| Studies have shown that mycotoxins such as patulin?PAT?,ochratoxin A?OTA?,and alternaria toxins?ATX?that exist in fruits are directly or potentially harmful.Since fruits and their products are complex polar liquid foods,the mycotoxins in fruits are also far from the mycotoxins contaminating the grain and oil.The water content of the fruit is relatively large,so the dilution of the water will decrease the concentration of mycotoxins.When several kinds of mycotoxins are present at the same time,it's very hard to detect them.Therefore,it is very important to develop mycotoxin detection methods with higher sensitivity and better specificity.The aim of this study was to research and develop enzyme-assisted target recycling strategies and efficient luminescent systems.Through which we canimprove the detection sensitivity of PAT,OTA,and TA by using an aptamer-based fluorescence sensor and chemiluminescence immunoassay.The main findings are as follows:?1?In this study,the enzyme-assisted target recycling strategy was used to increase the sensitivity of PAT detection,and a PAT aptamer fluorescence sensor was constructed.Studies have shown that 0.08 mg/mL of rGO-Fe3O4 could quench the labeled fluorescence on the PAT aptamer almost completely in 5 minutes,and the specific binding of PAT to the aptamer restored the fluorescence intensity.In the fluorescence recovery phase,75U/mL of DNase I was used to enzymatically target the system for signal amplification and enzymatic reaction for 1 h,which maximized the detection sensitivity of PAT.In the standard fluorescence detection method without signal amplification strategy,the limit of detection?LOD?was 3.61?g/L;in the fluorescence signal amplification strategy,the LOD was 0.28?g/L,and the sensitivity?LOD?was increased about 13 times.Based on this principle,a PAT aptamer fluorescence sensor was constructed.The linear correlation coefficient R in the range of 0.5-30?g/L was0.9966.In the actual sample spike recovery experiment,the average recoveries of PAT in apple juice and grape juice were between 77.34%96.65%?97.14%103.89%,respectively.Compared with the HPLC method in GB 5009.185-2016,the results showed that there was no significant difference between this method and the national standard method.The method showed no specific cross-reaction with AFB1,DON,TA and T-2 toxins.?2?In this study,peroxy oxalate chemiluminescence system was used to improve the detection sensitivity of OTA,and an indirect competitive chemiluminescence enzyme immunoassay?icCLEIA?was established based on this.Studies have shown that the coating concentration was 1?g/mL,the monoclonal antibody dilution factor was 10000 times,the enzyme-labeled secondary antibody was diluted 4,000 times,the enzyme reaction buffer pH was 6.0,and the concentration of CH4N2O·H2O2 and PHPPA in the enzyme reaction was 7.0×10-33 mol/L,enzyme reaction time 10 min,TCPO acetonitrile solution concentration of 0.01 mol/L,CH4N2O·H2O2 acetonitrile solution concentration of 1 mol/L,can maximize the detection sensitivity of OTA.An icCLEIA method for OTA was constructed based on this luminescent immune system.The linear regression equation for OTA detection was y=45.418-35.33x,the IC500 was 554.60 ng/L,the detection range was 48.94-6084.45 ng/L,and the LOD was 14.69 ng/L..In the spiked recovery experiment,the average recoveries of OTA in raisins and grape juice were between 84.55%91.36%and 73.32%87.64%,respectively.The average intra-and inter-assay coefficient of variation of this method is less than 10%,and the precision is better.The cross-reactivity rates of AFB1,DON,T-2,PAT,ZEN and TA were less than 1%and the specificity was high.The icCLEIA method was compared with the HPLC method,and there was no significant difference between the two test results.By studying the universality of the method,it was found that this method has a good detection effect on mycotoxins containing hydroxyl groups in polar samples such as fruits and their products,milk,etc.,and is not applicable to the detection of non-polar mycotoxins such as AFB1.?3?In this study,a monoclonal antibody of TA with high specificity prepared by in vivo murine induction was used as a recognition element,and the sensitivity of TA chemiluminescence immunoassaywasimproved by using a peroxyoxalate chemiluminescence system combined with a monoclonal antibody.Through research,the original concentration of the coating was 0.5?g/mL,tthe monoclonal antibody dilution factor was 26000 times,the enzyme-conjugated secondary antibody was diluted6,000 times,the pH of the enzyme reaction buffer was 6.0,and the concentration of CH4N2O·H2O2 and PHPPA in the enzyme reaction was 8.0×10-33 mol/L,7.0×10-33 mol/L,enzyme reaction time 10 min,TCPO acetonitrile solution concentration 5×10-33 mol/L,CH4N2O·H2O2 acetonitrile solution concentration 2 mol/L,The detection sensitivity of TA was maximized,and it was found that the chemiluminescence system has better sensitivity improvement in the detection of mycotoxins with a keto acid structure.The icCLEIA method for TA was constructed based on this luminescent immune system.The standard curve for TA detection was y=23.67-26.06x,IC500 was 95.2 ng/L,detection range was 12.20-1562.5 ng/L,and LOD was 2.53 ng/L.In the spiked recovery experiment,the average recoveries of the actual samples were between 76.24%and93.28%.The mean intra-assay coefficient of variation was 8.59%and the inter-assay coefficient of variation was 11.24%.The cross-reaction rates with ZEN,OTA,AFB1,DON,T-2,PAT,AOH,and AME were all less than 1%..Compared with the traditional ELISA and luminol icCLEIA methods,the sensitivity?LOD?increased by about 5 times and 2 times,respectively. |
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