CN115219716A

CN115219716A - Method for detecting carbendazim residue based on personal blood glucose meter

Info

Publication number: CN115219716A
Application number: CN202210781736.1A
Authority: CN
Inventors: 何悦; 刘浩然; 王成秋; 焦必宁
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2022-10-21

Abstract

The invention discloses a method for detecting carbendazim residue based on a personal blood glucose meter, which is an improvement on the traditional ELISA method, and comprises the steps of simultaneously coupling sAb and ALP on AuNPs, catalyzing G-1-P dephosphorylation by the ALP to generate glucose molecules, and using PGM as a signal reading tool to realize portable quantitative detection of CBD. The method realizes effective amplification of detection signals by using a double signal amplification strategy of high specific surface area of AuNPs and TdT catalysis single-stranded DNA extension, so that the method has higher sensitivity. The method is a detection method constructed based on the specific recognition function of the antigen and the antibody, so that the method has better specificity and strong anti-interference capability. The method realizes portable quantitative detection of PGM on CBD, and overcomes the defect that the traditional ELISA can only carry out accurate quantitative analysis by means of large-scale instruments such as an enzyme-linked immunosorbent assay (ELISA) instrument. Therefore, the method is very suitable for on-site rapid detection and analysis and has higher application potential in resource-poor areas.

Description

Method for detecting carbendazim residue based on personal blood glucose meter

Technical Field

The invention belongs to the technical field of pesticide residue detection, and particularly relates to a method for detecting carbendazim residue based on a personal blood glucose meter.

Background

Carbendazim (CBD) is a multi-purpose broad-spectrum benzimidazole fungicide, and is widely used for controlling various fruit and vegetable diseases caused by fungi. However, CBD has a half-life of 12 months in the environment, and 2-aminobenzimidazole formed by the degradation of CBD is a highly toxic component and poses a certain potential threat to the health of organisms. Many researchers have investigated the toxic effects of different levels of CBD on different animals. It was observed that CBD could inhibit cell division, rendering mice infertile or stunted. In addition, CBD may cause hepatocyte dysfunction, affect hematopoiesis, increase estrogen levels, disturb the endocrine system, and have other potential threats. Based on the multiple toxic effects of CBD, CBD is classified as a dangerous chemical by the world health organization, as an endocrine disruptor by the european union, and the maximum residual limit of CBD in agricultural products is regulated by many countries. Therefore, it is very important to detect and analyze the CBD residue in agricultural products and food. 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 specialized training of operators. These deficiencies have led to limited use in rapid screening for CBD residuals.

In the prior art, many researchers have developed a novel ELISA method based on a Personal Glucose Meter (PGM), a portable pH meter, a thermometer, a potentiometer, and a barometer by controlling an output detection signal by using different enzyme-labeled antibodies based on a conventional ELISA so that the detection signal can be read by a small-sized and commercialized detection device, and thus, a portable quantitative detection of a target object has been achieved. Among them, PGM is widely used in the development of methods for detecting various targets, in addition to the measurement of personal blood glucose, because of its small size, simple operation, low cost, and stable and reliable results. For example, li et al couple antibodies and invertase (sucrase) to AuNPs, resulting in antibody/invertase-AuNPs probes. The introduction of AuNPs will amplify the detection signal significantly due to the high specific surface area of AuNPs, which can couple multiple antibodies and convertases on their surface. Then, they use the probe for ELISA construction, invertase on the antibody/invertase-AuNPs probe catalyzes sucrose hydrolysis to generate glucose, and glucose concentration in the detection solution is determined by PGM, so that quantitative detection of the PGM on Torrencol in pork is realized, and LOD of the method is as low as 0.1ng/mL. In addition, li et al synthesized an antibody-converting enzyme nanoparticle containing a large amount of antibody and converting enzyme by the reverse micelle method. According to ELISA constructed by the nano-particles, after a target antigen is captured by a double-antibody sandwich method, invertase catalyzes sucrose hydrolysis to generate glucose, PGM is used for measuring the concentration of the glucose in a detection solution, and then the quantitative detection of a tumor marker alpha fetoprotein antigen can be realized, and LOD is 5.4pg/mL. As can be seen from the above examples, when developing an ELISA method using PGM as a signal readout device, it is necessary to convert the change in the concentration of the analyte into the change in the concentration of glucose, and therefore, the selection of the cognate enzyme in ELISA is very important. In addition to invertase, both glucose oxidase and ALP can achieve this goal. The glucose oxidase can specifically catalyze glucose to generate gluconic acid, so that the concentration of the glucose is reduced along with the increase of the concentration of the glucose oxidase; ALP efficiently removes phosphate groups in the substrate G-1-P to generate glucose, so that the glucose concentration increases as the ALP concentration increases. Wherein, the enzyme catalysis signal amplification function of ALP can be conveniently combined with an SA-biotin signal amplification system, and the detection sensitivity of ELISA can be obviously improved. Therefore, ALP has great application potential in constructing ELISA detection method based on PGM, and only relevant research is not reported yet.

CN105572347A discloses a chemiluminescence enzyme-linked immunoassay method for detecting carbendazim, which comprises coating carbendazim-OVA complete antigen on an enzyme label plate, and then sealing with a sealing liquid for later use. In the detection process, 50 mu L of carbendazim with different concentrations and 50 mu L of polyclonal antibody (1. However, the whole reaction time of the technology is too long to achieve the purpose of rapid detection. Secondly, the adopted luminescent solvent is a reaction system of the luminol solution and the hydrogen peroxide, the signal conversion system can only generate single blue, human eyes are insensitive to the change of single color, so that a professional instrument is needed for measuring and accurate quantification, the luminol luminescence time is very limited, the grasping time is needed for measuring, and higher requirements are provided for field detection.

In an enzyme linked immunosorbent assay kit for detecting carbendazim and a detection method thereof disclosed in CN105510589A, a set of rapid detection methods for detecting carbendazim residues are also constructed by utilizing a traditional enzyme linked immunosorbent assay method. The principle of the method is basically similar to that of CN105572347A, and the method is that carbendazim in a sample and antigen specificity competitive antibodies fixed on an enzyme label plate are added, horseradish peroxidase (HRP) -labeled secondary antibodies are added to react with the antibodies, a chromogenic reagent is catalyzed by the enzymes to develop color, and the content of the carbendazim in the sample is judged according to the color depth. Although the reaction time is greatly shortened and only about 45-50min is needed as a whole, the final color of the developing solution is yellow with different shades, and human eyes cannot obviously perceive the shade change of a single color, so that an enzyme-labeling instrument is still needed for quantitative analysis, and the on-site detection is difficult.

Based on the analysis, the method for detecting the residual of the ganoderma lucidum is high in response speed, short in detection time, simple in operation steps, high in sensitivity, good in specificity and strong in anti-interference capability, and can overcome the defect that the traditional ELISA can only carry out accurate quantitative analysis by means of large instruments such as an enzyme-linked immunosorbent assay (ELISA) instrument.

Disclosure of Invention

In view of the above disadvantages, the present invention improves the signal output mode of the conventional enzyme-linked immunosorbent assay (ELISA), and finally realizes the rapid detection of carbendazim using a portable personal blood glucose meter. Meanwhile, in previous researches and patents, most of the tests of the object to be tested by using a personal glucometer generate glucose by hydrolyzing sucrose with invertase, and the research uses alkaline phosphatase (ALP) to catalyze the dephosphorylation of glucose-1-disodium phosphate to generate glucose, and applies the scheme to the detection of CBD for the first time.

The application aims to couple a secondary antibody (sAb) and alkaline phosphatase (ALP) on AuNPs simultaneously, wherein the ALP can catalyze dephosphorylation of glucose-1-disodium phosphate (G-1-P) to generate glucose molecules, and finally, a Personal Glucometer (PGM) is adopted to read the glucose concentration in the solution so as to realize portable quantitative detection of CBD. In the present application, in order to modify ALP to the surface of AuNPs by a strategy of SA-biotin interaction to retain the dual signal amplification function of the enzymatic catalysis of ALP and the SA-biotin signal amplification system, the present application intends to first modify sAb and single-stranded DNA on the surface of AuNPs (fig. 1A) to obtain sAb/DNA-AuNPs. And then extending the single-stranded DNA by using terminal deoxynucleotidyl transferase (TdT), adding biotin-modified cytosine triphosphate deoxynucleotide (biotin-16-dCTP) in the extension process to obtain biotin modification of multiple sites on the extended DNA, and finally modifying a large amount of streptavidin-labeled alkaline phosphatase (SA-ALP) to the surface of AuNPs through interaction of biotin-streptavidin (SA-biotin), thereby realizing high-efficiency amplification of detection signals. As shown in FIG. 1B, when the sample does not contain CBD, the monoclonal antibody (mAb) binds specifically to the envelope antigen CBD-BSA to form a complex, and after the sAb/DNA-AuNPs probe is added, the sAb on the probe binds to the mAb efficiently. TdT, biotin-16-dCTP and SA-ALP are added in sequence, a large amount of ALP is successfully modified on the surface of AuNPs, phosphate groups in a substrate G-1-P are efficiently removed through ALP, glucose is generated, and the PGM signal of the solution is increased. When the sample contains CBD, the competition between CBD and CBD-BSA for mAb binding results in a decrease of the amount of sAb/DNA-AuNPs probe captured on the microplate, and ultimately a decrease of the PGM signal of the solution. Based on the detection principle, portable quantitative detection of PGM on CBD can be realized.

In order to realize the technical purpose, the invention is realized by the following technical scheme:

a method for detecting carbendazim residue based on a personal blood glucose meter, comprising:

firstly, synthesizing AuNPs:

reduction of HAuCl with sodium citrate ₄ To synthesize AuNPs. All glassware was treated with aqua regia (HCl: HNO) prior to synthesis ₃ = 3) for 15-20 min to remove impurities, and then washed with ultrapure water and dried for later use. 100mL of 1.0mM HAuCl were added under magnetic stirring ₄ The solution was heated to boiling, and 1mL of a pre-prepared 400mM sodium citrate solution was then added rapidly. And continuously heating the mixed solution under the magnetic stirring state, keeping the boiling state for 8-10 min, stopping heating after the color of the solution gradually changes into mauve, naturally cooling to room temperature to obtain a stable AuNPs solution, and finally storing the stable AuNPs solution in a refrigerator at 4 ℃ for later use.

(II) preparation of sAb/DNA-AuNPs Probe:

as shown in FIG. 1A, the sAb/DNA-AuNPs probe was prepared according to the method described in the literature:

(1) And (3) purifying the sAb by a dialysis mode:

adding 1mL of sAb into a dialysis bag, sealing the bag by using a sealing clamp, putting the sealed dialysis bag into a beaker containing 10mM PB (note that PB needs to completely submerge the dialysis bag), dialyzing in a refrigerator at 4 ℃, replacing dialysate (10 mM PB) every 4-6 h, and after replacing the dialysate for 5-6 times, placing the obtained sAb in the refrigerator at 4 ℃ for later use;

(2) Pretreatment of SH-DNA:

mu.L of 1M Na was added to 30. Mu.L of 100. Mu.M SH-DNA ₃ PO ₄ The solution and 2 mu L of 30mM TCEP solution are mixed evenly and reacted for 2h at 25 ℃; adding the reaction solution into an ultrafiltration tube, centrifuging for 20min under the condition of 10000r/min, washing for 8 times, collecting SH-DNA, and storing in a refrigerator at 4 ℃ for later use;

(3) Taking 100 mu L of AuNPs, and using 0.1M K ₂ CO ₃ Adjusting the pH value to 9, adding 4 mu L of SH-DNA product obtained in the step (2) into the mixture, and reacting the mixture for 16 hours at 4 ℃;

(4) To the above solution was added 27.5. Mu.L of PB containing 10% PEG20000, 2. Mu.L of dialyzed sAb, incubated at room temperature for 30min, added 15. Mu.L of NaCl solution, and equilibrated at 4 ℃ overnight. The resulting solution was centrifuged at 12000r/min for 30min, the supernatant was discarded, and the supernatant was washed 3 times to remove excess SH-DNA and sAb. Finally, the prepared sAb/DNA-AuNPs probe was dispersed in PBS and stored in a refrigerator at 4 ℃ until use.

(III) establishment of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on AuNPs:

(1) Coating: diluting CBD-BSA by 32000 times by CBS, adding 100 mu L/hole into a 96-hole enzyme label plate, and placing the plate in a constant-temperature incubator at 37 ℃ for incubation for 3h or overnight at 4 ℃;

(2) And (3) sealing: washing the coated enzyme label plate for 3-5 times by PBST, patting to dry, adding 5% skim milk powder with 300 mu L/hole, incubating at 37 ℃ for 40min, taking out, washing for 3-5 times by PBST, patting to dry, and storing in a refrigerator at-50 ℃ for later use;

(3) Sample adding: taking out the enzyme label plate which is sealed in advance, returning to room temperature, adding mAb and CBD standard substance diluted by PBS at 50 mu L/hole, mixing uniformly, and incubating at constant temperature of 37 ℃ for 30min;

(4) Adding the sAb/DNA-AuNPs probe: taking out the enzyme label plate in the step (3), washing the plate for 3-5 times by using PBST, beating to dry, adding PBS (95 mu L/hole) and sAb/ALP-AuNPs (6 mu L/hole) probes, uniformly mixing, and incubating at the constant temperature of 37 ℃ for 30min;

(5) TdT reaction: taking out the ELISA plate obtained in the step (4), washing the plate for 3-5 times by using PBST, beating the plate to be dry, adding 0.3 mu L of TdT,1.2 mu L of biotin-16-dCTP and 98.5 mu L of TdT reaction buffer solution into the plate, uniformly mixing, and reacting for 30min at 37 ℃;

(6) SA-biotin reaction: taking out the ELISA plate obtained in the step (5), washing the plate for 3-5 times by using PBST, beating the plate to be dry, adding 1 mu L of SA-ALP and 99 mu L of Tris-HCl buffer solution, and reacting for 10min at room temperature;

(7) Adding a reaction substrate: taking out the ELISA plate obtained in the step (6), washing the plate for 3-5 times by using PBST, beating the plate to be dry, and adding 100 mu L of 60mg/mLG-1-P solution;

(8) And (3) determination: the above solution was reacted at 37 ℃ for 30min, and the glucose content was measured using PGM, and the PGM value was recorded.

(IV) portable detection of CBD:

a series of CBD standard solutions were prepared in PBS at different concentrations, 0, 0.13, 0.41, 1.23, 3.70, 11.1, 33.3 and 100ng/mL. Adding the sample as a sample into the sample adding in the step (3) for detection, carrying out 3 groups of parallel experiments at each concentration, and carrying out strict experiments under other conditions according to optimized optimal conditions (namely all conditions in the establishment of the AuNPs-based indirect competitive enzyme-linked immunosorbent assay). The PGM signal values exhibited by the test solutions at different concentrations of CBD were finally recorded.

The invention has the beneficial effects that:

1. the method improves the traditional ELISA, simultaneously couples sAb and ALP on AuNPs, catalyzes G-1-P dephosphorylation to generate glucose molecules by ALP, and uses PGM as a signal reading tool to realize portable quantitative detection of CBD.

2. The method realizes effective amplification of detection signals by using a double signal amplification strategy of high specific surface area of AuNPs and TdT catalysis single-stranded DNA extension, so that the method has higher sensitivity.

3. The method is a detection method constructed based on the specific recognition effect of the antigen and the antibody, so that the method has better specificity. In addition, the method has strong anti-interference capability, and a satisfactory result is obtained in the detection of the actual sample.

4. The method realizes portable quantitative detection of PGM to CBD, and overcomes the defect that the traditional ELISA can only carry out accurate quantitative analysis by means of large-scale instruments such as an enzyme-linked immunosorbent assay (ELIAS). Therefore, the method is very suitable for on-site rapid detection and analysis and has higher application potential in resource-poor areas.

Drawings

FIG. 1 (A) is a schematic diagram showing the preparation of a sAb/DNA-AuNPs probe; FIG. 1 (B) is a schematic diagram of AuNPs-based portable ELISA for CBD detection.

Fig. 2 shows the PGM 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.

FIG. 3 is an agarose gel electrophoresis analysis of the TdT product of DNA: lane A represents the product of the reaction of TdT and low-concentration Co < 2+ >; lane B represents the product of the reaction of TdT with high concentration Co2 +.

FIG. 4 is a schematic representation of the detection of CBD by PGM-based ELISA.

Fig. 5 shows PGM signal values for PGM-based ELISA method: (A) does not contain CBD; (B) CBD concentration 1000ng/mL.

Fig. 6 shows PGM signal values of the constructed portable AuNPs-based ELISA method: (A) indicates that no CBD is present; (B) shows that the CBD concentration was 1000ng/mL.

FIG. 7 is a standard curve of PGM signal as the CBD concentration increases from 0.13ng/mL to 100 ng/mL; error bars represent standard deviation (n = 3).

FIG. 8 is a portable ELISA assay using AuNPs; from left to right the compounds: bromothalonil, methamidophos, metalaxyl, spirotetramat, acetamiprid, (methyl) kresoxim-methyl, kasugamycin, captan, thiophanate-methyl, benomyl, prochloraz, imazalil, phoxim, abamectin, PBS and CBD.

FIG. 9 is a standard curve of PGM signals at 10% cabbage substrate with increasing CBD concentration from 0.13ng/mL to 100 ng/mL; error bars represent standard deviation (n = 3).

FIG. 10 is a standard curve of PGM signal at 10% citrus substrate with increasing CBD concentration from 0.13ng/mL to 100 ng/mL; error bars represent standard deviation (n = 3).

FIG. 11 is a calibration curve of the PGM signal at 10% orange peel can base as the CBD concentration increases from 0.13ng/mL to 100 ng/mL; error bars represent standard deviation (n = 3).

Detailed Description

synthesis of AuNPs:

reduction of HAuCl with sodium citrate ₄ To synthesize AuNPs. All glassware was treated with aqua regia (HCl: HNO) prior to synthesis ₃ 1) soaking for 15-20 min to remove impurities, and then washing and drying with ultrapure water for later use. 100mL of 1.0mM HAuCl were added under magnetic stirring ₄ The solution was heated to boiling and 1mL of a pre-prepared 400mM sodium citrate solution was immediately added rapidly. And continuously heating the mixed solution under the magnetic stirring state, keeping the boiling state for 8-10 min, stopping heating after the color of the solution gradually changes into mauve, naturally cooling to room temperature to obtain a stable AuNPs solution, and finally storing the stable AuNPs solution in a refrigerator at 4 ℃ for later use.

(II) preparation of sAb/DNA-AuNPs Probe:

as shown in FIG. 1A, the sAb/DNA-AuNPs probe was prepared according to the method described in the prior art:

(1) And (3) purifying the sAb by a dialysis mode:

adding 1mL of sAb into a dialysis bag, sealing the bag by using a sealing clamp, putting the sealed dialysis bag into a beaker containing 10mM PB (note that PB needs to completely submerge the dialysis bag), putting the beaker into a refrigerator at 4 ℃ for dialysis, replacing dialysate (10 mM PB) every 4-6 h, and after replacing the dialysate for 5-6 times, putting the obtained sAb into the refrigerator at 4 ℃ for standby;

(2) Pretreatment of SH-DNA:

mu.L of 1M Na was added to 30. Mu.L of 100. Mu.M SH-DNA ₃ PO ₄ The solution and 2 mu L of TCEP solution with the concentration of 30mM are mixed evenly and reacted for 2h at the temperature of 25 ℃; adding the reaction solution into an ultrafiltration tube, centrifuging for 20min under the condition of 10000r/min, washing for 8 times, collecting SH-DNA, and storing in a refrigerator at 4 ℃ for later use;

(3) Taking 100. Mu.L of AuNPs, and adding 0.1M K ₂ CO ₃ Adjusting the pH value to 9, adding 4 mu L of SH-DNA product obtained in the step (2) into the solution, and reacting the solution at 4 ℃ for 16h;

(4) To the above solution, 27.5. Mu.L of PB containing 10% PEG20000 was added, 2. Mu.L of sAb after dialysis was added, and after incubation at room temperature for 30min, 15. Mu.L of NaCl solution was added, and the mixture was allowed to equilibrate at 4 ℃ overnight. The resulting solution was centrifuged at 12000r/min for 30min, the supernatant was discarded, and the supernatant was washed 3 times to remove excess SH-DNA and sAb. Finally, the prepared sAb/DNA-AuNPs probe was dispersed in PBS and stored in a refrigerator at 4 ℃ until use.

(7) Adding a reaction substrate: taking out the enzyme label plate in the step (6), washing the plate for 3-5 times by using PBST, beating to dry, and adding 100 mu L of 60mg/mL G-1-P solution;

(8) And (3) determination: the above solution was reacted at 37 ℃ for 30min, and then the glucose content was measured using PGM, and the PGM value was recorded.

(IV) Portable detection of CBD:

a series of CBD standard solutions were prepared in PBS at different concentrations, 0, 0.13, 0.41, 1.23, 3.70, 11.1, 33.3 and 100ng/mL. Adding the sample into the detection step of step 3, performing 3 groups of parallel experiments for each concentration, and performing experiments under other conditions strictly according to the operation steps and the optimized optimal conditions. The PGM signal exhibited by the test solutions at different concentrations of CBD was finally recorded.

Test example 1

Specificity analysis

The specificity of the method was verified using some common non-target pesticides. Non-target pesticides include: bromothalonil, methamidophos, metalaxyl, spirotetramat, acetamiprid, kresoxim-methyl, kasugamycin, captan, thiophanate-methyl, benomyl, prochloraz, imazalil, phoxim and abamectin. All assays were performed under identical conditions, with CBD concentrations of 100ng/mL and other non-target pesticides at 1000ng/mL. Each pesticide was subjected to 3 parallel experiments, and the other conditions were strictly tested according to the optimized optimal conditions (i.e., all conditions in the establishment of indirect competitive enzyme-linked immunosorbent assay based on AuNPs).

Test example 2

Determination of CBD under complex substrates

The orange, orange peel can and Chinese cabbage samples used in the test example are all taken from Chongqing city. The CBD in citrus, canned orange segments and cabbage was first analyzed by LC-MS/MS and these samples were confirmed to be free of any CBD residue. To obtain the above sample matrix, the samples were all homogenized using a beater. 5g of homogenate was taken, and 5mL of acetonitrile was added thereto. Extracting under shaking for 10min, freezing the obtained mixture at-50 deg.C for 10min, and adding 2g anhydrous MgSO ₄ And 0.5g NaCl, shaking continuously for 1min. Finally, centrifuging the mixture at room temperature at 10000r/min for 5min, and taking supernatant for later use. The obtained sample matrix was treated with ddH ₂ O was diluted 10-fold to perform the following experiment.

And finally, preparing CBD standard solutions with different concentrations by using the different food matrix diluents, and performing experiments according to the operation step of sample loading in the step (3) in the establishment of the AuNPs-based indirect competitive enzyme-linked immunosorbent assay. A linear standard curve was obtained for the different matrix cases and used for subsequent calculation of CBD addition recovery in different food matrices.

Test example 3

Determination of CBD in actual samples:

the test example verifies the accuracy of the established method in the actual sample detection by adding a recovery mode. The orange, orange peel can and Chinese cabbage are taken from Chongqing city. The above samples were analyzed using LC-MS/MS and proved to be free of any CBD residue. CBD standard solutions (0.01, 0.1, 0.5 and 5 mu g/mL) with different concentrations are added into the fruit and vegetable samples to simulate positive samples.

Homogenizing the fruit and vegetable samples by a wall breaking machine. Thereafter, 5g of each homogenate was added to 50. Mu.L of standard CBD solutions of different concentrations, and the mixture was allowed to stand for 30min to mix CBD with the sample. Subsequently, CBD was extracted using acetonitrile. To the mock positive sample was added 5mL of acetonitrile. Extracting under shaking for 10min, freezing the obtained mixture at-50 deg.C for 10min, and adding 2g anhydrous MgSO ₄ And 0.5g NaCl, shaking continuously for 1min. Finally, all samples were centrifuged at 10000r/min at room temperature for 5min. The supernatant was obtained and stored at-20 ℃ until use.

ddH for mock-positive samples with addition levels of 0.01 and 0.1. Mu.g/mL CBD ₂ O diluted 10-fold. The resulting solution was then assayed by established PGM-based ELISA. However, the additional two samples that simulated positive samples with the addition of 0.5 and 5 μ g/mL CBD were outside the detection range of the standard curve. Thus, the present application begins with ddH ₂ O diluted the two samples 10-fold. The obtained solution was further diluted 10-fold and 50-fold with the corresponding 10-fold diluted food substrates, and then measured by the established ELISA method based on PGM, respectively. Then, the sample was used at 10%The standard curve obtained in the sample matrix was used to calculate the CBD concentration in the actual sample. Finally, the addition recovery of CBD in different matrixes is calculated according to the addition level and the detected concentration.

In addition, LC-MS/MS will also be used to verify the accuracy of the methods developed by the present application. Before LC-MS/MS analysis, the supernatant obtained from the acetonitrile extraction was filtered through a 0.2 μm organic membrane, and the content of CBD was determined using LC-MS/MS to obtain its addition recovery rate.

And (4) analyzing results:

1. feasibility analysis

To confirm the feasibility of this approach, the present application was validated through a series of experiments. Since the experiment was performed based on the removal of phosphate groups in G-1-P by ALP to generate glucose, the present application needs to confirm whether other substances in the entire detection system can affect PGM signals, and also to verify that only ALP can remove phosphate groups of G-1-P to generate glucose. The range of glucose concentration that can be measured by the PGM used in this experiment is 1.1 to 33.3mM, indicating that the glucose concentration in the test solution is less than 1.1mM when the PGM is represented by L0, and more than 33.3mM when the PGM is represented by H1. As shown in Table 1, when each substance in the detection system was measured with PGM alone, it was revealed that no PGM signal was generated from all the substances. As shown in FIG. 2, only when G-1-P and SA-ALP reacted, a glucose signal was detected and was greater than 33.3mM, and when other substances reacted with G-1-P, no glucose was detected in the test solution. The above results show that only SA-ALP catalyzes G-1-P to generate glucose in the detection system, and that no other substance in the detection system affects the detection signal of PGM.

TABLE 1 investigating the influence of various substances of the system on the PGM signal

SA-ALP

G-1-P

TdT

biotin-16-dCTP

sAb

AuNPs

mAb

sAb/DNA-AuNPs

Buffer

L0

In this protocol, signal amplification was achieved by DNA sequence extension of TdT ends to form a biotin label, co ²⁺ Is a necessary cofactor for catalyzing DNA terminal extension by TdT, and Co with different concentrations ²⁺ May affect the catalytic efficiency of TdT. Therefore, the present application is directed to TdT at different Co ²⁺ The DNA end extension products at concentration were analyzed by agarose gel electrophoresis. As shown in FIG. 3, the original DNA strand has 36 bases, and the DNA size is distributed in the range of 150 to 300bp after TdT elongation. By agarose gel electrophoresis analysisIt is shown that TdT can achieve DNA elongation and thus function as signal amplification. And the application can observe Co at low concentration ²⁺ Under the condition of better DNA prolonging effect, low-concentration Co is selected in subsequent experiments ²⁺ As reaction conditions.

The method utilizes a strategy of combining AuNPs high specific surface area and TdT catalytic DNA chain extension to amplify the detection signal so as to improve the sensitivity of PGM to CBD detection. In order to prove the feasibility and the necessity of the signal amplification strategy, a series of experiments are designed and verified. First, the present application constructed an ELISA that did not pass the above two signal amplification modes. As shown in FIG. 5, CBD-BSA as the coating antigen competitively binds mAb with CBD in the sample. When the sample does not contain CBD, the mAb binds to CBD-BSA to form a complex, biotin-sAb is added thereto, and the sAb is captured by the mAb to form a CBD-BSA-mAb-sAb-biotin complex. ALP is attached under the action of SA-biotin specific binding, and the phosphate group of G-1-P detached by ALP generates glucose, resulting in an increase in PGM signal. When a sample contains a certain concentration of CBD, the resulting decrease in the PGM signal in solution is due to the competition of CBD and CBD-BSA for mAb binding. When the CBD was detected by this method, the results are shown in fig. 5. The PGM signal was 1.1mM when the sample contained no CBD, but was not detected when the sample contained CBD. This is because the glucose concentration range that can be measured by the PGM used in this experiment is 1.1 to 33.3mM, and excessively low glucose concentration cannot be measured. Next, the present application used ELISA methods established in this work with large specific surface areas of AuNPs and amplification of TdT enzyme catalyzed signals to determine the same concentration of CBD. As shown in FIG. 6, the mAb was fully bound to CBD-BSA and the captured sAb/DNA-AuNPs probe was more abundant with a final PGM signal of 14.9mM when the sample contained no CBD, whereas the captured sAb/DNA-AuNPs probe was also reduced with a final PGM signal of 3.2mM when the sample contained 1000ng/mL CBD, which competed for mAb binding with CBD-BSA.

The experimental results show that the detection sensitivity is improved by 15 times through double signal amplification of AuNPs and TdT. Moreover, since the reading range of PGM signal is 1.1-33.3 mM, ELISA without AuNPs and TdT signal amplification is not suitable for the detection of CBD using PGM as a signal output tool.

2. Determination of CBD:

through the optimization experiment, the optimal concentration of each reagent and the optimal reaction time among various substances in the detection system are determined. Under optimized experimental conditions, the present application quantitatively analyzes CBD at different concentrations by observed PGM. The experiment is a detection method constructed by changing a signal output strategy on the basis of the traditional ELISA. Therefore, the data processing and standard curve plotting for this experiment are the same as for the conventional ELISA. As shown in fig. 7, the PGM signal is maximum in the absence of CBD, and gradually decreases as the CBD concentration increases. The CBD concentration is therefore inversely proportional to the PGM signal. In the concentration range of 0.13-100 ng/mL, a good linear relationship is shown between the concentration of CBD and PGM signal, and the detection limit of the method is calculated to be 0.37ng/mL, and the linear fitting equation is (wherein x represents the concentration of CBD and y represents the PGM signal):

compared with the traditional ELISA method, the method keeps the advantages of higher sensitivity, higher detection speed, simple operation steps and the like. Meanwhile, the method also makes up the defect that the traditional method can be accurately and quantitatively analyzed only by a microplate reader, and provides a reliable method for quickly and accurately quantitatively analyzing the CBD on site.

To evaluate the specificity of the developed portable AuNPs-based ELISA for CBD detection, the present application used this method for the detection of other commonly used pesticides. Such as: bromothalonil, methamidophos, metalaxyl, spirotetramat, acetamiprid, kresoxim-methyl, kasugamycin, captan, thiophanate-methyl, benomyl, prochloraz, imazalil, phoxim and abamectin. Wherein the concentration of CBD is 100 mug/mL, and the concentration of other pesticides is 1000 mug/mL. As shown in fig. 8, the PGM signal was lowest when CBD was present and the PGM signal was consistent with the signal detected in the blank buffer (PBS) when other pesticides were present, indicating that no other pesticides were detected by this method. When benomyl and thiophanate-methyl are detected, PGM signals are between the benomyl and the thiophanate-methyl, and the reason for the phenomenon is that the benomyl and the thiophanate-methyl are easily decomposed into CBD. The above results indicate that the detection method established in the present application has good specificity for CBD.

3. Determination of the actual sample:

in order to prove whether the portable ELISA detection method based on AuNPs established by the application has the potential of being applied to actual sample detection, CBD with different concentrations is added into the actual sample for detection. The practical samples selected for the application include: mandarin orange, canned orange segments, and Chinese cabbage. Notably, these sample matrices were confirmed to not contain any CBD residue by LC-MS/MS detection prior to use. These matrices contain complex components that may affect the specific binding of antigen-antibody, so to eliminate this effect, the present application establishes standard curves in 3 different matrices, respectively. The recovery of the subsequent addition was determined by calculation from a standard curve obtained for the corresponding matrix.

As shown in fig. 9, 10 and 11. In 3 different matrices, the PGM signal decreased for all test solutions as the CBD concentration increased from 0ng/mL to 100ng/mL. In different matrix solutions, the application obtains a linear fit between CBD concentration and PGM signal. These results indicate that the PGM-based ELISA developed in the present application can detect CBD with high sensitivity under a variety of complex matrices.

Next, the present application performed CBD addition recovery tests in the sample matrices of 3 above, based on the standard curves established in the 3 matrices. The recovery rate of CBD in different samples ranges from 70.4% to 109.4%, and the relative standard deviation ranges from 5.8% to 12.8%. In order to evaluate the accuracy of the portable ELISA based on AuNPs established in the present application, the above spiked samples were measured by LC-MS/MS method and compared with the recovery obtained by the method established in the present application, and the detailed data of spiked recovery are shown in table 2. The RSD of the novel ELISA method established in the application is slightly higher than that of LC-MS/MS, but the recovery rate presented by the two methods is basically consistent. Therefore, the accuracy of the portable AuNPs-based ELISA established in this application is acceptable. Based on the above results, the portable ELISA based on AuNPs developed by the present application has a great application potential in the field detection of CBD.

TABLE 2 recovery rate of carbendazim in actual sample

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims

1. A method for detecting carbendazim residue based on a personal blood glucose meter, comprising:

reduction of HAuCl with sodium citrate ₄ Synthesizing an AuNPs solution;

preparing a sAb/DNA-AuNPs probe;

establishing indirect competitive enzyme-linked immunosorbent assay based on AuNPs; and

and (4) detecting quickly.

2. The method of claim 1, wherein:

the AuNPs solution is synthesized by the following method:

100mL of 1.0mM HAuCl were added under magnetic stirring ₄ Heating the solution to boiling, and adding 1mL of 400mM sodium citrate solution to obtain a first mixed solution;

continuously heating the first mixed solution under magnetic stirring, continuously boiling for 8-10 min, and stopping heating; and naturally cooling the solution to room temperature, and storing the solution in a refrigerator at 4 ℃ to obtain the AuNPs solution.

3. The method of claim 2, wherein:

before the synthesis of AuNPs solution, soaking the used glassware for 15-20 min by using aqua regia, then washing by using ultrapure water and drying for later use.

4. The method of claim 1, wherein:

the preparation of the sAb/DNA-AuNPs probe comprises the following steps:

(1) And (3) purifying the sAb by a dialysis mode:

adding 1mL of sAb into a dialysis bag, putting the sealed dialysis bag into a beaker containing 10mM PB, and dialyzing in a refrigerator at 4 ℃ to obtain purified sAb for later use;

(2) Pretreatment of SH-DNA:

to 30. Mu.L of 100. Mu.M SH-DNA was added 2. Mu.L of 1M Na ₃ PO ₄ The solution and 2 mu L of TCEP solution with the concentration of 30mM are mixed evenly and reacted for 2h at the temperature of 25 ℃; adding the reaction solution into an ultrafiltration tube, centrifuging for 20min under the condition of 10000r/min, washing for 8 times to obtain pretreated SH-DNA, and storing in a refrigerator at 4 ℃ for later use;

(3) Taking 100 mu L of AuNPs, and using 0.1M K ₂ CO ₃ After the pH value is adjusted to 9, 4 mu L of pretreated SH-DNA is added into the solution, and the solution reacts for 16 hours at the temperature of 4 ℃ to obtain a first mixed solution;

(4) Adding 27.5. Mu.L of PB containing 10% PEG20000 and 2. Mu.L of purified sAb to the first mixed solution, incubating at room temperature for 30min, adding 15. Mu.L of NaCl solution, and equilibrating at 4 ℃ overnight to obtain a second mixed solution;

(5) Centrifuging the second mixed solution at 12000r/min for 30min, discarding supernatant, washing for 3 times to obtain sAb/DNA-AuNPs probe, dispersing the probe in PBS, and storing in refrigerator at 4 deg.C.

5. The method of claim 4, wherein:

in the dialysis treatment in the step (1), the dialysate is replaced every 4 to 6 hours for 5 to 6 times.

6. The method of claim 1, wherein:

the establishment of the indirect competitive enzyme-linked immunosorbent based on the AuNPs comprises the following steps:

(2) And (3) sealing: washing the coated ELISA plate for 3-5 times by PBST, beating to dry, adding 5% skim milk powder with 300 mu L/hole, incubating at 37 ℃ for 40min, taking out, washing for 3-5 times by PBST, beating to dry, and storing in a refrigerator at-50 ℃ for later use;

(3) Sample adding: taking out the sealed enzyme label plate, returning to room temperature, adding 50 mu L/hole of mAb and CBD standard substance diluted by PBS, mixing uniformly, and incubating at 37 ℃ for 30min;

(4) Adding the sAb/DNA-AuNPs probe: taking out the elisa plate obtained in the step (3), washing the plate for 3-5 times by using PBST, beating the plate to be dry, adding PBS and a sAb/ALP-AuNPs probe, uniformly mixing, and incubating at the constant temperature of 37 ℃ for 30min;

(7) Adding a reaction substrate: taking out the ELISA plate obtained in the step (6), washing the plate for 3-5 times by using PBST, patting the plate dry, and adding 100 mu L of 60mg/mLG-1-P solution;

(8) And (3) determination: the solution obtained in step (7) was reacted at 37 ℃ for 30min, the glucose content thereof was determined using PGM, and the PGM value was recorded.

7. The method of claim 6, wherein:

adding PBS according to the step (4) with the standard of 95 mu L/hole; the sAb/ALP-AuNPs addition standard was 6. Mu.L/well.

8. The method of claim 1, wherein:

the rapid detection comprises the following steps:

and (3) preparing CBD standard solutions with different concentrations by using PBS, carrying out sample loading treatment on the CBD standard solutions as samples, carrying out 3 groups of parallel experiments on each concentration, and recording PGM signal values presented in the detection solutions under the CBD with different concentrations.

9. The method of claim 8, wherein:

the CBD standard solutions with different concentrations comprise:

CBD standard solutions with concentrations of 0, 0.13, 0.41, 1.23, 3.70, 11.1, 33.3 and 100ng/mL, respectively.