Research Of Aptamer-based Molecular Beacon Method For Rapid Detection Of Aflatoxin B1

Aflatoxin B1(AFB1)is a highly toxic natural mycotoxin,commonly found in moldy and rotting cereals and their products.Due to its severe carcinogenicity,AFB1 has been listed as a Class I carcinogen by the World Health Organization’s Agency for Cancer Research(IARC),posing a serious threat to human food safety and health.AFB1 detection is of great significance to prevent the occurrence of related pollution and hazards.The traditional AFB1 detection method is limited by complex sample pretreatment procedures and large instruments and equipment,and the analysis time is long and the cost is high,so it is not suitable for point-of-care testing.Therefore,it is necessary to develop a new AFB1 detection method.In this paper,the emerging aptamer is used as the AFB1 molecular recognition element and the molecular beacon sensing technology is combined to construct a simple method suitable for the field rapid detection of AFB1.The main research contents are as follows:(1)Functional structural characterization and optimization of AFB1 aptamer.The functional structure of AFB1 aptamer was studied by circular dichroism spectroscopy,and its key functional structure and non-essential nucleotide sequences were clarified,and the sequence and structure of the original aptamer were tailored and optimized.The optimized aptamer has a simpler structure and higher affinity,and can be used for the rapid detection of AFB1 by the circular dichroism method with a detection limit of 0.6 n M.(2)Aptamer-based molecular beacon method for rapid detection of AFB1.With the assistance of complementary DNA(c DNA)chains,molecular beacons consisting of DNA aptamers labeled fluorescein FAM and fluorescent quencher BHQ1 on both sides showed greater fluorescence response to AFB1.Optimizing some experimental factors,the method was used to achieve the rapid detection of AFB1 in the range of 1 n M～3 μM within 20 min.The detection limit was 1 n M,lower than that of 8 n M obtained without c DNA.This aptamer-based molecular beacon detection method has the advantages of simple operation,fast analysis,and large signal response.(3)Rapid detection of AFB1 based on the structural changes of the label-free aptamer.In the absence of AFB1,the unlabeled aptamer can hybridize with two short complementary DNA strands at the same time to form a DNA double-stranded structure.At this time,the fluorescein(FAM)labeled at the 3’ end of one complementary DNA was close to the fluorescent quencher(BHQ1)labeled at the 5’end of the other complementary DNA,the fluorescence resonance energy transfer occurred,and the FAM fluorescence was quenched.When AFB1 is present,the aptamer binds to AFB1 without hybridizing with the complementary DNA.At this time,FAM was far away from BHQ1,and FAM fluorescence could not be quenched.AFB1 was quantitatively detected by measuring the fluorescence intensity of the system.The detection concentration ranges from 1 n M to 4 μM,and the detection limit was 1 n M.This method did not require labeling of the aptamer,and with lower cost and background,and fluorescence intensity increased with increasing concentration of AFB1.