Studies On Kanamycin And Chloramphenicol Residues Analysis Based On Structure-switching Aptamers

With the increasing abuse of antibiotics,the detection of antibiotic residues in food has become increasingly important.The traditional detection methods mainly include high performance liquid chromatography and liquid chromatography-mass spectrometry,but they require skilled operators and the operation is complicated and time-consuming,which make them difficult to meet the requirements of on-site rapid detection.Nucleic acid aptamers have become a new type of biosensor identification element,taking advantages of their specificity,high affinity,good stability,and low price.Structure-switching aptamers are versatile in principle and have a wide range of applications.In this study,we established two structure-switching aptamers with different structure.The main aim of detecting the residues of kanamycin and chloramphenicol in milk was to combine nucleic aptamers with fluorescence resonance energy transfer(FRET),realizing a fast,sensitive detection of the target.The main contents of the study include:1.A method for detecting kanamycin using a structure-switching aptamers,which is characterized in that an aptamer labeled with FAM and complementary DNA chain labeled with DABCYL,are designed using the principle of fluorescence resonance energy transfer.The structure of the two chains constitutes a switch.In the absence of target,the aptamer binds to complementary strand via base pairs forming a structural switch,thus the fluorescence signal value is lower.When the target is present,the high affinity of kanamycin and its aptamer causes the structure-switching aptamers to open,releasing the complementary strand,resulting the fluorophore and the quenching group away,thus the fluorescence intensity increases.Based on the changes of fluorescence intensity,sensitive detection of kanamycin residues is detected.After a series of experimental optimizations,the structure-switching aptamers of the second group was found to be the best system.Under the optimal conditions,kanamycin showed linear relationship with the kanamycin in the range of 100~700 n M.The detection limit was 12.01 n M,and the recovery rate was 101.3%~109.1%.At the same time,this method has also been successfully applied to the detection of real samples with good stability,and this method was consistent with the results obtained by the national standard method.The structure-switching aptamers detection system provides a new method for the detection of kanamycin.2.Since the aptamer labeled fluorophore will reduce its binding affinity to the target,we established the three chains structure-switching aptamers to detect chloramphenicol,which contains a label-free aptamer,the FDNA labeled with a fluorophore,and QDNA labeled with a quenching group.This method is based on fluorescence analysis to achieve the target testing,which is almost in agreement with the ditection of kanamycin.Under optimal reaction conditions,chloramphenicol has a good linear relationship in the range of 1ng/m L~100ng/m,with the detection limit 0.75 ng/m L and the recovery rate 89.1%~106.6%.The method is simple,rapid,and can be used for on-site detection of chloramphenicol residues.At the same time,this method has been successfully applied to the detection of real samples of milk,providing a new idea for the detection of chloramphenicol.