Nano-enzyme, detection kit containing nano-enzyme and application of nano-enzyme in heavy metal detection

Abstract

The invention discloses a nano-enzyme, a detection kit containing the nano-enzyme and application of the nano-enzyme in heavy metal detection. The zirconium-doped cerium dioxide nano-enzyme is prepared by a chemical synthesis method, and has the activity of alkaline phosphatase. The nano-enzyme can catalyze hydrolysis of a substrate to generate chemiluminescence, so that the nano-enzyme is applied to the technical field of chemiluminescence analysis and detection, and the problems that natural enzyme is easily influenced by the external environment and is poor in stability are solved. The invention also discloses a chemiluminescence immunodetection kit for detecting heavy metals. The kit realizes automatic quantitative analysis of heavy metals based on the zirconium-doped cerium dioxide nano-enzyme and antigen-antibody specific binding property, has good specificity and sensitivity, and effectively reduces the heavy metal detection cost.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid heavy metal detection, and more specifically, to a nanozyme, a detection kit containing the nanozyme, and an application in detecting heavy metals. Background Art

[0002] The detection of heavy metals has always been a problem that people pay more attention to. The detection of heavy metal residues in grain and oil food is one of the hot topics of current social concern. The existing detection methods for heavy metals in grain and oil food include atomic absorption spectroscopy, plasma mass spectrometry, etc., but the instruments are expensive and require professional technicians to operate and maintain. In recent years, rapid detection methods for heavy metals have been developed on a large scale, such as electrochemical method, colloidal gold test strip method, enzyme-linked immunosorbent assay, etc., but there are also disadvantages such as low detection efficiency and manual operation throughout the process. In comparison, the fully automatic detection method can effectively reduce personnel operation and improve efficiency. It is the research direction with the most development potential and development prospects. Among them, the immunoassay method based on the immune principle and chemiluminescence principle has received more and more attention due to its advantages of high sensitivity, high specificity, convenience and speed, and no need for human intervention. At present, the magnetic separation automatic chemiluminescence immunoassay method is widely used in the field of in vitro diagnosis. On the one hand, through the antigen-antibody specific reaction, the accurate detection of the detection target is improved. On the other hand, the chemiluminescent substrate catalyzes the substrate to luminesce under a specific light source. The efficient catalytic effect improves the detection sensitivity of the method. In addition, with the help of magnetic solid phase carriers, the degree of automation is greatly improved compared with plate and tube types. Among them, the difference in luminescent substrates and reaction mechanisms has a greater impact on the detection performance. With the development of this method, many new luminescent systems have been widely studied and applied.

[0003] However, the quantum yield of the chemiluminescent reagent currently relied on by the chemiluminescence method is limited, and the luminescence intensity produced by most luminescence systems is weak and the sensitivity is relatively low. In order to increase its quantum yield, the traditional chemiluminescence immunoassay method often uses natural enzymes (horseradish peroxidase, alkaline phosphatase) to enhance the luminescence effect, thereby achieving the effect of improving the stability and sensitivity of the detection method. However, natural enzymes are biological proteases, and their activity is easily affected by changes in pH conditions, temperature, concentration, storage, etc., and their application and storage are limited, which seriously restricts their wide application in the automatic chemiluminescence immunoassay detection of heavy metals.

[0004] Therefore, it is of great practical significance to develop a highly stable nano-mimetic enzyme, a rapid detection kit and a detection method that are not restricted by the use conditions of natural enzymes. Summary of the invention

[0005] The first object of the present invention is to provide a nanozyme which has alkaline phosphatase activity, is simple to prepare, can be adapted to large-scale production, and has high application value.

[0006] The second object of the present invention is to provide a chemiluminescent immunoassay kit for detecting heavy metals.

[0007] The third object of the present invention is to provide a method for detecting heavy metals, which has high sensitivity and strong specificity, and is combined with the characteristics of automatic magnetic separation and sorting, does not require manual operation, can effectively realize automatic detection of heavy metals, and effectively reduce detection costs.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a zirconium-doped ceria nanozyme, the method comprising the following steps:

[0010] 1-3 g of cerium dioxide is ultrasonically dispersed in deionized water, 1-5 g of zirconium oxynitrate is added, stirred for 10-20 min, anhydrous sodium carbonate solution is added to adjust the pH to 9-10 and the reaction is continued for 1-5 h. After the reaction is completed, centrifugation, water washing, drying, and then crushing are carried out, and calcination is carried out at 450-550° C. for 2-4 h to obtain zirconium-doped cerium dioxide nanoparticles.

[0011] Furthermore, the particle size of the zirconium-doped ceria nanoparticles is 100-1000 nm.

[0012] Furthermore, the zirconium-doped ceria nanoparticles are spherical, cubic, rod-shaped, conical, triangular or polygonal.

[0013] The present invention also claims protection for the zirconium-doped ceria nanozyme prepared by the above method. The nanozyme has alkaline phosphatase activity and can catalyze substrate hydrolysis to produce chemiluminescence, so it can be used in the field of chemiluminescence analysis and detection technology, overcoming the shortcomings of natural enzymes that are easily affected by the external environment and have poor stability.

[0014] In a second aspect, the present invention provides a chemiluminescent immunoassay kit for detecting heavy metals, comprising: a heavy metal antigen-nanozyme conjugate, an anti-heavy metal antibody-magnetic particle conjugate, a heavy metal chelator, a sample diluent, a sample washing solution, and an enzymatic chemiluminescent substrate solution;

[0015] Wherein, the nanozyme is the above-mentioned zirconium-doped cerium dioxide nanozyme.

[0016] Furthermore, the heavy metal antigen is formed by coupling a heavy metal, a heavy metal chelator, and a carrier protein; the heavy metal chelator can be selected from one or more of ethylenediaminetetraacetic acid (EDTA), diethyltriaminepentaacetic acid (DTPA), and 1-(4-isothiocyanatobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid (ITCBE); and the carrier protein can be ovalbumin, serum albumin, or lock-key blue protein.

[0017] Furthermore, the anti-heavy metal antibody is a monoclonal antibody or a polyclonal antibody purchased commercially or prepared by a known method. Preferably, the anti-heavy metal antibody is a monoclonal antibody.

[0018] Further, in the kit, the heavy metal chelator is selected from one or more of ethylenediaminetetraacetic acid (EDTA), diethyltriaminepentaacetic acid (DTPA) and 1-(4-isothiocyanatobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid (ITCBE); preferably, the heavy metal chelator is ITCBE.

[0019] Furthermore, the enzymatic chemiluminescent substrate solution is (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacridinium methylene) phosphate disodium salt solution (APS-5) or 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)benzene-1,2-dioxetane solution (AMPPD); preferably, the enzymatic chemiluminescent substrate solution is APS-5.

[0020] Further, the sample diluent is selected from one or more of PBS, MES, PBST and Tris-HCl buffer; according to a specific embodiment of the present invention, the concentration of the sample diluent may be 0.01 to 0.1 mol / L, and the pH may be 6 to 9;

[0021] Furthermore, the sample washing solution is a Tris-HCl buffer solution with a concentration of 0.01 to 0.02 mol / L and containing 0.1 to 0.2% Tween-20.

[0022] Furthermore, the heavy metal antigen-nanozyme conjugate is prepared by modifying the surface of the nanozyme with carboxyl, amino, p-toluenesulfonyl, streptavidin or anti-fluorescein isothiocyanate antibody, and then coupling it with the heavy metal antigen; generally, the coupling reaction between the heavy metal antigen and the nanozyme can be completed using commonly used coupling reagents or coupling systems.

[0023] According to a specific embodiment of the present invention, the heavy metal antigen-nanozyme conjugate is prepared by the following method:

[0024] (1) 30-80 mg of nanozyme was dispersed in 80-150 mL of ethanol, stirred at room temperature for 5-20 min, 0.5-1.2 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred for 3-5 h. After the reaction, ethanol and water were used for alternating washing, and 100-200 mg of succinic anhydride was added, and the mixture was stirred for 2-4 h. The mixture was dried and cooled to obtain a carboxylated nanozyme.

[0025] (2) Take 20-70 mg of carboxylated nanozyme and disperse it in PBS or MES solution, add an equal mass of heavy metal antigen, incubate at room temperature for 2-5 h, wash with PBS or MES solution, and resuspend in PBS or MES solution to obtain heavy metal antigen-nanozyme conjugate.

[0026] Furthermore, the anti-heavy metal antibody-magnetic particle conjugate is prepared by modifying the surface of the magnetic particle with carboxyl, amino, p-toluenesulfonyl, streptavidin or anti-fluorescein isothiocyanate antibody, and then coupling it with the anti-heavy metal antibody; generally, the coupling reaction between the anti-heavy metal antibody and the magnetic particle can be completed using commonly used coupling reagents or coupling systems.

[0027] According to a specific embodiment of the present invention, the anti-heavy metal antibody-magnetic particle conjugate is prepared by the following method:

[0028] (1) Dissolve 1-3 g of ferric chloride and 0.3-1.5 g of ferrous chloride in 40-80 mL of water, stir under nitrogen, react at 80° C. for 10 min, adjust the pH to 10-11 with alkaline solution, continue the reaction for 40 min, wash with deionized water after the reaction, dry and cool to obtain magnetic Fe3O4 particles.

[0029] (2) Take 30-80 mg of magnetic Fe3O4 particles and disperse them in 80-150 mL of ethanol. After stirring at room temperature for 10 min, add 0.5-1.2 mL of 3-aminopropyltriethoxysilane and continue stirring for 3-5 h. After the reaction is completed, wash with ethanol and water alternately, then add a mixture of 0.1-0.5 mL of polyacrylic acid, 5-10 mg of EDC, and 5-10 mg of NHS, stir and react for 3-5 h, separate by magnetic separation, dry, and cool to obtain carboxylated magnetic particles.

[0030] (3) Dispersing 20-70 mg of carboxylated magnetic microparticles in PBS or MES solution, adding an equal amount of heavy metal antibody, incubating at room temperature for 2-5 h, performing magnetic separation and discarding the supernatant, washing with PBS or MES solution, and resuspending in PBS or MES solution to obtain anti-heavy metal antibody-magnetic microparticle conjugates;

[0031] The coupling ratio of the anti-heavy metal antibody to the magnetic particles is 10-50. Specifically, the coupling ratio is 10, 20, 30, 40, 50, etc. or any range between these coupling ratios.

[0032] In a third aspect, the present invention provides the use of the above chemiluminescent immunoassay kit in the detection of heavy metals.

[0033] When the chemiluminescent immunoassay kit of the present invention is applied to the detection of heavy metals, the indirect competitive reaction principle is adopted, that is, the heavy metals in the sample to be tested compete with the heavy metal antigen-nanozyme conjugate to bind to the anti-heavy metal antibody to form a magnetic particle-anti-heavy metal antibody-heavy metal antigen-nanozyme complex, and then the nanozyme on the complex catalyzes the substrate to cause the substrate to produce chemiluminescence, and the luminescence intensity is detected. The detected luminescence intensity is negatively correlated with the content of heavy metals in the sample to be tested, and the luminescence intensity-concentration curve drawn by the concentration gradient detection of heavy metal standard substances is used to calculate the heavy metal content in the sample to be tested.

[0034] Furthermore, the present invention provides a method for detecting heavy metals using the above-mentioned kit, the method comprising the following steps:

[0035] (1) adding a heavy metal chelating agent to the sample to be tested to carry out a chelation reaction to form a heavy metal chelated state;

[0036] Preferably, the temperature of the chelation reaction is 10 to 40°C;

[0037] Preferably, the chelation reaction time is 0.5 to 20 minutes;

[0038] Preferably, the concentration of the heavy metal chelating agent is 0.01 to 0.1 mmol / L;

[0039] (2) adding heavy metal antigen-nanozyme conjugates and anti-heavy metal antibody-magnetic particle conjugates in sequence and incubating, wherein the heavy metals in the sample to be tested compete with the heavy metal antigen-nanozyme conjugates for binding to the anti-heavy metal antibodies coupled to the surface of the magnetic particles to form a complex;

[0040] Preferably, the incubation temperature is 0 to 40°C;

[0041] Preferably, the incubation time is 5 to 60 min;

[0042] (3) Wash with sample washing solution to remove unbound substances and reduce nonspecific interference;

[0043] (4) utilizing magnetic effects to transfer the complex formed in step (2) into an enzymatic chemiluminescent substrate solution, wherein the nanozyme on the complex exerts alkaline phosphatase activity, causing the substrate to generate chemiluminescence, and detecting the luminescence intensity;

[0044] (5) The luminescence intensity is negatively correlated with the content of heavy metals in the sample to be tested. The heavy metal content in the sample to be tested can be calculated based on the luminescence intensity-concentration standard curve drawn by the concentration gradient detection of heavy metal standard substances.

[0045] Further, in step (2), the heavy metal antigen-nanozyme conjugate is diluted with a sample diluent to a concentration of 100 to 1000 ng / mL. Specifically, the concentration may be 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL, etc. or any range between these concentrations;

[0046] The anti-heavy metal antibody-magnetic particle conjugate is diluted with a sample diluent to a concentration of 100 to 1000 ng / mL. Specifically, the concentration can be 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL, etc. or any range between these concentrations;

[0047] Furthermore, in step (4), the concentration of the enzymatic chemiluminescent substrate solution is 0.5 to 2 mmol / mL.

[0048] Further, the sample to be tested is selected from food or oil products;

[0049] Furthermore, the food includes rice, wheat, corn or food products;

[0050] Further, the oil includes peanut oil, corn oil, soybean oil, rapeseed oil, sunflower oil, rapeseed oil, rice bran oil, sesame oil or olive oil;

[0051] Furthermore, the heavy metal is selected from one or more of cadmium, lead and mercury.

[0052] In addition, unless otherwise specified, the raw materials used in the present invention can be obtained from commercial sources, and any range recorded in the present invention includes the end value and any value between the end values ​​and any sub-range formed by the end value or any value between the end values. The percentages are all mass percentages unless otherwise specified, and the solutions are all aqueous solutions unless otherwise specified.

[0053] The beneficial effects of the present invention are as follows:

[0054] (1) The nanozyme of the present invention can effectively replace natural enzymes, has regulated catalytic activity, is stable to heat, acid, and alkali, is more suitable for complex environments, is simple to prepare, has good stability and reproducibility, can adapt to large-scale mass production, is easy to store and transport, and has high application value.

[0055] (2) The automatic chemiluminescence immunoassay detection kit of the present invention is based on the specific binding of antigen and antibody, has good specificity and good sensitivity.

[0056] (3) The chemiluminescent immunoassay detection kit of the present invention can be used in conjunction with a fully automatic chemiluminescent detector. Combined with the magnetic automatic separation and sorting characteristics, it can effectively realize automatic detection of heavy metals without manual operation, effectively reduce the detection cost, and especially realize highly automated and direct detection of heavy metals such as lead, cadmium and mercury in food and oils. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0058] Figure 1 A schematic diagram showing the detection method of the present invention.

[0059] Figure 2 Nanozyme morphology characterization is shown.

[0060] Figure 3 Validation of the alkaline phosphatase activity of the nanozyme is shown.

[0061] Figure 4 Shows the performance of the kit under unusual environmental conditions. DETAILED DESCRIPTION

[0062] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0063] Example 1: Preparation of zirconium-doped ceria nanozyme

[0064] 1.5 g of cerium dioxide was ultrasonically dispersed in 20 mL of deionized water, 1 g of zirconium oxynitrate was added, stirred for 15 min, anhydrous sodium carbonate solution was added dropwise to adjust the pH to 9-10, and the reaction was continued for 2 h. After the reaction was completed, it was centrifuged and washed with water three times and then dried at 60 ° C. After crushing, it was calcined at 500 ° C in a muffle furnace for 2 h to obtain spherical zirconium-doped cerium dioxide nanozymes with a particle size of about 400-800 nm. Figure 2 .

[0065] Example 2: Nanozyme activity verification

[0066] At room temperature, 100 μL of (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacridinium methylene) disodium phosphate solution (APS-5, 1 mmol / L) was added to 500 μL of phosphate buffer solution, followed by 50 μL of 0.1 mg / mL of the nanozyme prepared in Example 1. After rapid mixing, the luminescence intensity was monitored at 430 nm. Under the same conditions, a control group without the addition of APS-5 was set up, and a control group without the addition of nanozyme was also set up. After monitoring, it was found that no luminescence intensity was detected at 430 nm in the control group with only nanozyme or only APS-5, while in the experimental group with both nanozyme and substrate APS-5, a higher luminescence signal value was detected at 430 nm, as shown in FIG. Figure 3 . This indicates that the nanozyme prepared in Example 1 can effectively catalyze the substrate to produce chemiluminescence.

[0067] Example 3: Preparation of heavy metal antigens

[0068] 2 mg of chelating agent 1-(4-isothiocyanobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid (ITCBE) was dissolved in 200 μL DMSO and slowly added to 500 μL phosphate buffer containing 20 mg BSA, stirred at room temperature for 24 hours, and dialyzed with phosphate buffer solution for 2 days. Then 1 mL of 1 mg / mL cadmium / lead / mercury standard solution was slowly added to the ITCBE-BSA solution, stirred at room temperature for 3 hours, and dialyzed with phosphate buffer solution for 2 days to obtain heavy metal antigens. After UV scanning identification, the absorption peak of the heavy metal antigen at 280 nm was significantly offset from the absorption peak of BSA, indicating that the heavy metal antigen was successfully prepared.

[0069] Example 4: Preparation of heavy metal antigen-nanozyme conjugates

[0070] (1) 80 mg of the nanozyme prepared in Example 1 was dispersed in 80 mL of ethanol, stirred at room temperature for 10 min, and then 1 mL of 3-aminopropyltriethoxysilane (APTES) was added, and the reaction was continued for 3 h. After the reaction, ethanol and water were alternately washed 3 times, and 200 mg of succinic anhydride was added, stirred for 4 h, dried at 60 ° C, and cooled to obtain a carboxylated nanozyme.

[0071] (2) Take 40 mg of carboxylated nanozyme and disperse it in PBS or MES solution, add an equal mass of heavy metal antigen, incubate with rotation at room temperature for 2 h, wash with PBS or MES solution three times, and resuspend in PBS or MES solution to obtain heavy metal antigen-nanozyme conjugate.

[0072] Example 5: Preparation of anti-heavy metal antibody-magnetic particle conjugates

[0073] (1) Dissolve 1 g of ferric chloride and 0.6 g of ferrous chloride in 50 mL of water, stir mechanically with nitrogen, react at 80°C for 10 min, adjust the pH to 10-11 with aqueous ammonia, continue the reaction for 40 min, wash with deionized water three times after the reaction, dry at 60°C, and cool to obtain the product Fe3O4.

[0074] (2) 80 mg of magnetic Fe3O4 particles were dispersed in 80 mL of ethanol, stirred at room temperature for 10 min, and then 0.6 mL of APTES was added, and the reaction was continued for 3 h. After the reaction, the particles were washed alternately with ethanol and water for 3 times, and then a mixture of 0.5 mL of polyacrylic acid, 5 mg of EDC, and 5 mg of NHS was added, stirred for 5 h, magnetic separation was performed, and the particles were dried at 60 ° C and cooled to obtain carboxylated magnetic particles.

[0075] (3) Take 50 mg of carboxylated magnetic microparticles and disperse them in PBS or MES solution, add an equal amount of heavy metal antibody, incubate with rotation at room temperature for 2 h, perform magnetic separation and discard the supernatant, wash with PBS or MES solution for 3 times, add PBS or MES solution and resuspend to obtain anti-heavy metal antibody-magnetic microparticle conjugate.

[0076] Example 6: Kit Preparation (Lead, Cadmium, Mercury Kit)

[0077] (1) The heavy metal lead / cadmium / mercury antigen-nanozyme conjugate prepared in Example 4 was diluted with 0.01 mol / L phosphate buffer solution (PBS, pH 7.4) to obtain a 200 ng / mL heavy metal antigen-nanozyme conjugate working solution;

[0078] (2) diluting the lead / cadmium / mercury antibody-magnetic particle conjugate prepared in Example 5 with 0.01 M phosphate buffer solution (PBS, pH 7.4) to obtain 300 ng / mL of anti-heavy metal antibody-magnetic particle conjugate working solution;

[0079] (3) Preparation of sample diluent: 0.01 M phosphate buffer solution (PBS, pH 7.4);

[0080] (4) Preparation of washing solution: 0.01 M Tris-HCl buffer (containing 0.1% Tween-20);

[0081] (5) Preparation of chelating agent: 0.01 mmol / L ITCBE solution;

[0082] (6) Preparation of substrate solution: 1 mmol / L (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacridinium methylene) phosphate solution;

[0083] (7) Assembling: assembling the above reagents into a kit.

[0084] Example 7: Performance test and application of lead quantitative analysis kit

[0085] The kit prepared in Example 6 was subjected to detection method performance testing and specific application.

[0086] (1) Investigation of detection limit and quantification limit: Twenty independent negative tap water samples were measured. The detection limit of the method was calculated as the average value of the 20 measurement results plus 3 times the standard deviation, which was 0.05 ng / mL. The quantification limit was calculated as the average value of the 20 measurement results plus 10 times the standard deviation, which was 0.15 ng / mL.

[0087] (2) Specificity investigation: The lead quantitative analysis kit was used to detect common metal ions such as cadmium, mercury, copper, zinc, and manganese. The results are shown in Table 1. There was no cross reaction with other metal ions (cross reaction rate was less than 1%), indicating that the lead quantitative analysis kit had good specificity.

[0088] Table 1 Cross-reaction table

[0089]

[0090]

[0091] (3) Stability study: The lead quantitative analysis kit was subjected to an accelerated stability test at 37°C and continuously monitored for 12 days. The signal value change within 12 days was less than 5% (Table 2). According to experience, storage at 37°C for one day is equivalent to storage at 4°C for two months, that is, the lead kit can be stored at 4°C for 12 months without affecting its use.

[0092] Table 2 Stability test table

[0093] time Signal value Day 1 118561 the next day 115682 Day 3 120220 Day 4 128874 Day 5 113587 Day 6 130021 Day 7 113864 Day 8 122658 Day 9 127894 Day 10 128563 Day 11 119835 Day 12 125793 Relative coefficient of variation (%) 4.9

[0094] (4) Accuracy investigation: The lead quantitative analysis kit was used for the lead spike recovery test in rice, wheat, corn and other grain samples and peanut oil, soybean oil, corn oil and other vegetable oil samples. Three concentration levels were added and seven parallel samples were added. The recovery rate, mean recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 3. The recovery rate was between 91.4% and 111.2%, and the RSD was less than 5.8%.

[0095] Table 3 Accuracy inspection table

[0096]

[0097]

[0098] Example 8: Performance test and application of cadmium quantitative analysis kit

[0099] The kit prepared in Example 6 was subjected to detection method performance testing and specific application.

[0100] (1) Investigation of detection limit and quantification limit: Twenty independent soybean oil negative samples were measured. The detection limit of the method was calculated as the average value of the 20 measurement results plus 3 times the standard deviation, and the result was 0.03 ng / mL. The quantification limit was calculated as the average value of the 20 measurement results plus 10 times the standard deviation, and the result was 0.09 ng / mL.

[0101] (2) Specificity investigation: The cadmium quantitative analysis kit was used to detect common metal ions such as cadmium, mercury, copper, zinc, and manganese. The results are shown in Table 4. There was no cross reaction with other metal ions (cross reaction rate was less than 1%), indicating that the cadmium quantitative analysis kit had good specificity.

[0102] Table 4 Cross-reaction table

[0103] Metal ions IC50(ng / mL) CR(%) Cd 0.20 100 Mn ＞1000 ＜0.02 Zn ＞1000 ＜0.02 Cu ＞1000 ＜0.02 Mg ＞1000 ＜0.02 Ca ＞1000 ＜0.02 Co ＞1000 ＜0.02 Fe ＞1000 ＜0.02 Ni ＞1000 ＜0.02 Hg ＞1000 ＜0.02 Pb ＞1000 ＜0.02

[0104] (3) Stability study: The cadmium quantitative analysis kit was subjected to an accelerated stability test at 37°C and continuously monitored for 12 days. The signal value change within 12 days was less than 5% (Table 5). According to experience, storage at 37°C for one day is equivalent to storage at 4°C for two months, that is, the cadmium kit can be stored at 4°C for 12 months without affecting its use.

[0105] Table 5 Stability investigation table

[0106]

[0107]

[0108] (4) Accuracy investigation: The cadmium quantitative analysis kit was used for the spike recovery test of cadmium in rice, wheat, corn, peanut oil, soybean oil, and corn oil. Three concentration levels were added, and seven parallel experiments were performed. The recovery rate, mean recovery rate, and relative standard deviation (RSD) were calculated. The results are shown in Table 6. The recovery rate was between 90.2% and 115.6%, and the RSD was less than 7.3%.

[0109] Table 6 Accuracy inspection table

[0110]

[0111] Example 9: Performance test and application of mercury quantitative analysis kit

[0112] The kit prepared in Example 6 was subjected to detection method performance testing and specific application.

[0113] (1) Investigation of detection limit and quantification limit: Twenty independent negative rice samples were measured. The detection limit of the method was calculated as the average value of the 20 measurement results plus three times the standard deviation, and the result was 0.003 mg / kg. The quantification limit was calculated as the average value of the 20 measurement results plus ten times the standard deviation, and the result was 0.01 mg / kg.

[0114] (2) Specificity investigation: The mercury quantitative analysis kit was used to detect common metal ions such as cadmium, lead, copper, zinc, and manganese. The results are shown in Table 7. There was no cross reaction with other metal ions (cross reaction rate was less than 1%), indicating that the mercury quantitative analysis kit had good specificity.

[0115] Table 7 Cross-reaction table

[0116] Metal ions IC50(ng / mL) CR(%) Hg 0.02 100 Mn ＞1000 ＜0.01 Zn ＞1000 ＜0.01 Cu ＞1000 ＜0.01 Mg ＞1000 ＜0.01 Ca ＞1000 ＜0.01 Co ＞1000 ＜0.01 Fe ＞1000 ＜0.01 Ni ＞1000 ＜0.01 Pb ＞1000 ＜0.01 Cd ＞1000 ＜0.01

[0117] (3) Stability study: The mercury quantitative analysis kit was subjected to an accelerated stability test at 37°C and continuously monitored for 12 days. The signal value change within 12 days was less than 5% (Table 8). According to experience, storage at 37°C for one day is equivalent to storage at 4°C for two months, that is, the mercury kit can be stored at 4°C for 12 months without affecting its use.

[0118] Table 8 Stability Investigation Table

[0119]

[0120]

[0121] (4) Accuracy investigation: The mercury quantitative analysis kit was used for the spike recovery test of mercury in rice, wheat, corn, peanut oil, soybean oil, and corn oil. Three concentration levels were added, and seven parallels were used. The recovery rate, mean recovery rate, and relative standard deviation (RSD) were calculated. The results are shown in Table 9. The recovery rate was between 90.2% and 115.6%, and the RSD was less than 7.3%.

[0122] Table 9 Accuracy inspection table

[0123]

[0124] Example 10: Application under unconventional experimental environment conditions

[0125] In actual use, affected by transportation conditions and climatic conditions, the working temperature of the test kit and the instrument cannot usually be maintained at 37°C all the time. In addition, due to differences in the acidity and alkalinity of the sample itself, the pH value of the reagent reaction will usually fluctuate. Therefore, the present invention simulates several unconventional experimental conditions similar to the actual situation (40°C, 18°C, pH 9.0, pH 6.0), tests and applies the test kit prepared in Example 6, and compares it with a commercially available test kit using APS-5 as a substrate and alkaline phosphatase as a corresponding catalyst. A certain concentration of lead (0.2 mg / kg), cadmium (0.2 mg / kg), and mercury (0.02 mg / kg) were added to rice, and the test kit prepared in Example 6 of the present invention and the comparison test kit were used to test under 4 unconventional experimental conditions, 3 of which were parallel. The recovery rate and RSD were as follows: Figure 4 As shown, the kit of the present invention can still maintain high accuracy and stability under unconventional experimental environment conditions, while the accuracy and stability of the comparative kit are reduced.

[0126] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing zirconium-doped ceria nanozyme, characterized in that: The method comprises the following steps: 1-3 g of cerium dioxide is ultrasonically dispersed in deionized water, 1-5 g of zirconium oxynitrate is added, stirred for 10-20 min, anhydrous sodium carbonate solution is added to adjust the pH to 9-10, and the reaction is continued for 1-5 h. After the reaction is completed, the mixture is centrifuged, washed with water, dried, and then crushed, and calcined at 450-550° C. for 2-4 h to obtain zirconium-doped cerium dioxide nanoparticles; Preferably, the particle size of the zirconium-doped ceria nanoparticles is 100 to 1000 nm; Preferably, the zirconium-doped ceria nanoparticles are spherical, cubic, rod-shaped, conical, triangular or polygonal.

2. The zirconium-doped cerium dioxide nanozyme obtained by the preparation method as claimed in claim 1.

3. A chemiluminescent immunoassay kit for detecting heavy metals, characterized in that: include: Heavy metal antigen-nanozyme conjugate, anti-heavy metal antibody-magnetic particle conjugate, heavy metal chelator, sample diluent, sample washing solution and enzymatic chemiluminescent substrate solution; Wherein, the nanozyme is the zirconium-doped ceria nanozyme as described in claim 2; Preferably, the heavy metal antigen is formed by coupling heavy metal, heavy metal chelator and carrier protein; and the anti-heavy metal antibody is a monoclonal antibody or a polyclonal antibody.

4. The chemiluminescent immunoassay kit according to claim 3, characterized in that: The heavy metal chelating agent is selected from one or more of ethylenediaminetetraacetic acid, diethyltriaminepentaacetic acid and 1-(4-isothiocyanatobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid; The enzymatic chemiluminescent substrate solution is (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacridinium methylene) phosphate disodium salt solution or 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)benzene-1,2-dioxetane solution; The sample diluent is selected from one or more of PBS, MES, PBST and Tris-HCl buffer; preferably, the concentration of the sample diluent is 0.01-0.1 mol / L, and the pH is 6-9; The sample washing solution is a Tris-HCl buffer solution with a concentration of 0.01-0.02 mol / L and containing 0.1-0.2% Tween-20.

5. The chemiluminescent immunoassay kit according to claim 3, characterized in that: The heavy metal antigen-nanozyme conjugate is prepared by modifying the surface of the nanozyme with carboxyl, amino, p-toluenesulfonyl, streptavidin or anti-fluorescein isothiocyanate antibody, and then coupling it with the heavy metal antigen; Preferably, the heavy metal antigen-nanozyme conjugate is prepared by the following method: (1) 30-80 mg of nanozyme was dispersed in 80-150 mL of ethanol, stirred at room temperature for 5-20 min, 0.5-1.2 mL of 3-aminopropyltriethoxysilane was added, and the mixture was stirred for 3-5 h. After the reaction, ethanol and water were used for alternating washing, and 100-200 mg of succinic anhydride was added, and the mixture was stirred for 2-4 h. The mixture was dried and cooled to obtain a carboxylated nanozyme. (2) Take 20-70 mg of carboxylated nanozyme and disperse it in PBS or MES solution, add an equal mass of heavy metal antigen, incubate at room temperature for 2-5 h, wash with PBS or MES solution, and resuspend in PBS or MES solution to obtain heavy metal antigen-nanozyme conjugate.

6. The chemiluminescent immunoassay kit according to claim 3, characterized in that: The anti-heavy metal antibody-magnetic particle conjugate is prepared by modifying the surface of the magnetic particle with carboxyl, amino, p-toluenesulfonyl, streptavidin or anti-fluorescein isothiocyanate antibody, and then conjugating with the anti-heavy metal antibody; Preferably, the anti-heavy metal antibody-magnetic particle conjugate is prepared by the following method: (1) Dissolve 1-3 g of ferric chloride and 0.3-1.5 g of ferrous chloride in 40-80 mL of water, stir with nitrogen, react at 80° C. for 10 min, adjust the pH to 10-11 with alkaline solution, continue the reaction for 40 min, wash with deionized water after the reaction, dry and cool to obtain magnetic Fe3O4 particles; (2) 30-80 mg of magnetic Fe3O4 particles were dispersed in 80-150 mL of ethanol, stirred at room temperature for 10 min, and then 0.5-1.2 mL of 3-aminopropyltriethoxysilane was added. The reaction was continued for 3-5 h. After the reaction, the mixture was washed alternately with ethanol and water, and then 0.1-0.5 mL of polyacrylic acid, 5-10 mg of EDC, and 5-10 mg of NHS were added. The mixture was stirred for 3-5 h, magnetic separation was performed, drying, and cooling were performed to obtain carboxylated magnetic particles. (3) Disperse 20-70 mg of carboxylated magnetic microparticles in PBS or MES solution, add an equal amount of heavy metal antibody, incubate at room temperature for 2-5 h, perform magnetic separation and discard the supernatant, wash with PBS or MES solution, add PBS or MES solution and resuspend to obtain anti-heavy metal antibody-magnetic microparticle conjugate; the coupling ratio of anti-heavy metal antibody to magnetic microparticle is 10-50.

7. Use of the chemiluminescent immunoassay kit according to any one of claims 3 to 6 in detecting heavy metals.

8. A method for detecting heavy metals, characterized in that: Using the chemiluminescent immunoassay kit according to any one of claims 3 to 6 comprises the following steps: (1) Adding a heavy metal chelating agent to the sample to be tested to carry out a chelation reaction; Preferably, the temperature of the chelation reaction is 10 to 40°C; Preferably, the chelation reaction time is 0.5 to 20 minutes; Preferably, the concentration of the heavy metal chelating agent is 0.01 to 0.1 mmol / L; (2) adding heavy metal antigen-nanozyme conjugates and anti-heavy metal antibody-magnetic particle conjugates in sequence and incubating, wherein the heavy metals in the sample to be tested compete with the heavy metal antigen-nanozyme conjugates for binding to the anti-heavy metal antibodies coupled to the surface of the magnetic particles to form a complex; Preferably, the incubation temperature is 0 to 40°C; Preferably, the incubation time is 5 to 60 min; (3) Wash with sample washing solution; (4) utilizing magnetic effects to transfer the complex formed in step (2) into an enzymatic chemiluminescent substrate solution, wherein the nanozyme on the complex exerts alkaline phosphatase activity, causing the substrate to generate chemiluminescence, and detecting the luminescence intensity; (5) The luminescence intensity is negatively correlated with the content of heavy metals in the sample to be tested. The heavy metal content in the sample to be tested can be calculated based on the luminescence intensity-concentration standard curve drawn by the concentration gradient detection of heavy metal standard substances.

9. The method according to claim 8, characterized in that In step (2), the heavy metal antigen-nanozyme conjugate is diluted with a sample diluent to a concentration of 100 to 1000 ng / mL; The anti-heavy metal antibody-magnetic particle conjugate is diluted with a sample diluent to a concentration of 100 to 1000 ng / mL; Preferably, in step (4), the concentration of the enzymatic chemiluminescent substrate solution is 0.5 to 2 mmol / mL.

10. The method according to claim 8, characterized in that The sample to be tested is selected from food or oil products; Preferably, the food includes rice, wheat, corn or food products; Preferably, the oil comprises peanut oil, corn oil, soybean oil, rapeseed oil, sunflower oil, rapeseed oil, rice bran oil, sesame oil or olive oil; Preferably, the heavy metal is selected from one or more of cadmium, lead and mercury.

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