution; S2: The solid product is dispersed and redissolved in phosphate buffer solution, and one of the cadmium antibody or the secondary antibody is added, and the reaction is carried out at 30-37°C for 1-1.5 h, and BSA is added and the reaction is continued for 30-40 min, and the solid material is collected after centrifugation, and is added to a marker storage solution, and a cadmium antibody fluorescent microsphere or a secondary antibody fluorescent microsphere containing solution is obtained, respectively; wherein 500 μL of phosphate buffer solution is added per 4 mg of solid product, and the mass ratio of the solid product, the cadmium antibody or the secondary antibody, and BSA is 4:0.1:5, and 500 μL of marker storage solution is added per 4 g of solid material.

4. The method of claim 3, wherein, The marker storage solution is: 0.5 g of BSA, 0.2 g of Pc300, and 0.5 mL of Tween-20 are added to 100 mL of phosphate buffer solution.

5. The method of claim 4, wherein, The pH of the phosphate buffer solution is 7.8, the phosphate ion concentration is 50 mmol / L, the mass concentration of BSA is 0.2-1.6 wt%, and the mass concentration of Tween-20 is 0.2-1.6 wt%.

6. The method of claim 1, wherein, The fluorescent microspheres have an emission light of 615 nm and an excitation spectrum of 360-410 nm; the carboxyl content on the surface of the microspheres is 205-350 μmol / g.

7. The method of claim 1 wherein, The secondary antibody is goat anti-mouse or rabbit anti-mouse.

8. The method of claim 1, wherein, In step 2, the coating concentration of the cadmium antigen-fluorescent microspheres on the T line is 0.2 mg / mL-1.6 mg / mL.

9. The method of claim 1 wherein, In step 2, the chromatography temperature is 20-25°C, the chromatography time is 10-20 min, and the liquid volume dropped on the reagent card is 60-120 μL.

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