Method for detecting heavy metal cadmium in fresh vegetables by using immune colloidal gold method

Abstract

The invention relates to the technical field of food safety rapid detection, in particular to a method for detecting heavy metal cadmium in fresh vegetables by using an immune colloidal gold method, which comprises the following steps: 1, preparing a liquid to be detected, and 2, detecting the liquid to be detected by using a detection card. The method for preparing the to-be-detected liquid specifically comprises the following steps: S1, weighing a to-be-detected sample, putting the to-be-detected sample into a centrifugal tube M, slowly adding a reagent A into the centrifugal tube M, uniformly mixing, adding a reagent B, oscillating for 0.5-3 minutes, uniformly mixing, and standing to form clear liquid; s2, sucking clear liquid in the centrifugal tube M into a centrifugal tube N by using a dropper, adding a reagent C, and uniformly mixing to form to-be-detected liquid. The method is high in detection sensitivity, small in error and low in probability of false positive, false negative and invalid detection; meanwhile, the method is simple to operate, does not need expensive instruments, has low requirements on operators, experimental environments and the like, is low in detection cost, is suitable for real-time detection on a base layer and a site, and is wider in application range.

Description

Technical Field

[0001] The invention relates to the technical field of rapid food safety detection, and in particular to a method for detecting heavy metal cadmium in fresh vegetables by utilizing an immune colloidal gold method. Background Art

[0002] Cadmium (Cd) is a non-essential element for the human body and one of the most toxic heavy metal elements. Cadmium is cumulative and difficult to eliminate. Cadmium can enter the body through air, water, food, etc., and can accumulate in organs such as bones, kidneys and liver, eventually inducing orthopedic diseases such as osteomalacia, osteoporosis, humeral pain, etc. It can also induce renal function decline or damage, anemia, and even cause cancer in severe cases.

[0003] At present, the main detection methods for heavy metal cadmium include atomic fluorescence spectrometry (AFS), atomic absorption spectrophotometry (AAS), inductively coupled plasma emission spectrometry, etc. Among them, atomic fluorescence spectrometry has high sensitivity, a wide linear range of the calibration curve, and can perform simultaneous determination of multiple elements; however, the intensity of atomic fluorescence is low, and a high-intensity excitation light source is required to obtain the fluorescence intensity required for determination; in addition, due to weak fluorescence, stray light has a greater impact on the analysis and detection process, which reduces the accuracy and reliability of the test results. Atomic absorption spectrophotometry can only analyze the content of inorganic elements, and cannot be directly used for content analysis and structural analysis of organic compounds; and, for each element measured by conventional atomic absorption spectrophotometry, the hollow cathode lamp light source must be replaced, and multi-element analysis cannot be performed simultaneously. Inductively coupled plasma emission spectrometry has the advantages of fast analysis speed, high sensitivity, high accuracy and precision, and a wide range of determination, but this method has high requirements for the equipment used and high operating costs. In addition, the requirements for standard reference components are also relatively high. The above traditional methods require high instrument and detection costs, and have high requirements for operators, experimental environment, and pre-treatment. They are not suitable for real-time online detection at the grassroots level and cannot meet the needs of grassroots hospitals, grassroots environmental monitoring agencies, and food testing agencies.

[0004] Colloidal gold is a solution composed of nanoparticles, whose surface easily adsorbs some compounds or biological molecules to form the so-called "colloidal gold marker". Based on this principle, the immunocolloidal gold detection method detects the target substance in the sample through immune reaction by covalently binding specific antibodies and other molecules to colloidal gold particles, forming nanoparticle aggregates with visible colors, thereby achieving rapid, sensitive and visual detection of certain biological molecules.

[0005] At present, the immunocolloidal gold method can not only be used for the detection of heavy metal lead in dairy products, but also for the rapid quantitative detection of heavy metal cadmium residues in food such as grains. However, the following problems often occur in the process of using the existing immunocolloidal gold method to detect heavy metal cadmium in fresh vegetables: 1. The test results of fresh vegetables (except leafy vegetables, legumes, root and tuber vegetables, stem vegetables, and day lily) cannot be unified with the detection limit specified in "GB2762-2022 National Food Safety Standard Limits of Contaminants in Food", that is, the detection sensitivity is poor; 2. The test results of different matrices with the same detection limit are different, that is, the false negative and false positive rates of the test results of different matrices with the same method are high, especially for ginger, the probability of false positive test results is very high; 3. Invalid cards may appear during acid matrix testing.

[0006] According to the contents recorded in "GB2762 2022 National Food Safety Standard-Limits of Contaminants in Food", the detection limits for vegetables are shown in Table 1.

[0007] Table 1. Detection limits of different types of vegetables

[0008] Therefore, there is an urgent need to develop a method that can be fast, convenient and suitable for detecting whether the heavy metal element cadmium remains in fresh vegetables.

[0009] The invention provides a method for detecting heavy metal cadmium in fresh vegetables by utilizing an immune colloidal gold method, so as to solve the problems of poor sensitivity, high probability of false negative or false positive in detection results and high probability of invalid detection in the detection methods in the prior art. Summary of the invention

[0010] The purpose of the present invention is to provide a method for detecting heavy metal cadmium in fresh vegetables by using the immunocolloidal gold method, so as to solve the problems of poor sensitivity, high false negative and false positive rates of the detection results, many invalid cards and small application range of the detection methods in the prior art.

[0011] The technical scheme of the present invention is: a method for detecting heavy metal cadmium in fresh vegetables by using immunocolloidal gold method, comprising: step one, preparing a liquid to be tested; step two, using standby consumables to detect the liquid to be tested; The step of preparing the liquid to be tested specifically includes the following steps: S1. Weigh the sample to be tested and place it in a centrifuge tube M. Slowly add reagent A to the centrifuge tube M. After mixing evenly, add reagent B and shake for 0.5-3 minutes. After mixing evenly, let it stand to form a clear liquid. The reagent A is an acidic solution; the reagent B is a chelating agent solution; S2. Use a dropper to draw the clarified liquid in centrifuge tube M into centrifuge tube N, add reagent C, mix well, and prepare the liquid to be tested; The reagent C is a neutralizing reagent; the neutralizing reagent is an alkaline solution or a neutral buffer solution.

[0012] Preferably, the acidic solution is any one of concentrated sulfuric acid solution, concentrated nitric acid solution, acetic acid solution, polybasic organic acid solution, and a mixture of a polybasic organic acid solution and an optimized reagent; the polybasic organic acid solution is any one of citric acid solution, tartaric acid solution, and malic acid solution; and the optimized reagent is ultrapure water or an inorganic weak base salt solution.

[0013] Preferably, the optimization reagent is an inorganic weak alkaline salt solution; in the mixed solution of the polybasic organic acid solution and the optimization reagent, the volume ratio of the polybasic organic acid solution to the optimization reagent is (2.0-4.0):1; The inorganic weak alkaline salt solution is an aqueous solution of any one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, and sodium bicarbonate.

[0014] Preferably, in the mixed solution of the polybasic organic acid solution and the optimization reagent, the concentration of the polybasic organic acid solution is 0.03-0.1 mol / L, and the concentration of the optimization reagent is 0.05-0.4 mol / L.

[0015] Preferably, the chelating agent solution is a DMSO solution of Isothiocyanobenzyl-EDTA or an aqueous solution of EDTASOLUTINON and sodium hydroxide.

[0016] Preferably, the neutralizing agent is an alkaline solution; the alkaline solution is any one of 0.01-0.2 mol / L sodium hydroxide solution, 0.01-0.5 mol / L dipotassium hydrogen phosphate solution, 0.01-0.5 mol / L potassium dihydrogen phosphate solution, saturated sodium bicarbonate solution, 0.01-0.4 mol / L potassium carbonate solution, and 0.01-0.4 mol / L sodium carbonate solution.

[0017] Preferably, the neutralizing agent is a neutral buffer solution; the neutral buffer solution is composed of any one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, sodium chloride in combination with a surfactant and a preservative.

[0018] Preferably, in step S1, the volume of the reagent A added is 1-3 mL, and the volume of the reagent B added is 20-500 μL; In the step S2, the volume of the clear liquid sucked is 25-300 μL; the volume of the reagent C added is 0-1.5 mL.

[0019] Preferably, in step S2, the pH value of the prepared liquid to be tested is 6-7.5.

[0020] Compared with the prior art, the advantages of the present invention are: (1) The present application provides a method for detecting heavy metal cadmium in fresh vegetables using the immunocolloidal gold method. The method first prepares a test liquid using a suitable scheme according to the type and characteristics of the vegetables, and then uses the standby consumables packaged in an aluminum foil bag to detect the test liquid; the method is simple to operate and does not require the use of expensive instruments. It has low requirements on operators, experimental environment, etc., and has a low detection cost. It is suitable for real-time detection at the grassroots level and on-site, and has a wider range of applications; at the same time, when the method is used to detect whether fresh vegetables contain the heavy metal element cadmium, the detection result has high sensitivity, small error, and extremely low probability of false positive, false negative, and invalid detection. It can solve the problems of poor sensitivity, high probability of false negative or false positive in the detection result, and high probability of invalid detection in the detection method in the prior art.

[0021] (2) The present application provides a method for detecting heavy metal cadmium in fresh vegetables using the immunocolloidal gold method. This method can be used according to the type and characteristics of the vegetables. When preparing the liquid to be tested, the concentration of the acidic substance in the liquid to be tested or the content of reagent B can be regulated by selecting and using appropriate reagents A, B, and C, and by adjusting the concentrations of reagents A, B, and C and the amount of reagents B and C added, so that the test results have high accuracy; improve the sensitivity of the test, and reduce the probability of false positives, false negatives, and invalid tests in the test results; at the same time, the test results of different matrices with the same detection limit are basically consistent, thereby making the method have excellent stability. In addition, the detection method can also quickly and conveniently detect whether various vegetables contain the heavy metal element cadmium, expanding the scope of application of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below in conjunction with the accompanying drawings and embodiments: Figure 1 A flow chart of preparing the liquid to be tested according to the present invention; Figure 2 This is a display diagram of the test results in Example 40 of the present invention; DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments: A method for detecting heavy metal cadmium in fresh vegetables by using an immune colloidal gold method comprises: step one, preparing a liquid to be tested, and step two, using standby consumables to test the liquid to be tested; wherein the liquid to be tested needs to be restored to room temperature before testing.

[0024] The preparation of the liquid to be tested specifically includes the following: Figure 1 As shown, S1. Weigh an appropriate amount of the vegetable sample to be tested and place it in a centrifuge tube M. Slowly add 1-3 mL of reagent A to the centrifuge tube M. After shaking and mixing, slowly add 20-500 μL of reagent B to the centrifuge tube M, shake for 0.5-3 min, and let stand after mixing to form a clear liquid; wherein reagent A is an acidic solution, and the acidic solution can be an inorganic acid solution, a monobasic organic acid solution, a polybasic organic acid solution, or a mixture of a polybasic organic acid solution and an optimized reagent; the inorganic acid solution is one of concentrated sulfuric acid solution, concentrated nitric acid solution, hydrochloric acid, etc., the monobasic organic acid solution is preferably an acetic acid solution, and the polybasic organic acid solution is one of citric acid solution, tartaric acid solution, malic acid solution, etc.; the optimized reagent is ultrapure water, an aqueous solution of an inorganic salt, etc.; reagent B is a chelating agent solution; S2. Use a dropper to draw 25-300 μL of the clear liquid in the centrifuge tube M, and drop it into the centrifuge tube N. Then use a dropper to draw 0-1.5 mL of reagent C, and slowly drop the reagent C into the centrifuge tube N, shake it to mix it evenly, and prepare a liquid to be tested; wherein the reagent C is a neutralizing reagent, and the neutralizing reagent is preferably an alkaline solution or a neutral buffer solution; the pH value of the prepared liquid to be tested is 6-7.5.

[0025] Place the prepared liquid to be tested on the operating table, wait for it to cool naturally and return to room temperature, and then test it. The testing operation specifically includes the following steps: a. Place the consumables packaged in aluminum foil bags on the operating table and wait for them to return to room temperature (20°C-30°C). Then take out the consumables from the aluminum foil bags and place them horizontally in front of the observer. The consumables packaged in aluminum foil bags include test cards, gold-labeled microwells, droppers, etc. The test cards are also provided with sample addition holes and test areas, and the test areas are provided with control lines (C lines) and test lines (T lines). b. Use a dropper to draw 120 μL of the liquid to be tested, and drop the drawn liquid into the gold-labeled microwell. Slowly draw the liquid to be tested to fully mix with the red substance at the bottom of the gold-labeled microwell, and let it stand horizontally for 2 minutes to wait for the reaction. c. After the reaction is over, aspirate the solution in the gold-labeled microwell, gently blow for 10 seconds, and then drop it into the sample well on the test card for sample addition detection. Start timing after sample addition; d. Within 8 minutes after the start of the timing, read the result based on the color of the control line (C line) and the test line (T line) in the test card.

[0026] The specific judgment method of the results on the test card is: if the color of the T line is stronger than that of the C line or there is no obvious difference with the color of the C line, or the T / C ratio of the gold label card reader is ≥1.0, it means that the sample does not contain the analyte or its concentration is lower than the detection limit, that is, the test result of the sample is negative; if the color of the T line is significantly weaker than that of the C line or the T line does not show color, or the T / C ratio of the gold label card reader is <1.0, it means that the concentration of the analyte in the sample is equal to or higher than the detection limit, that is, the test result of the sample is positive; if no purple-red strips appear on the C line or the T line, it means that the operation process is incorrect or the test card has deteriorated and damaged. In this case, you should read the instructions carefully again and retest with a new test card.

[0027] 1. Preparation of Reagent B There are many kinds of chelating agents currently on the market. Since chelating agents have the ability to selectively bind to certain metal ions, the chelating agent solutions prepared by different chelating agents have great differences in the complexing ability with different metal atoms or ions. Therefore, the present invention first needs to prepare a chelating agent solution with good binding ability with heavy metal cadmium in ultrapure water as reagent B. In order to reduce experimental errors, each experimental group prepares the liquid to be tested and a blank sample for detection; at the same time, in order to avoid interference of other metal ions in pure water on the experimental results, ultrapure water is used in the following specific embodiments. Example 1

[0028] According to the characteristics of the chelating agent, EDTA SOLUTION powder is selected as the chelating agent in this embodiment, and ultrapure water is used as the solvent to prepare a chelating agent solution as reagent B; wherein the chelating agent solution is formed by adding 0.5 mmol of the chelating agent to 500 mL of ultrapure water and mixing; but a small amount of chelating agent is precipitated in the chelating agent solution.

[0029] Take 1 mL of ultrapure water into centrifuge tube M, add 20 μL of heavy metal cadmium standard (10 μg / ml) into centrifuge tube M, shake and mix evenly, then add 50 μl of reagent B into centrifuge tube M; shake and mix for 1 minute and let it stand to form the liquid to be tested, P1.

[0030] Take 1 mL of ultrapure water into centrifuge tube M, add 50 μl of reagent B into centrifuge tube M; shake and mix for 1 minute and let stand to form a blank sample, Q1.

[0031] Place the liquid P1 to be tested and the consumables to be used packaged in an aluminum foil bag on the operating table. When the liquid P1 to be tested and the consumables to be used have returned to room temperature, take out the consumables to be used from the aluminum foil bag and place them horizontally in front of the observer; use a dropper to draw 120 μL of the liquid P1 to be tested, and drop the drawn liquid P1 into the gold-labeled microwell, slowly aspirate to allow the liquid to be tested to fully mix with the red substance at the bottom of the gold-labeled microwell, and let it stand horizontally for 2 minutes to wait for the reaction; after the reaction is completed, draw the solution in the gold-labeled microwell, gently blow for 10 seconds, and then drop it into the sample well on the test card for detection, and start timing after the sample is added; at the 8th minute after the timing starts, read the result based on the color development of the C line and the test line T line in the test card. At the same time, the blank sample Q1 is detected using the above detection method.

[0032] The test results show that the blank sample Q1 has an invalid card phenomenon. After retesting, the results are the same, which can rule out the problem of the test card. It is speculated that there is a small amount of chelating agent powder in the blank sample Q1, which will interfere with the colloidal gold, thus causing the test result to be invalid. Since the blank sample Q1 has an invalid card phenomenon, the test result of the liquid P1 to be tested has no reference significance.

[0033] EDTA SOLUTION powder is used as a chelating agent, and an aqueous solution of sodium hydroxide is used as a solvent; EDTA2Na powder is used as a chelating agent, and an ultrapure aqueous solution is used as a solvent; Isothiocyanobenzyl-EDTA powder is used as a chelating agent, and DMSO (dimethyl sulfoxide) is used as a solvent, etc. to prepare a chelating agent solution as reagent B, such as Example 2-4; and the chelating agent solution prepared, wherein the chelating agent is completely dissolved in the solvent. The liquid to be tested and the blank sample are prepared in the same manner as in Example 1, and the test is performed in the same manner as in Example 1; the test results are shown in Table 2.

[0034] Table 2. Comparison of test results of Examples 1-4

[0035] As shown in Table 2, embodiment 2 is compared with embodiment 1, and the phenomenon of invalid card does not occur in embodiment 2, indicating that the dissolution degree of chelating agent in solvent will cause certain influence on detection result, confirming the above-mentioned speculation. The test result of blank sample in embodiment 2 and embodiment 3 is all negative, and the detection result of liquid to be inspected is shown as weak positive or weak negative, indicating that the chelating agent solution prepared by EDTA SOLUTION and EDTA 2Na is as reagent B, for metal cadmium in complexing ultrapure water, and its complexing ability is all poor, but EDTA SOLUTION is as chelating agent, and the complexing ability of its chelating agent solution is slightly better than the complexing ability of chelating agent solution prepared by EDTA 2Na as chelating agent; and it is found by research that the concentration of EDTASOLUTION chelating agent solution is appropriately increased, and the complexing ability of chelating agent solution can be improved; embodiment 4 is compared with embodiment 1-3, and the complexing ability of its chelating agent solution is better; therefore, the DMSO solution of Isothiocyanobenzyl-EDTA is used as chelating agent solution in specific embodiment.

[0036] 2. Preparation of Reagent A The following examples all use the chelating reagent solution prepared in the above-mentioned Example 4 as reagent B; at least two vegetables with the same detection limit and no heavy metal cadmium are selected as matrices to prepare the liquid to be tested and the blank sample, and heavy metal cadmium standards are added when preparing the liquid to be tested, and comparative detection experiments are performed. Comparative experiments are also helpful in determining whether the test results of different vegetables with the same detection limit are consistent when the liquid to be tested is prepared using the same solution. The following examples all use broccoli, cowpea and potato as matrices, respectively, and 50 μl of a heavy metal cadmium standard with a concentration of 1 μg / ml is added when preparing the liquid to be tested. Example 5

[0037] Weigh 1 ml of concentrated sulfuric acid and prepare it with ultrapure water to form a 1 mol / L sulfuric acid solution as reagent A.

[0038] Weigh 1g of broccoli into centrifuge tube M, and add 50μl of heavy metal cadmium standard (1μg / ml) into centrifuge tube M; then, add 2ml of reagent A, shake and mix, then add 0.1ml of reagent B, shake and mix for 1 minute and let stand to form a clear liquid; take 100μl of the clear liquid in the centrifuge tube into centrifuge tube N, add 500μl of 0.2mol / L sodium hydroxide, mix well, and form the liquid to be tested, P5-1.

[0039] Weigh 1g of broccoli into centrifuge tube M, add 2ml of reagent A, shake and mix, then add 0.1ml of reagent B, shake and mix for 1 minute and let stand to form a clear liquid; take 100μl of the clear liquid in the centrifuge tube into centrifuge tube N, add 500μl of 0.2mol / L sodium hydroxide, mix well, and form a blank sample, Q5-1.

[0040] Weigh 1 g of cowpea respectively, and prepare the test liquid P5-2 and blank sample Q5-2 using the same scheme as the preparation of the test liquid P5-1 and blank sample Q5-1.

[0041] Weigh 1 g of potato respectively, and prepare the test liquid P5-3 and blank sample Q5-3 using the same scheme as the preparation of the test liquid P5-1 and blank sample Q5-1.

[0042] The blank sample and the liquid to be tested were tested respectively by the same detection method as in Example 1; the test results showed that an invalid card appeared in the blank sample, and the results were the same after retesting, so the problem of the test card could be ruled out; it was speculated that the content of sulfuric acid in the blank sample was relatively high, which made the acidity of the blank sample too high, resulting in the inactivation of the colloidal gold, and thus the invalid test result.

[0043] In Examples 6-16, the difference from Example 5 in preparing the liquid to be tested and the blank sample is that in Examples 6-16, different acidic substances are used to prepare acidic solutions, or the same acidic substance is used to prepare acidic solutions of different concentrations as reagent A; sodium hydroxide solutions of different concentrations are used as reagent C, or different doses of reagent C are added. After the preparation of the liquid to be tested and the blank sample is completed, the liquid to be tested and the blank sample are tested using the same detection method as in Example 5. The test results are shown in Table 3.

[0044] Table 3 Comparison of test results of Examples 6-16

[0045] As shown in Table 3, compared with Examples 7-5, it can be seen that the content of sulfuric acid in blank samples is high in Examples 8 and 9, which will lead to the phenomenon of invalid cards in the test results; increasing the dosage of reagent C or reducing the concentration of reagent A can reduce the content of sulfuric acid in the blank sample, and can weaken the influence of sulfuric acid in the blank sample on colloidal gold, thereby reducing the probability of invalid cards in the test results; compared with Example 9, Example 10 reduces the concentration of acidic substances in reagent A, which can reduce the content of acidic substances in the blank sample, and its test results do not have the phenomenon of invalid cards, confirming the above inference. Similarly, it can be deduced that the acidic matrix will also affect the test results during the detection process, and the phenomenon of invalid cards will appear during the detection, and the interference caused by the matrix itself can be eliminated by increasing the dosage of reagent C.

[0046] In addition, when Example 10 is compared with Examples 5-9, when a highly acidic or highly concentrated inorganic acid solution, such as a sulfuric acid solution, is selected as Reagent A, a highly alkaline solution should be used as Reagent C when preparing the liquid to be tested and the blank sample, and more Reagent C should be added to adjust the content of acidic substances in the liquid to be tested and the blank sample. The amount of Reagent C added is difficult to control, which leads to the difficulty in adjusting the pH value of the liquid to be tested and the blank sample, and the accuracy and stability of the test results are also poor. The reason why the test results of the liquid to be tested in Example 10 are both negative and positive at the same time may be that when preparing the sulfuric acid solution, part of the concentrated sulfuric acid will remain on the pipette, resulting in a deviation in the concentration of the prepared sulfuric acid solution, which leads to uncertainty in the content of sulfuric acid in the liquid to be tested, and uncertainty in the pH value of the liquid to be tested, which causes fluctuations in the test results, resulting in poor consistency in the test results of different vegetables in the same detection limit. In order to improve the consistency of the test results, further research was conducted through experiments. The experimental results showed that the concentration of sulfuric acid solution should be in the range of 0.01-0.05mol / L; Reagent C preferably uses a strong alkaline solution, such as sodium hydroxide solution, potassium hydroxide solution, etc., and the concentration of the alkaline solution is 0.05-0.1mol / L, and the addition amount is 50-1000μl. Through experimental research, it can be seen that other inorganic acid solutions, such as nitric acid solution, hydrochloric acid solution, etc., as reagent A, have the same characteristics as concentrated sulfuric acid solution; and in order to improve the sensitivity and consistency of the test results, the concentration of nitric acid solution should also be in the range of 0.01-0.05mol / L.

[0047] Compared with Examples 7-10, Examples 11-13 and 14-16 use citric acid as reagent A to prepare the liquid to be tested and the blank sample, and the sensitivity and stability of the test results are significantly better than those using sulfuric acid solution and glacial acetic acid solution as reagent A, and this conclusion can be verified by multiple groups of experiments; it can be seen that the liquid to be tested and the blank sample prepared using polybasic organic acid solution as reagent A are more sensitive and stable than inorganic acid solution or monobasic organic acid solution as reagent A. Through experimental research, it can be seen that other polybasic organic acid solutions, such as malic acid solution, tartaric acid solution, etc., as reagent A, have the same characteristics as citric acid solution. At the same time, through experimental research, it can be deduced that the concentration of the organic polybasic acid solution is preferably 0.01-0.1 mol / L; and the study found that when the organic polybasic acid solution is used as reagent A, the selection of reagent C, the concentration of reagent C, and the amount of addition have a greater impact on the pH value of the blank sample and the liquid to be tested.

[0048] 3. Effect of Reagent C on Test Results At least two vegetables with the same detection limit and no heavy metal cadmium are selected as matrices, and 0.05 mol / L citric acid solution is used as reagent A, and the chelating reagent solution prepared in Example 4 is used as reagent B. Sodium hydroxide solution of different concentrations, potassium dihydrogen phosphate solution of different concentrations, and neutral buffer solution are used as reagent C to prepare the liquid to be tested and blank samples for detection. The following examples all use broccoli, cowpea, and potato as matrices, and 50 μl of heavy metal cadmium standard with a concentration of 1 μg / ml is added when preparing the liquid to be tested. Among them, the neutral buffer solution is an aqueous solution formed by disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, a surfactant, and a preservative. Embodiment 17

[0049] 1 g of sodium hydroxide was weighed and prepared with ultrapure water to form 0.1 mol / L sodium hydroxide, which was used as reagent C.

[0050] Weigh 1g of broccoli into centrifuge tube M, and add 50μl of heavy metal cadmium standard to centrifuge tube M; then, add 2ml of reagent A, shake and mix, then add 0.1ml of reagent B, shake and mix for 1 minute and let stand to form a clear liquid; take 100μl of the clear liquid in the centrifuge tube into centrifuge tube N, add 30μl of reagent C, mix evenly to form the liquid to be tested, P17-1.

[0051] Weigh 1g of broccoli into centrifuge tube M, add 2ml of reagent A, shake and mix, then add 0.1ml of reagent B, shake and mix for 1 minute and let stand to form a clear liquid; take 100μl of the clear liquid in the centrifuge tube into centrifuge tube N, add 30μl of reagent C, mix well to form a blank sample, Q17-1.

[0052] Weigh 1 g of cowpea respectively, and prepare the test liquid P17-2 and blank sample Q17-2 using the same scheme as the preparation of the test liquid P17-1 and blank sample Q17-1.

[0053] Weigh 1 g of potato respectively, and prepare the test liquid P17-3 and blank sample Q17-3 using the same scheme as the preparation of the test liquid P17-1 and blank sample Q17-1.

[0054] The same detection method as in Example 1 was used to detect the blank sample and the liquid to be tested. The test results showed that the blank samples were all negative, while the test results of the liquid to be tested showed both positive and weak positive results. The results were the same after retesting, and the problem with the test card could be ruled out. The results showed that the test results of different vegetables with the same detection limit for the liquid to be tested were poorly reproducible.

[0055] In Examples 18-24, the difference from Example 17 in preparing the test liquid and blank sample is that the reagent C and / or the amount of reagent C used are different. After the test liquid and blank sample are prepared, the test liquid and blank sample are tested using the same detection method as in Example 5. The test results are shown in Table 4.

[0056] Table 4. Comparison of test results of Examples 17-24

[0057] As shown in Table 4, the test results of the liquid to be tested and the blank sample prepared with alkaline solution and neutral buffer as reagent C are accurate to a certain extent; however, the test results of the liquid to be tested prepared with different vegetables with the same detection limit as the matrix using 0.02mol / L and 0.05mol / L sodium hydroxide solution as reagent C are inconsistent; and the test results of the liquid to be tested prepared with 0.1mol / L sodium hydroxide solution as reagent C are poor in repeatability of the test results of the same vegetable; the liquid to be tested and the blank sample prepared with weakly alkaline potassium hydrogen phosphate solution and neutral buffer as reagent C do not have the above problems; it is speculated that the alkalinity of sodium hydroxide solution is relatively strong, and the pH value of the prepared liquid to be tested is unstable when sodium hydroxide solution is used as reagent C to neutralize reagent A, which leads to unstable test results. It can be seen from this that when citric acid solution is used as reagent A, weakly alkaline solution and neutral buffer are preferred as reagent C; and the concentration and dosage of reagent C can be optimized according to the reagent A used. At the same time, compared with Example 24, Example 23 shows that when the amount of neutral buffer added is less, the phenomenon of invalid cards will appear in the test results. Increasing the amount of neutral buffer added can effectively avoid the phenomenon of invalid cards and improve the consistency of the test results; it can be seen that the more reagent C is added, the lower the concentration of acidic substances in the liquid to be tested, which also helps to weaken the influence of different vegetables on the colloidal gold test card, improve the stability and sensitivity of the test results of different vegetables with the same detection limit, and reduce the difference of the test results of different vegetables with the same detection limit. At the same time, in order to further improve the sensitivity of the test results, it is also necessary to optimize the citric acid solution. Among them, the neutral buffer can also be an aqueous solution formed by other inorganic salts, surfactants and preservatives.

[0058] 4. Optimization of polybasic organic acid solution In the following examples, vegetables with the same detection limit, such as water spinach, leek, cabbage, etc., which do not contain heavy metal cadmium, are selected as detection matrices to prepare the liquid to be tested and the blank sample, and 50 μl of a heavy metal cadmium standard with a concentration of 2 μg / ml is added when preparing the liquid to be tested; at the same time, the chelating reagent solution prepared in Example 4 is used as reagent B, the neutral buffer is used as reagent C, and a mixed solution of 0.05 mol / L citric acid solution and the optimized reagent is used as reagent A to prepare the liquid to be tested and the blank sample for detection; the detection results are shown in Table 5.

[0059] Table 5. Comparison of test results of Examples 25-36

[0060] Examples 25-36 use ultrapure water, disodium hydrogen phosphate solution, and sodium chloride solution as optimized reagents, respectively, and mix them with citric acid solution in a certain proportion as reagent A to prepare liquids to be tested and blank samples. As shown in Table 5, the test results of the liquids to be tested and blank samples prepared by using disodium hydrogen phosphate solution and sodium chloride solution as optimized reagents and mixing them with citric acid solution in a certain proportion as reagent A have good stability and accuracy, and high sensitivity; and the stability of the test results of disodium hydrogen phosphate solution as the optimized reagent is higher; at the same time, it can be verified through experiments that the test results of the liquids to be tested and blank samples prepared by using dipotassium hydrogen phosphate solution, sodium dihydrogen phosphate solution, potassium dihydrogen phosphate solution, sodium carbonate solution, sodium bicarbonate solution and other inorganic weak alkaline salt solutions as optimized reagents and mixing them with citric acid solution in a certain proportion as reagent A also have good stability, accuracy and high sensitivity. It can be seen that using ultrapure water and inorganic weak base salt solution as optimization reagents to adjust the concentration of citric acid can effectively improve the sensitivity and stability of the detection results; and when citric acid is mixed with an inorganic weak base salt solution, the concentration of the citric acid solution is preferably 0.03-0.1 mol / L, and the concentration of the weak alkaline solution is preferably 0.05-0.4 mol / L; the volume ratio of the citric acid solution to the inorganic weak base salt solution is preferably (2-4): 1. Among them, the weak base solution can also be an aqueous solution containing two or more weak base salts, for example, an aqueous solution containing dipotassium hydrogen phosphate and potassium dihydrogen phosphate. In addition, through experimental studies, it can be seen that the optimization methods and characteristics of other polybasic organic acid solutions, such as malic acid solution, tartaric acid solution, etc., are similar to those of citric acid solution.

[0061] 5. Factors affecting the sensitivity of test results Because ginger is prone to false positive or false negative during the detection process, the following specific examples use ginger that does not contain the heavy metal element cadmium as the debugging basic matrix; and a 0.05 mol / L citric acid solution and a 0.2 mol / L potassium dihydrogen phosphate solution with a volume ratio of 2.5:1 are evenly mixed as reagent A, the chelating reagent solution prepared in Example 4 is used as reagent B, and a neutral buffer solution is used as reagent C to prepare the liquid to be tested and a blank sample, and 50 μl of a heavy metal cadmium standard with a concentration of 1 μg / ml is added when preparing the liquid to be tested, and a detection experiment is carried out. Embodiment 37

[0062] Weigh 0.5g of the ginger sample to be tested into centrifuge tube M, and add 50μl of heavy metal cadmium standard with a concentration of 1μg / ml into centrifuge tube M; then add 2mL of reagent A to centrifuge tube M, and then add 25μL of reagent B, shake and mix for 1 minute and let it stand to form a clear liquid; take 200μL of the clear liquid in centrifuge tube M into centrifuge tube N, and add 100μL of reagent C to centrifuge tube N, mix evenly, and form the liquid to be tested, P37.

[0063] Weigh 0.5 g of the ginger sample to be tested into a centrifuge tube M, add 2 mL of reagent A to the centrifuge tube M, then add 25 μL of reagent B, shake and mix for 1 minute and let stand to form a clear liquid; take 200 μL of the clear liquid in the centrifuge tube M into a centrifuge tube N, and add 100 μL of reagent C to the centrifuge tube N, mix well to form a blank sample, Q37.

[0064] The same detection method as in Example 1 is used to detect the liquid to be tested P37 and the blank sample Q37, respectively, and the results displayed on the detection card are read. At the same time, the detection results are read using a gold label detector.

[0065] The difference between Examples 38-42 and Example 37 is that different amounts of reagent B and reagent C are added when preparing the liquid to be tested and the blank sample; the test results are shown in Table 6.

[0066] Table 6. Comparison of test results of Examples 37-42

[0067] As shown in Table 6, Example 38 increases the dosage of reagent C on the basis of Example 37, and Example 39 further increases the dosage of reagent C on the basis of Example 38; Comparing the test results of Example 39 with those of Example 37 and Example 38, it can be seen that the higher the citric acid content in the blank sample and the liquid to be tested, the greater the difference between the T / C value of the blank sample and the T / C value of the liquid to be tested, and the higher the sensitivity of the test result, that is, the higher the T / C value of the blank sample and the smaller the T / C value of the liquid to be tested, the higher the sensitivity of the test; and, through a large number of experimental studies, it can be seen that when the T / C value of the blank sample is not less than 1.6 and the T / C value of the liquid to be tested is not higher than 0.7, the consistency of the test results is good and the sensitivity is high. At the same time, comparing the test results of Example 39 with those of Example 37 and Example 38, it can be seen that the concentration of the acidic substance in the blank sample and the sample to be tested, the stronger the T line color, the stronger the negative signal displayed on the test card, and the sensitivity of the test result will also decrease accordingly.

[0068] Examples 40-42 gradually increase the amount of reagent B on the basis of Example 37. Compared with the test results of Example 37, it can be seen that as the amount of reagent B added gradually increases, the difference between the T / C value of the blank sample and the T / C value of the liquid to be tested gradually decreases, and the sensitivity of the test result also gradually decreases; at the same time, it can be seen that as the amount of reagent B added gradually increases, the T / C value of the blank sample decreases significantly, and even becomes less than 1, that is, a false positive phenomenon occurs in the test result; and when detecting the spiked liquid to be tested, the T / C value of the liquid to be tested also gradually decreases; further, it can be explained that the higher the concentration of reagent B in the sample to be tested within a certain range, the weaker the T line color, the stronger the positive signal displayed on the test card, and the worse the sensitivity of the test result. Among them, the T / C value obtained from the test and the Figure 2 As shown, the liquid to be tested prepared in Example 40 has the highest sensitivity in the test results.

[0069] In summary, when using the immunocolloidal gold method to detect whether vegetables contain the heavy metal element cadmium, the stability and sensitivity of the test results can be adjusted by adjusting the content of acidic substances and the content of chelating reagents in the liquid to be tested.

[0070] Vegetables other than ginger were randomly selected as matrices, and 0.05 mol / L citric acid solution and 0.2 mol / L potassium dihydrogen phosphate solution in a volume ratio of 3:1 were evenly mixed and used as reagent A; the chelating reagent solution prepared in Example 4 was used as reagent B; a neutral buffer solution was used as reagent C; a liquid to be tested and a blank sample were prepared, and 50 μl of a heavy metal cadmium standard was added when preparing the liquid to be tested, and a verification experiment was carried out; and repeated experiments and analyses were carried out on different matrices with the same detection limit.

[0071] For matrices that are fresh vegetables (leafy vegetables, legumes, root and tuber vegetables, stem vegetables, and day lilies except), such as green peppers and tomatoes, prepare the test liquid and blank samples according to the following scheme; Weigh 1g of the sample to be tested into centrifuge tube M, add 2mL of reagent A to centrifuge tube M, then add 0.1mL of reagent B, shake and mix for 1 minute, take 100μL of the clear liquid in centrifuge tube M to centrifuge tube N, add 500μL of reagent C, mix well to form the liquid to be tested.

[0072] For the matrix vegetables broccoli, cowpea and potato belonging to legume vegetables, root and tuber vegetables, and stem vegetables (except ginger and celery), prepare the liquid to be tested and the blank sample according to the following scheme; Weigh 0.5 g of the sample to be tested into centrifuge tube M, add 2 mL of reagent A to centrifuge tube M, then add 50 μL of reagent B, shake and mix for 1 minute, take 50 μL of the clear liquid in centrifuge tube M into centrifuge tube N, add 500 μL of reagent C, mix evenly to form the liquid to be tested.

[0073] For leafy vegetables, celery, day lily matrix, water spinach, leek, cabbage and celery, prepare the liquid to be tested and blank samples according to the following scheme; Weigh 0.5 g of the sample to be tested into a 5 mL centrifuge tube M, add 2 mL of reagent A to the centrifuge tube M, then add 50 μL of reagent B, shake and mix for 1 minute, take 50 μL of the clear liquid in the centrifuge tube M into the centrifuge tube N, add 1 mL of reagent C, mix well to form the liquid to be tested.

[0074] The liquid to be tested and the blank sample were tested; the test results are shown in Table 7.

[0075] Table 7. Test results of different matrices

[0076] It can be seen from Table 7 that the test results in Examples 43-51 are: the blank samples are all negative, with T / C values ​​between 1.6 and 3.5; the liquids to be tested are all positive, with T / C values ​​between 0.2 and 0.7; and there are no invalid cards, false positives, false negatives, etc. in all experiments; at the same time, the test results have good stability, accuracy, and high sensitivity; which means that the test liquid is prepared by the above scheme, and its test results have good stability, accuracy, and high sensitivity; and its test results can reach the detection limit specified in "GB2762-2022 National Food Safety Standard Limits of Contaminants in Food".

[0077] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the scope of protection of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. A method for detecting heavy metal cadmium in fresh vegetables using immunocolloidal gold method, characterized in that: include: Step 1, preparing the liquid to be tested; Step 2, using the consumables to be used to test the liquid to be tested; The step of preparing the liquid to be tested specifically includes the following steps: S1. Weigh the sample to be tested and place it in a centrifuge tube M. Slowly add reagent A to the centrifuge tube M. After mixing evenly, add reagent B and shake for 0.5-3 minutes. After mixing evenly, let it stand to form a clear liquid. The reagent A is an acidic solution; the reagent B is a chelating agent solution; S2. Use a dropper to draw the clarified liquid in centrifuge tube M into centrifuge tube N, add reagent C, mix well, and prepare the liquid to be tested; The reagent C is a neutralizing reagent; the neutralizing reagent is an alkaline solution or a neutral buffer solution.

2. The method for detecting heavy metal cadmium in fresh vegetables by using immunocolloidal gold method according to claim 1, characterized in that: The acidic solution is any one of concentrated sulfuric acid solution, concentrated nitric acid solution, acetic acid solution, polybasic organic acid solution, and a mixture of a polybasic organic acid solution and an optimized reagent; the polybasic organic acid solution is any one of citric acid solution, tartaric acid solution, and malic acid solution; and the optimized reagent is ultrapure water or an inorganic weak base salt solution.

3. A method for detecting heavy metal cadmium in fresh vegetables using immunocolloidal gold method according to claim 2, characterized in that: The optimization reagent is an inorganic weak base salt solution; in the mixed solution of the polybasic organic acid solution and the optimization reagent, the volume ratio of the polybasic organic acid solution to the optimization reagent is (2.0-4.0):1; the inorganic weak base salt solution is an aqueous solution of any one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, and sodium bicarbonate.

4. The method for detecting heavy metal cadmium in fresh vegetables by utilizing immunocolloidal gold method according to claim 3, characterized in that: In the mixed solution of the polybasic organic acid solution and the optimization reagent, the concentration of the polybasic organic acid solution is 0.03-0.1 mol / L, and the concentration of the optimization reagent is 0.05-0.4 mol / L.

5. The method for detecting heavy metal cadmium in fresh vegetables by utilizing immunocolloidal gold method according to claim 2, characterized in that: The chelating agent solution is a DMSO solution of Isothiocyanobenzyl-EDTA or an aqueous solution of EDTA SOLUTINON and sodium hydroxide.

6. The method for detecting heavy metal cadmium in fresh vegetables by utilizing immunocolloidal gold method according to claim 2, characterized in that: The neutralizing agent is an alkaline solution; the alkaline solution is any one of 0.01-0.2 mol / L sodium hydroxide solution, 0.01-0.5 mol / L dipotassium hydrogen phosphate solution, 0.01-0.5 mol / L potassium dihydrogen phosphate solution, saturated sodium bicarbonate solution, 0.01-0.4 mol / L potassium carbonate solution, and 0.01-0.4 mol / L sodium carbonate solution.

7. The method for detecting heavy metal cadmium in fresh vegetables by utilizing immunocolloidal gold method according to claim 2, characterized in that: The neutralizing agent is a neutral buffer solution; the neutral buffer solution is composed of any one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium bicarbonate, and sodium chloride in combination with a surfactant and a preservative.

8. A method for detecting heavy metal cadmium in fresh vegetables by using immunocolloidal gold method according to claim 6 or 7, characterized in that: In step S1, the volume of the reagent A added is 1-3 mL, and the volume of the reagent B added is 20-500 μL; In the step S2, the volume of the clear liquid sucked is 25-300 μL; the volume of the reagent C added is 0-1.5 mL.

9. The method for detecting heavy metal cadmium in fresh vegetables by using immunocolloidal gold method according to claim 8, characterized in that: In the step S2, the pH value of the prepared liquid to be tested is 6-7.5.

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