A method for detecting carbendazim residue based on personal blood glucose meter

Abstract

The present invention discloses a method for detecting carbendazim residues based on a personal blood glucose meter. The present application is an improvement on the traditional ELISA method. sAb and ALP are coupled to AuNPs at the same time. ALP catalyzes the dephosphorylation of G‑1‑P to generate glucose molecules. PGM is used as a signal reading tool to realize the portable quantitative detection of CBD. The present method utilizes the high specific surface area of ​​AuNPs and the dual signal amplification strategy of TdT catalyzing the extension of single-stranded DNA to realize the effective amplification of the detection signal, so that the present method has a high sensitivity. Since the present method is a detection method constructed based on the specific recognition of antigens and antibodies, it has good specificity and strong anti-interference ability. The present method realizes the portable quantitative detection of CBD by PGM, overcoming the disadvantage that the traditional ELISA can only be accurately quantitatively analyzed with the help of large instruments and equipment such as microplate readers. Therefore, it is very suitable for on-site rapid detection and analysis, and has a high application potential in resource-scarce areas.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticide residue detection, and in particular relates to a method for detecting carbendazim residue based on a personal blood glucose meter. Background Art

[0002] Carbendazim1., CBD is a multi-purpose broad-spectrum benzimidazole fungicide, which is widely used to prevent and treat various fruit and vegetable diseases caused by fungi. However, the half-life of CBD in the environment is as long as 12 months, and the 2-aminobenzimidazole formed by the degradation of CBD is a highly toxic component, which poses a certain potential threat to the health of organisms. Many researchers have studied the toxic effects of different levels of CBD on different animals. It has been observed that CBD can inhibit cell division and make mice infertile or stunted. In addition, CBD may cause liver cell dysfunction, affect hematopoiesis, increase estrogen levels, disrupt the endocrine system, and have other potential threats. Based on the multiple toxic effects of CBD, the World Health Organization has listed CBD as a hazardous chemical, the European Union has listed it as an endocrine disruptor, and many countries have stipulated the maximum residue limit of CBD in agricultural products. Therefore, it is of great significance to detect and analyze CBD residues in agricultural products and foods. Traditional detection methods, such as ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and capillary electrophoresis (CE), have high accuracy and sensitivity, but these methods require the use of large amounts of organic solvents, cumbersome sample pretreatment processes, expensive equipment, and require professional training for operators. These shortcomings have limited their application in rapid screening of CBD residues.

[0003] In previous literature reports, many researchers have used different enzyme-labeled antibodies on the basis of traditional ELISA to regulate the output detection signal, so that the detection signal can be read by small commercial detection equipment, and finally developed a new ELISA method based on personal glucose meter (PGM), portable pH meter, thermometer, potentiometer and barometer, realizing portable quantitative detection of the target. Among them, PGM is widely used in the development of various target detection methods in addition to its small size, simple operation, low cost, stable and reliable results. For example, Li et al. coupled antibodies and invertase (sucrase) to AuNPs to obtain antibody / invertase-AuNPs probes. Due to the high specific surface area of ​​AuNPs, multiple antibodies and invertases can be coupled on its surface, so the introduction of AuNPs will significantly amplify the detection signal. Subsequently, they used the probe to construct ELISA. The invertase on the antibody / invertase-AuNPs probe catalyzed the hydrolysis of sucrose to produce glucose, and then the concentration of glucose in the detection solution was determined by PGM, thereby achieving the quantitative detection of trencro in pork by PGM, and the LOD of this method was as low as 0.1 ng / mL. In addition, Li et al. synthesized an antibody-invertase nanoparticle using the reverse micelle method, which contained a large amount of antibodies and invertase. In the ELISA constructed with this nanoparticle, after the target antigen was captured by the double antibody sandwich method, the invertase catalyzed the hydrolysis of sucrose to produce glucose, and the glucose concentration in the detection solution was measured by PGM to achieve the quantitative detection of the tumor marker alpha-fetoprotein antigen, with an LOD of 5.4 pg / mL. From the above examples, it can be seen that the development of an ELISA detection method with PGM as a signal readout device requires the conversion of changes in the concentration of the substance to be tested into changes in the glucose concentration, so the selection of the associated enzyme in ELISA is particularly important. In addition to invertase, glucose oxidase and ALP can achieve this goal. Glucose oxidase can specifically catalyze glucose to produce gluconic acid, so that the glucose concentration decreases with the increase of glucose oxidase concentration; ALP can efficiently remove the phosphate group in the substrate G-1-P to generate glucose, so that the glucose concentration increases with the increase of ALP concentration. Among them, the enzyme-catalyzed signal amplification function of ALP can also be conveniently combined with the SA-biotin signal amplification system, which can significantly improve the detection sensitivity of ELISA. Therefore, ALP has great application potential in the construction of PGM-based ELISA detection methods, but related research has not been reported.

[0004] In a chemiluminescent enzyme-linked immunosorbent assay for detecting carbendazim disclosed in CN105572347A, carbendazim-OVA complete antigen is coated on ELISA plate, and then used for standby after closing with blocking solution. During detection, 50 μL of carbendazim 50 μL and polyclonal antibody (1:1000 times dilution) 50 μL of concentrations are added to the coated ELISA plate, room temperature reaction 2-4h, freshly diluted horseradish peroxidase-sheep anti-rabbit IgG antibody 100 μL / holes are added after washing, room temperature reaction 1.5h, 100 μL / holes of luminescent solution of temporary configuration are added in each hole after washing, then immediately measured with chemiluminescent analyzer, standard curve is made according to luminescent value, the luminescent value measured by unknown concentration carbendazim is substituted into the equation of standard curve, the carbendazim concentration in target object is calculated. But the overall reaction time of the technology is too long, and the purpose of rapid detection can not be achieved. Secondly, the luminescent solvent it uses is a reaction system of luminol solution and hydrogen peroxide. This signal conversion system can only generate a single blue color. The human eye is not sensitive to changes in a single color, so professional instruments are needed to measure and perform accurate quantification. In addition, the luminescence time of luminol is very limited and needs to be measured in a hurry, which puts higher requirements on on-site testing.

[0005] In a kind of enzyme-linked immunosorbent assay kit and its detection method for detecting carbendazim disclosed in CN105510589A, a set of rapid detection methods for detecting carbendazim residues is also constructed using traditional enzyme-linked immunosorbent assay. The principle of the method is substantially similar to CN105572347A, which is the carbendazim in the sample and the antigen-specific competitive antibody fixed on the ELISA plate, and the second antibody and antibody reaction labeled with horseradish peroxidase (HRP) are added, and the color is developed by enzyme-catalyzed developer, and the content of carbendazim in the sample is judged according to the depth of color. Although its reaction time is greatly shortened, the whole only needs about 45-50min, but the final color of the application color developing solution is a yellow of different shades, and the human eye can not obviously perceive the depth change of a single color, so it is still necessary for an ELISA instrument to carry out quantitative analysis, so it is difficult to carry out field detection.

[0006] Based on the above analysis, a method for detecting polymicrobial residues with fast response speed, short detection time, simple operation steps, high sensitivity, good specificity, and strong anti-interference ability is urgently needed in the industry, which can overcome the defect that traditional ELISA can only be used for accurate quantitative analysis with the help of large instruments such as enzyme markers. Summary of the invention

[0007] In view of the above shortcomings, the present invention improves the signal output mode of the traditional enzyme-linked immunosorbent assay (ELISA), and finally realizes the rapid detection of carbendazim using a portable personal blood glucose meter. At the same time, in previous studies and patents, when using a personal blood glucose meter to detect the analyte, sucrose is mostly hydrolyzed by invertase to produce glucose. This study uses alkaline phosphatase (ALP) to catalyze the dephosphorylation of glucose-1-phosphate disodium salt to produce glucose, and this scheme is applied to the detection of CBD for the first time.

[0008] The present application intends to simultaneously couple a secondary antibody (sAb) and alkaline phosphatase (ALP) to AuNPs. ALP can catalyze the dephosphorylation of glucose-1-phosphate disodium salt (G-1-P) to generate glucose molecules. Finally, a personal blood glucose meter (PGM) is used to read the glucose concentration in the solution to achieve portable quantitative detection of CBD. In the present application, in order to modify ALP to the surface of AuNPs through the SA-biotin interaction strategy to retain the dual signal amplification functions of the enzyme catalysis of ALP and the SA-biotin signal amplification system, the present application intends to first modify the surface of AuNPs with sAb and single-stranded DNA ( Figure 1 A) to obtain sAb / DNA-AuNPs. The single-stranded DNA is then extended by terminal deoxynucleotidyl transferase (TdT). During the extension process, biotin-modified cytosine triphosphate deoxynucleotide (biotin-16-dCTP) is added to obtain biotin modification of multiple sites on the extended DNA. Finally, a large amount of streptavidin-labeled alkaline phosphatase (SA-ALP) is modified to the surface of AuNPs through biotin-streptavidin (SA-biotin) interaction, thereby achieving efficient amplification of the detection signal. The detection principle of CBD is as follows Figure 1 As shown in B, when the sample does not contain CBD, the monoclonal antibody (mAb) specifically binds to the coated antigen CBD-BSA to form a complex. After the sAb / DNA-AuNPs probe is added, the sAb on the probe efficiently binds to the mAb. TdT, biotin-16-dCTP, and SA-ALP are added in sequence, and a large amount of ALP is successfully modified to the surface of AuNPs. ALP efficiently removes the phosphate group in the substrate G-1-P to generate glucose, resulting in an increase in the solution PGM signal. When the sample contains CBD, the sAb / DNA-AuNPs probe captured on the ELISA plate decreases due to the competition between CBD and CBD-BSA for mAb, which ultimately leads to a decrease in the solution PGM signal. Based on this detection principle, portable quantitative detection of CBD by PGM can be achieved.

[0009] In order to achieve the above technical objectives, the present invention is implemented through the following technical solutions:

[0010] A method for detecting carbendazim residue based on a personal blood glucose meter, comprising:

[0011] (I) Synthesis of AuNPs:

[0012] AuNPs were synthesized by reducing HAuCl4 with sodium citrate. Before the synthesis, all glassware needed to be soaked in aqua regia (HCl:HNO3=3:1) for 15-20 minutes to remove impurities, then washed with ultrapure water and dried for use. Under magnetic stirring, 100mL of 1.0mM HAuCl4 solution was heated to boiling, and then 1mL of 400mM sodium citrate solution prepared in advance was quickly added to it. The mixed solution was heated under magnetic stirring and kept boiling for 8-10 minutes. When the color of the solution gradually turned purple-red, the heating was stopped and allowed to cool naturally to room temperature to obtain a stable AuNPs solution, which was finally stored in a 4°C refrigerator for use.

[0013] (II) Preparation of sAb / DNA-AuNPs probe:

[0014] like Figure 1 As shown in A, sAb / DNA-AuNPs probes were prepared according to the method described in previous literature:

[0015] (1) Purification of sAb by dialysis:

[0016] Take 1 mL of sAb and add it to the dialysis bag, seal it with a sealing clip, put the sealed dialysis bag into a beaker containing 10 mM PB (note that PB needs to completely cover the dialysis bag), and place it in a 4°C refrigerator for dialysis. Replace the dialysate (10 mM PB) every 4 to 6 hours. After the dialysate is replaced 5 to 6 times, the obtained sAb is placed in a 4°C refrigerator for standby use;

[0017] (2) Pretreatment of SH-DNA:

[0018] Add 2 μL of 1M Na3PO4 solution and 2 μL of 30mM TCEP solution to 30 μL of 100 μM SH-DNA, mix well and react at 25°C for 2 hours; add the reaction solution to an ultrafiltration tube and centrifuge at 10000 r / min for 20 minutes, wash 8 times, collect SH-DNA, and store in a 4°C refrigerator for later use;

[0019] (3) Take 100 μL of AuNPs, adjust its pH to 9 with 0.1 M K2CO3, add 4 μL of the SH-DNA product obtained in step (2), and react at 4°C for 16 h;

[0020] (4) Add 27.5 μL of PB containing 10% PEG20000 to the above solution, add 2 μL of dialyzed sAb, incubate at room temperature for 30 min, add 15 μL of NaCl solution, and equilibrate overnight at 4°C. The resulting solution was centrifuged at 12000 r / min for 30 min, the supernatant was discarded, and washed three times to remove excess SH-DNA and sAb. Finally, the prepared sAb / DNA-AuNPs probe was dispersed in PBS and stored in a 4°C refrigerator for later use.

[0021] (III) Establishment of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on AuNPs:

[0022] (1) Coating: Dilute CBD-BSA 32,000 times with CBS, add 100 μL / well to a 96-well ELISA plate, and incubate in a 37°C incubator for 3 h or 4°C overnight;

[0023] (2) Blocking: The coated ELISA plate was washed 3 to 5 times with PBST, patted dry, and then 300 μL / well of 5% skim milk powder was added. After incubation at 37°C for 40 min, the plate was taken out and washed 3 to 5 times with PBST. After patting dry, the plate was stored in a -50°C refrigerator for later use.

[0024] (3) Sample addition: Take out the pre-sealed ELISA plate, restore it to room temperature, add 50 μL / well of mAb and CBD standards diluted with PBS, mix well, and incubate at 37°C for 30 min;

[0025] (4) Adding sAb / DNA-AuNPs probe: Take out the ELISA plate from step (3) and wash it 3-5 times with PBST. After patting dry, add PBS (95 μL / well) and sAb / ALP-AuNPs (6 μL / well) probe, mix well and incubate at 37°C for 30 min.

[0026] (5) TdT reaction: Take out the ELISA plate from step (4) and wash it 3 to 5 times with PBST. After patting dry, add 0.3 μL TdT, 1.2 μL biotin-16-dCTP, and 98.5 μL TdT reaction buffer, mix well, and react at 37°C for 30 min.

[0027] (6) SA-biotin reaction: Take out the ELISA plate from step (5) and wash it 3-5 times with PBST. After patting it dry, add 1 μL SA-ALP and 99 μL Tris-HCl buffer and react at room temperature for 10 min.

[0028] (7) Adding reaction substrate: Take out the ELISA plate from step (6) and wash the plate 3 to 5 times with PBST. After patting dry, add 100 μL of 60 mg / mL G-1-P solution;

[0029] (8) Determination: After the above solution is reacted at 37°C for 30 min, the glucose content therein is determined using PGM and the PGM value is recorded.

[0030] (IV) Portable detection of CBD:

[0031] A series of CBD standard solutions with different concentrations were prepared with PBS, with concentrations of 0, 0.13, 0.41, 1.23, 3.70, 11.1, 33.3 and 100 ng / mL, respectively. They were added as samples to the sample addition in step (3) for detection, with 3 sets of parallel experiments for each concentration, and other conditions were strictly tested according to the optimized optimal conditions (i.e., all conditions in the establishment of indirect competitive enzyme-linked immunosorbent based on AuNPs). Finally, the PGM signal values ​​presented in the detection solution under different CBD concentrations were recorded.

[0032] The beneficial effects of the present invention are:

[0033] 1. This method improves the traditional ELISA by simultaneously coupling sAb and ALP on AuNPs. ALP catalyzes the dephosphorylation of G-1-P to generate glucose molecules, and PGM is used as a signal reading tool to achieve portable quantitative detection of CBD.

[0034] 2. This method utilizes the high specific surface area of ​​AuNPs and the dual signal amplification strategy of TdT-catalyzed single-stranded DNA extension to achieve effective amplification of the detection signal, making this method have high sensitivity.

[0035] 3. Since this method is a detection method based on the specific recognition of antigen and antibody, it has good specificity. In addition, this method has strong anti-interference ability, and in the detection of actual samples, this application has also obtained satisfactory results.

[0036] 4. This method realizes the portable quantitative detection of CBD by PGM, overcoming the disadvantage that the traditional ELISA can only be accurately quantitatively analyzed with the help of large instruments such as microplate reader. Therefore, it is very suitable for rapid on-site detection and analysis, and has high application potential in resource-scarce areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 (A) is a schematic diagram of the preparation of sAb / DNA-AuNPs probe; Figure 1 (B) Schematic diagram of AuNPs-based portable ELISA for CBD detection.

[0038] Figure 2PGM signals under different conditions: a represents SA-ALP+G-1-P; b represents TdT+G-1-P; c represents biotin+G-1-P; d represents sAb+G-1-P; e represents AuNPs+G-1-P; f represents mAb+G-1-P; g represents sAb / DNA+G-1-P.

[0039] Figure 3 Agarose gel electrophoresis analysis of TdT products of DNA: Lane A represents the product of the reaction between TdT and low concentration Co2+; Lane B represents the product of the reaction between TdT and high concentration Co2+.

[0040] Figure 4 Schematic diagram of PGM-based ELISA detection of CBD.

[0041] Figure 5 The PGM signal values ​​of the PGM-based ELISA method: (A) indicates no CBD; (B) indicates a CBD concentration of 1000 ng / mL.

[0042] Figure 6 PGM signal values ​​of the constructed AuNPs-based portable ELISA method: (A) indicates no CBD; (B) indicates a CBD concentration of 1000 ng / mL.

[0043] Figure 7 Figure 2 is a standard curve of PGM signal as CBD concentration increased from 0.13 ng / mL to 100 ng / mL; error bars represent standard deviation (n=3).

[0044] Figure 8 For analysis using AuNPs-based portable ELISA; from left to right compounds: bromothionil, methamidophos, metalaxyl, spirotetramat, acetamiprid, kresoxim-methyl, kasugamycin, captan, methyl thiophanate, benomyl, prochloraz, imazalil, phoxim, avermectin, PBS and CBD.

[0045] Fig. 9 Figure 2 is a standard curve of PGM signal as CBD concentration increased from 0.13 ng / mL to 100 ng / mL in 10% Chinese cabbage matrix; error bars represent standard deviation (n=3).

[0046] Fig.10 Standard curve of PGM signal as CBD concentration increased from 0.13 ng / mL to 100 ng / mL in 10% citrus matrix; error bars represent standard deviation (n=3).

[0047] Fig.11Calibration curve of PGM signal as CBD concentration increased from 0.13 ng / mL to 100 ng / mL in 10% canned orange peel matrix; error bars represent standard deviation (n=3). DETAILED DESCRIPTION

[0048] A method for detecting carbendazim residue based on a personal blood glucose meter, comprising:

[0049] (I) Synthesis of AuNPs:

[0050] AuNPs were synthesized by reducing HAuCl4 with sodium citrate. Before the synthesis, all glassware needed to be soaked in aqua regia (HCl:HNO3=3:1) for 15-20 minutes to remove impurities, then washed with ultrapure water and dried for use. Under magnetic stirring, 100mL of 1.0mM HAuCl4 solution was heated to boiling, and then 1mL of 400mM sodium citrate solution prepared in advance was quickly added to it. The mixed solution was heated under magnetic stirring and kept boiling for 8-10 minutes. When the color of the solution gradually turned purple-red, the heating was stopped and allowed to cool naturally to room temperature to obtain a stable AuNPs solution, which was finally stored in a 4°C refrigerator for use.

[0051] (II) Preparation of sAb / DNA-AuNPs probe:

[0052] like Figure 1 As shown in A, sAb / DNA-AuNPs probes were prepared according to the method described in previous literature:

[0053] (1) Purification of sAb by dialysis:

[0054] Take 1 mL of sAb and add it to the dialysis bag, seal it with a sealing clip, put the sealed dialysis bag into a beaker containing 10 mM PB (note that PB needs to completely cover the dialysis bag), and place it in a 4°C refrigerator for dialysis. Replace the dialysate (10 mM PB) every 4 to 6 hours. After the dialysate is replaced 5 to 6 times, the obtained sAb is placed in a 4°C refrigerator for standby use;

[0055] (2) Pretreatment of SH-DNA:

[0056] Add 2 μL of 1M Na3PO4 solution and 2 μL of 30mM TCEP solution to 30 μL of 100 μM SH-DNA, mix well and react at 25°C for 2 hours; add the reaction solution to an ultrafiltration tube and centrifuge at 10000 r / min for 20 minutes, wash 8 times, collect SH-DNA, and store in a 4°C refrigerator for later use;

[0057] (3) Take 100 μL of AuNPs, adjust its pH to 9 with 0.1 M K2CO3, add 4 μL of the SH-DNA product obtained in step (2), and react at 4°C for 16 h;

[0058] (4) Add 27.5 μL of PB containing 10% PEG20000 to the above solution, add 2 μL of dialyzed sAb, incubate at room temperature for 30 min, then add 15 μL of NaCl solution and equilibrate overnight at 4°C. The resulting solution was centrifuged at 12000 r / min for 30 min, the supernatant was discarded, and the mixture was washed three times to remove excess SH-DNA and sAb. Finally, the prepared sAb / DNA-AuNPs probe was dispersed in PBS and stored in a 4°C refrigerator for later use.

[0059] (III) Establishment of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on AuNPs:

[0060] (1) Coating: Dilute CBD-BSA 32,000 times with CBS, add 100 μL / well to a 96-well ELISA plate, and incubate in a 37°C incubator for 3 h or 4°C overnight;

[0061] (2) Blocking: The coated ELISA plate was washed 3 to 5 times with PBST, patted dry, and then 300 μL / well of 5% skim milk powder was added. After incubation at 37°C for 40 min, the plate was taken out and washed 3 to 5 times with PBST. After patting dry, the plate was stored in a -50°C refrigerator for later use.

[0062] (3) Sample addition: Take out the pre-sealed ELISA plate, restore it to room temperature, add 50 μL / well of mAb and CBD standards diluted with PBS, mix well, and incubate at 37°C for 30 min;

[0063] (4) Adding sAb / DNA-AuNPs probe: Take out the ELISA plate from step (3) and wash it 3-5 times with PBST. After patting dry, add PBS (95 μL / well) and sAb / ALP-AuNPs (6 μL / well) probe, mix well and incubate at 37°C for 30 min.

[0064] (5) TdT reaction: Take out the ELISA plate from step (4) and wash it 3 to 5 times with PBST. After patting dry, add 0.3 μL TdT, 1.2 μL biotin-16-dCTP, and 98.5 μL TdT reaction buffer, mix well, and react at 37°C for 30 min.

[0065] (6) SA-biotin reaction: Take out the ELISA plate from step (5) and wash it 3-5 times with PBST. After patting it dry, add 1 μL SA-ALP and 99 μL Tris-HCl buffer and react at room temperature for 10 min.

[0066] (7) Adding reaction substrate: Take out the ELISA plate from step (6) and wash the plate 3 to 5 times with PBST. After patting dry, add 100 μL of 60 mg / mL G-1-P solution;

[0067] (8) Determination: After the above solution is reacted at 37°C for 30 min, the glucose content therein is determined using PGM and the PGM value is recorded.

[0068] (IV) Portable detection of CBD:

[0069] A series of CBD standard solutions with different concentrations were prepared with PBS, with concentrations of 0, 0.13, 0.41, 1.23, 3.70, 11.1, 33.3 and 100 ng / mL. They were added as samples to the detection step in step 3, and 3 groups of parallel experiments were performed for each concentration. Other conditions were optimized according to the best conditions and the experiment was carried out strictly according to the operating procedures. Finally, the PGM signal values ​​presented in the detection solution under different CBD concentrations were recorded.

[0070] Test Example 1

[0071] Specificity analysis

[0072] The specificity of the method was verified using some common non-target pesticides. Non-target pesticides include: bromothionil, methamidophos, metalaxyl, spirotetramat, acetamiprid, kresoxim-methyl, kasugamycin, captan, methyl thiophanate, benomyl, prochloraz, imazalil, phoxim and avermectin. All tests were performed under the same conditions, with the concentration of CBD at 100 ng / mL and the concentration of other non-target pesticides at 1000 ng / mL. Three parallel experiments were performed for each pesticide, and other conditions were strictly carried out according to the optimized best conditions (i.e., all conditions in the establishment of indirect competitive enzyme-linked immunosorbent based on AuNPs).

[0073] Test Example 2

[0074] Determination of CBD in complex matrices

[0075] The citrus, canned tangerine segments and cabbage samples used in this test example were all taken from Chongqing. LC-MS / MS was first used to analyze the CBD in citrus, canned tangerine segments and cabbage, proving that these samples did not contain any CBD residues. In order to obtain the above sample matrix, all samples were homogenized using a beater. Take 5 g of the homogenate and add 5 mL of acetonitrile to it. After oscillating and extracting for 10 minutes, the resulting mixture was frozen at -50°C for 10 minutes, and then 2 g of anhydrous MgSO4 and 0.5 g of NaCl were added, and the mixture was continuously oscillated for 1 minute. Finally, the mixture was centrifuged at 10,000 r / min at room temperature for 5 minutes, and the supernatant was taken for later use. The obtained sample matrix was diluted 10 times with ddH2O for the following experiments.

[0076] Finally, different concentrations of CBD standard solutions were prepared using the above-mentioned different food matrix dilutions, and the experiment was carried out according to the sample loading operation steps of step (3) in the establishment of indirect competitive enzyme-linked immunosorbent based on AuNPs. The linear standard curves under different matrix conditions were obtained and used for the subsequent calculation of the recovery rate of CBD addition in different food matrices.

[0077] Test Example 3

[0078] Determination of CBD in real samples:

[0079] This test example verifies the accuracy of the established method in actual sample detection by adding recovery. The citrus, canned orange segments and cabbage samples used were taken from Chongqing. The above samples were analyzed using LC-MS / MS, which proved that these samples did not contain any CBD residues. Different concentrations of CBD standard solutions (0.01, 0.1, 0.5, 5 μg / mL) were added to the above fruit and vegetable samples to simulate positive samples.

[0080] The above-mentioned fruit and vegetable samples were homogenized with a wall breaker. Afterwards, 5 g of different homogenates were taken, 50 μL of CBD standard solutions of different concentrations were added and mixed, and the mixture was allowed to stand for 30 minutes to allow CBD to be fully mixed with the sample. Subsequently, CBD was extracted using acetonitrile. 5 mL of acetonitrile was added to the above-mentioned simulated positive samples. After oscillating extraction for 10 minutes, the resulting mixture was frozen at -50°C for 10 minutes, 2 g of anhydrous MgSO4 and 0.5 g of NaCl were added, and the mixture was continuously oscillated for 1 minute. Finally, all samples were centrifuged at 10000 r / min for 5 minutes at room temperature. The supernatant was obtained and stored at -20°C until use.

[0081] The simulated positive samples with added levels of 0.01 and 0.1 μg / mL CBD were diluted 10 times with ddH2O. Then, the obtained solution was determined by the established PGM-based ELISA. However, the samples with added 0.5 and 5 μg / mL CBD of the other two simulated positive samples exceeded the detection range of the standard curve. Therefore, the present application first diluted these two samples 10 times with ddH2O. The obtained solution was further diluted 10 times and 50 times with the corresponding 10-fold diluted food matrix, and then detected by the above-mentioned established PGM-based ELISA detection method. Subsequently, the standard curve obtained in the 10% sample matrix was used to calculate the CBD concentration in the actual sample. Finally, the added recovery rate of CBD in different matrices was calculated based on the addition level and the detected concentration.

[0082] In addition, LC-MS / MS will be used to verify the accuracy of the method developed in this application. Before LC-MS / MS analysis, the supernatant obtained by acetonitrile extraction was filtered through a 0.2 μm organic membrane, and the CBD content was determined by LC-MS / MS to obtain its spike recovery rate.

[0083] Result analysis:

[0084] 1. Feasibility analysis

[0085] In order to confirm the feasibility of this scheme, this application is verified through a series of experiments. Because this experiment is conducted on the basis of ALP removing the phosphate group in G-1-P to generate glucose, this application needs to confirm whether other substances in the entire detection system can affect the PGM signal, and at the same time verify that only ALP can remove the phosphate group of G-1-P to produce glucose. The PGM used in this experiment can measure the glucose concentration range of 1.1~33.3mM. When PGM displays L0, it means that the glucose concentration in the test solution is less than 1.1mM. When PGM displays H1, it means that the glucose concentration in the test solution is greater than 33.3mM. As shown in Table 1, when PGM is used to measure each substance in the detection system separately, the results show that all substances will not generate PGM signals. As Figure 2 As shown, only when G-1-P and SA-ALP reacted, the glucose signal could be detected and was greater than 33.3 mM, while after other substances reacted with G-1-P, no glucose was detected in the test solution. The above results indicate that only SA-ALP can catalyze G-1-P to produce glucose in this detection system, and other substances in the detection system will not affect the detection signal of PGM.

[0086] Table 1 Effects of various substances in the detection system on PGM signals

[0087] SA-ALP G-1-P TdT biotin-16-dCTP sAb AuNPs mAbs sAb / DNA-AuNPs Buffer L0 L0 L0 L0 L0 L0 L0 L0

[0088] In this experimental protocol, signal amplification is achieved by extending the TdT end to form a biotin-labeled DNA sequence. 2+ It is an essential cofactor for TdT-catalyzed DNA end extension. Different concentrations of Co 2+ This may affect the catalytic efficiency of TdT. 2+ The DNA end extension products under the concentration were analyzed by agarose gel electrophoresis. The specific results are as follows Figure 3 As shown in the figure, the original DNA chain has 36 bases, and after being extended by TdT, the DNA size is distributed in the range of 150 to 300 bp. It can be seen from agarose gel electrophoresis analysis that TdT can extend DNA, thereby playing a role in signal amplification. And the present application can observe that at low concentrations of Co 2+ In this case, the DNA extension effect is better, so in subsequent experiments, low concentration Co 2+ as reaction conditions.

[0089] This application uses a strategy combining the high specific surface area of ​​AuNPs and TdT-catalyzed DNA chain extension to amplify the detection signal to improve the sensitivity of PGM to CBD detection. In order to prove the feasibility and necessity of this signal amplification strategy, this application designed a series of experiments for verification. First, this application constructed an ELISA that did not pass the above two signal amplification modes. Figure 5 As shown, CBD-BSA is used as the coating antigen, which competes with the CBD in the sample to bind to mAb. When the sample does not contain CBD, mAb binds to CBD-BSA to form a complex, and biotin-sAb is added thereto, and sAb is captured by mAb to form a CBD-BSA-mAb-sAb-biotin complex. Under the action of the specific binding of SA-biotin, ALP is connected, and the phosphate group of G-1-P removed by ALP generates glucose, resulting in an increase in the PGM signal. When the sample contains a certain concentration of CBD, CBD and CBD-BSA compete for binding to mAb, which ultimately leads to a decrease in the PGM signal in the solution. When this method is used to detect CBD, the results are as follows Figure 5 As shown. When the sample does not contain CBD, the PGM signal is 1.1mM, but when the sample contains CBD, no PGM signal is detected. This is because the PGM used in this experiment can measure the glucose concentration range of 1.1 to 33.3mM, and too low glucose concentration cannot be measured. Next, this application uses the ELISA method established in this work with the large specific surface area of ​​AuNPs and TdT enzyme-catalyzed signal amplification to measure the same concentration of CBD. The results are shown in Figure 6As shown in the figure, when the sample did not contain CBD, mAb was completely bound to CBD-BSA, more sAb / DNA-AuNPs probes were captured, and the final PGM signal was 14.9mM. When the sample contained 1000ng / mL CBD, CBD competed with CBD-BSA for binding to mAb, the captured sAb / DNA-AuNPs probes were also reduced, and the final PGM signal was 3.2mM.

[0090] The above experimental results show that the detection sensitivity of this method is improved by 15 times after dual signal amplification of AuNPs and TdT. In addition, since the PGM signal reading range is 1.1-33.3mM, ELISA without AuNPs and TdT signal amplification is not suitable for using PGM as a signal output tool for CBD detection.

[0091] 2. Determination of CBD:

[0092] Through the above optimization experiments, the optimal concentrations of various reagents in the detection system and the optimal reaction time between various substances were determined. Under the optimized experimental conditions, this application quantitatively analyzed CBD of different concentrations by observing PGM. Since this experiment is a detection method constructed by changing the signal output strategy based on traditional ELISA, the data processing and standard curve drawing methods of this experiment are the same as those of traditional ELISA. Figure 7 As shown in the figure, in the absence of CBD, the PGM signal is the largest, and when the CBD concentration increases, the PGM signal gradually decreases. Therefore, the CBD concentration is inversely proportional to the PGM signal. In the concentration range of 0.13 to 100 ng / mL, there is a good linear relationship between the CBD concentration and the PGM signal. The detection limit of this method is calculated to be 0.37 ng / mL, and its linear fitting equation is (where x represents the concentration of CBD and y represents the PGM signal):

[0093]

[0094] Compared with the traditional ELISA method, this method maintains the advantages of high sensitivity, fast detection speed, and simple operation steps. At the same time, it also makes up for the deficiency of the traditional method that requires an enzyme marker for accurate quantitative analysis, and provides a reliable method for rapid and accurate quantitative analysis of CBD on site.

[0095] In order to evaluate the specificity of the developed AuNPs-based portable ELISA for CBD detection, this application uses this method to detect other commonly used pesticides. Such as: bromothionil, methyl parathion, metalaxyl, spirotetramat, acetamiprid, kresoxim-methyl, kasugamycin, captan, methyl thiophanate, benomyl, prochloraz, imazalil, phoxim and avermectin. The CBD concentration is 100μg / mL, and the concentration of other pesticides is 1000μg / mL. Figure 8 As shown, when CBD is present, the PGM signal is the lowest. When other pesticides are present, the PGM signal is consistent with the signal when detecting blank buffer (PBS), indicating that the method cannot detect other pesticides. When benomyl and methyl thiophanate are detected, the PGM signal is between the two. The reason for this phenomenon is that benomyl and methyl thiophanate are easily decomposed into CBD. The above results show that the detection method established in this application has good specificity for CBD.

[0096] 3. Determination of actual samples:

[0097] In order to prove whether the portable ELISA detection method based on AuNPs established in this application has the potential to be applied to actual sample detection, this application adds CBD of different concentrations to actual samples for detection. The actual samples selected in this application include: citrus, canned orange segments and cabbage. It is worth noting that these sample matrices have been tested by LC-MS / MS before use to prove that they do not contain any CBD residues. These matrices contain complex components that may affect the specific binding of antigens and antibodies. Therefore, in order to eliminate this effect, this application established standard curves in 3 different matrices. The subsequent determination of the recovery rate of addition is calculated by the standard curve obtained by its corresponding matrix.

[0098] like Fig. 9 , Fig.10 and Fig.11 As shown. In 3 different matrices, as the CBD concentration increased from 0 ng / mL to 100 ng / mL, the PGM signal of all test solutions decreased. In different matrix solutions, the present application obtained a linear fitting relationship between the CBD concentration and the PGM signal. These results show that the PGM-based ELISA developed by the present application can detect CBD with high sensitivity in various complex matrices.

[0099] Next, based on the standard curves established in the three matrices, the present application conducted an additive recovery test of CBD in the above three sample matrices. The recovery rates of CBD in different samples ranged from 70.4% to 109.4%, and the relative standard deviations ranged from 5.8% to 12.8%. In order to evaluate the accuracy of the AuNPs-based portable ELISA established in this application, the LC-MS / MS method was used to measure the above-mentioned spiked samples, and then compared with the recovery rates obtained by the method established in this application. The detailed additive recovery data are shown in Table 2. The RSD of the new ELISA method established in this application is slightly higher than that of LC-MS / MS, but the recovery rates presented by the two methods are basically the same. Therefore, the detection accuracy of the AuNPs-based portable ELISA established in this application is acceptable. Based on the above results, the AuNPs-based portable ELISA developed in this application has great application potential in on-site detection of CBD.

[0100] Table 2 Recovery of carbendazim in actual samples

[0101]

[0102]

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting carbendazim residue based on a personal blood glucose meter, comprising: Preparation of sAb / DNA-AuNPs probes; Establishment of indirect competitive enzyme-linked immunosorbent assay based on AuNPs; as well as Rapid testing; The detection principle is: the secondary antibody and alkaline phosphatase are coupled to AuNPs at the same time, ALP can catalyze the dephosphorylation of glucose-1-phosphate disodium salt to generate glucose molecules, and finally a personal blood glucose meter is used to read the glucose concentration in the solution to achieve portable quantitative detection of carbendazim; wherein: In order to modify ALP onto the surface of AuNPs through the SA-biotin interaction strategy, in order to retain the dual signal amplification functions of the enzyme catalysis of ALP and the SA-biotin signal amplification system, sAb and single-stranded DNA were first modified on the surface of AuNPs to obtain sAb / DNA-AuNPs probes, and then the single-stranded DNA was extended by terminal deoxynucleotidyl transferase. During the extension process, biotin-modified cytosine triphosphate deoxynucleotides were added to obtain biotin modification of multiple sites on the extended DNA. Finally, a large amount of streptavidin-labeled alkaline phosphatase was modified onto the surface of AuNPs through the biotin-streptavidin interaction, thereby achieving efficient amplification of the detection signal. When the sample did not contain carbendazim, the monoclonal antibody specifically bound to the coated antigen carbendazim-BSA to form a complex. After the sAb / DNA-AuNPs probe was added, the sAb on the probe efficiently bound to the mAb. TdT, biotin-16-dCTP, and SA-ALP were added in sequence, and a large amount of ALP was successfully modified on the surface of AuNPs. ALP efficiently removed the phosphate group in the substrate G-1-P to generate glucose, resulting in an increase in the PGM signal of the solution. When carbendazim was present in the sample, the sAb / DNA-AuNPs probes captured on the ELISA plate decreased due to the competition between carbendazim and carbendazim-BSA for binding to mAb, which ultimately led to a decrease in the solution PGM signal.

2. The method according to claim 1, wherein: The AuNPs solution was synthesized by the following method: Under magnetic stirring, 100 mL of 1.0 mM HAuCl4 solution was heated to boiling, and 1 mL of 400 mM sodium citrate solution was added to obtain a first mixed solution; The first mixed solution was heated continuously under magnetic stirring and boiled for 8-10 minutes, and the heating was stopped; the solution was naturally cooled to room temperature and stored in a refrigerator at 4°C to obtain an AuNPs solution.

3. The method according to claim 2, wherein: Before the synthesis of the AuNPs solution, the glassware used was soaked in aqua regia for 15 to 20 minutes, then washed with ultrapure water and dried for later use.

4. The method according to claim 1, wherein: The preparation of the sAb / DNA-AuNPs probe comprises: (1) Purification of sAb by dialysis: Take 1 mL of sAb and add it to the dialysis bag. Place the sealed dialysis bag in a beaker containing 10 mM PB and place it in a 4°C refrigerator for dialysis to obtain the purified sAb for later use. (2) Pretreatment of SH-DNA: Add 2 μL of 1M Na3 PO4 solution and 2 μL of 30 mM TCEP solution to 30 μL of 100 μM SH-DNA, mix well and react at 25°C for 2 h; add the reaction solution to an ultrafiltration tube and centrifuge at 10000 r / min for 20 min. After washing 8 times, the pretreated SH-DNA was obtained and stored in a 4°C refrigerator for later use; (3) Take 100 μL of AuNPs, adjust its pH to 9 with 0.1 M K2 CO3, add 4 μL of pretreated SH-DNA, and react at 4 °C for 16 h to obtain the first mixed solution; (4) adding 27.5 μL of PB containing 10% PEG20000 to the first mixed solution, adding 2 μL of purified sAb, incubating at room temperature for 30 min, adding 15 μL of NaCl solution, and equilibrating at 4°C overnight to obtain a second mixed solution; (5) The second mixed solution was centrifuged at 12000 r / min for 30 min, the supernatant was discarded, and the mixture was washed three times to obtain sAb / DNA-AuNPs probe: The probe was dispersed in PBS and stored in a 4°C refrigerator for future use.

5. The method according to claim 4, wherein: In the dialysis treatment of step (1), the dialysis fluid is replaced every 4 to 6 hours, and the dialysis fluid is replaced 5 to 6 times.

6. The method according to claim 1, wherein: The establishment of the indirect competitive enzyme-linked immunosorbent based on AuNPs includes: (1) Coating: dilute carbendazim-BSA 32,000 times with CBS, add 100 μL / well to a 96-well ELISA plate, and incubate in a constant temperature incubator at 37°C for 3 h or at 4°C overnight; (2) Blocking: The coated ELISA plate was washed 3-5 times with PBST, patted dry, and then 300 μL / well of 5% skim milk powder was added. After incubation at 37°C for 40 min, the plate was taken out and washed 3-5 times with PBST. After patting dry, the plate was stored in a -50°C refrigerator for later use. (3) Sample addition: Take out the sealed ELISA plate, restore it to room temperature, add 50 μL / well of mAb and carbendazim standard diluted in PBS, mix well and incubate at 37°C for 30 min; (4) Adding sAb / DNA-AuNPs probe: Take out the ELISA plate obtained in step (3) and wash it with PBST for 3 to 5 times. After patting dry, add PBS and sAb / ALP-AuNPs probe, mix well and incubate at 37°C for 30 min. (5) TdT reaction: Take out the ELISA plate obtained in step (4) and wash it 3 to 5 times with PBST. After patting it dry, add 0.3 μL TdT, 1.2 μL biotin-16-dCTP, 98.5 μL TdT reaction buffer, mix well and react at 37°C for 30 min; (6) SA-biotin reaction: Take out the ELISA plate obtained in step (5) and wash it 3-5 times with PBST. After patting it dry, add 1 μL SA-ALP and 99 μL Tris-HCl buffer and react at room temperature for 10 min. (7) Adding reaction substrate: Take out the ELISA plate obtained in step (6) and wash the plate 3 to 5 times with PBST. After patting dry, add 100 μL of 60 mg / mL G-1-P solution; (8) Determination: The solution obtained in step (7) was reacted at 37° C. for 30 min, and the glucose content therein was determined using PGM. Record the PGM value.

7. The method according to claim 6, wherein: In step (4), the PBS addition standard is 95 μL / well; the sAb / ALP-AuNPs addition standard is 6 μL / well.

8. The method according to claim 1, wherein: The rapid test includes: Different concentrations of carbendazim standard solutions were prepared with PBS and used as sample addition treatment. Three groups of parallel experiments were performed for each concentration, and the PGM signal values ​​presented in the test solution at different concentrations of carbendazim were recorded.

9. The method according to claim 8, wherein: The carbendazim standard solutions of different concentrations include: The concentrations of carbendazim standard solutions are 0, 0.13, 0.41, 1.23, 3.70, 11.1, 33.3 and 100 ng / mL respectively.

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