Research Of Novel Lateral Immunochromatographic Assays For Ochratoxin (OTA)

Toxicogenic fungi such as Aspergillus and Penicillium were widely distributed in agricultural environment,which can produce OTA.OTA is one of the most toxic and polluting mycotoxins,which has been listed as?class B carcinogens by IARC(International Agency for Research on Cancer).Instrumental analysis and immunoassay were common OTA detection techniques.Lateral immunochromatography(LFA),as one of the typical immunoassay techniques,has many advantages,such as high sensitivity,simple,fast,low cost,on-site real-time,etc.,and has been widely concerned.In this paper,a new,semi-quantitative,quantitative,enhanced,highly sensitive,simultaneous LFA detection technology for OTA was studied,and applied to the detection of OTA pollution in agricultural environment.The main research results are as follows:1.OTA semi-quantification-quantitative colloidal gold LFA methodBased on the preparation of m Ab-GNPs probe and the assembly of test strip,the LFA method can be used for semi-quantitative OTA detection by direct visual interpretation and quantitative detection by card reader.The semi-quantitative detection limit of LFA for OTA is 0.5 ng/mL,and the quantitative detection limit is0.1 ng/mL,the real-time semi-quantification-quantitative detection of OTA can be achieved in the range of 0.1-10 ng/mL.The crossover rate of OTB was 85.8%,and there was almost no crossover reaction to other mycotoxins.The recoveries of the method were 97.0%-110.8%,and the standard deviations were 4.9%-12.2%.The results of 10 real samples showed that the OTA positive detection range was 1.7-101.3ng/g,and the LC-MS/MS positive detection range was 1.5-97.5 ng/g.The correlation coefficient between the two methods was 0.9928.The results show that the developed LFA method is simple,rapid,sensitive,specific and accurate,and can be applied to the semi-quantification and quantitative real-time detection of OTA in environment.2.OTA double nano gold enhanced LFA method based on biotinylated DNAThe biotinylated DNA double nano gold enhanced LFA method established in this study is the first time that applied double gold nanoparticles and streptavidin-biotin joint enlarge strategy to detect OTA,this method has a semi-quantitative detection limit of 0.06 ng/mL(8 times lower than that of ordinary LFA)and a quantitative detection limit of 0.03 ng/mL(3 times lower),which can achieve real-time semi-quantitative to quantitative detection in the range of 0.03-1.2ng/mL in the field.The recoveries were 87.4%-113.4%,and the standard deviations were 4.4%-11.6%.The correlation between the results of real samples and instrumental analysis was also very high.Results show that the development of enhanced LFA strip through the simple and feasible double gold nanoparticles and streptavidin-biotin joint enlarge strategy implements the OTA higher sensitivity of simple rapid,high specificity,high accuracy,semi-quantitative and quantitative detection,to expand the strip application has great reference value.3.OTA and AFB₁ simultaneous LFA method based on bacteria-gold growth enhancementThe LFA method was established for the detection of OTA and AFB₁ in agricultural environmental samples by using Escherichia coli as carrier.This is the first time that bacteria were used as carrier to enrich gold nanoparticles and then detected simultaneously by gold growth for OTA and AFB₁.The limits of semi-quantitative detection for OTA and AFB₁ were 0.03 ng/mL and 0.03 ng/mL,respectively(16 and 18 fold lower than conventional LFA),and the limits of quantitative detection were 0.01 ng/mL and 0.01 ng/mL(10 and 20 fold lower,respectively).It can realize the real-time semi-quantification and quantitative detection of 0.01-0.5ng/mL for OTA and 0.01-0.2ng/mL for AFB₁.The method has good specificity,high accuracy and high correlation with the results of real samples detected by instrument method.The results showed that the enhanced test strips were enriched with gold nanoparticles on the surface of Escherichia coli to significantly improve the sensitivity,and the simultaneous semi-quantification and quantitative detection of OTA and AFB₁ was achieved,which had important guiding significance and application value for the screening of agricultural environmental samples contaminated by multiple mycotoxins at the same time.