Label-free Fluorescence Aptasensors For Detection Of Oxytetracycline And Melamine In Milk

This research is affiliated with The National Natural Science Foundation Project-?The Analysis of Multiple Antibiotic Residues Based on Aptamer Switches and Signal Amplification of Enzymatic Cycle Strategy??31571919?.As a common food in people's life,milk is closely related to people's health.Aptamers are widely used in the construction of biosensors due to their advantages of high affinity,good specificity and easy modification.In this thesis,we established two novel aptamer-based fluorescent sensors utilizing the conformational changes of the binding of target and aptamer.This method was based on the specific recognition between aptamer and target,employing the DNA staining reagent and the optical properties of lanthanide rare earth elements,and will be adaptable for rapid detection of oxytetracycline?OTC?and melamine?MA?in milk.The main contents are as follows:The fluorescence sensor for detecting oxytetracycline was constructed by using the specific recognition ability of aptamer and the optical properties of SYBR GREEN?SGI?.In the absence of oxytetracycline,SGI incorporates a hydrogen bond between the oxytetracycline aptamer and its complementary sequence,giving a high fluorescence intensity at 495 nm of excitation light.When oxytetracycline is present in the system,the aptamer specifically binds to the target and peels away from the double-stranded DNA,resulting in the release of SGI from the hydrogen bond and a significant decrease in the fluorescence intensity of the system.In addition,the addition of exonuclease Exo I can cut aptamer,which can re-release the target,the formation of the role of enzymatic amplification cycle amplification.Under the optimum conditions,the linear range of the method for the detection of oxytetracycline was 1?g·mL^-1-10?g·mL^-1 with the detection limit of 0.157?g·mL^-1.This assay showed excellent selectivity towards oxytetracycline over other oxytetracycline analogues.In the real milk samples,the obtained recoveries of this method is similar to that of HPLC method.In summary,the method has the advantages of high sensitivity,simplicity,economy and so on,which is expected to be suitable for the preparation of a kit for the field test of an actual sample.We established a fluorescence probe for detection of Melamine in Milk,which taking advantages of terbium ions and special modified aptamer,as well as special modified aptamer complementary sequence.First,the aptamer complementary sequence is modified with six consecutive guanine bases?G6?and allowed to form its own hairpin structure.The guanine-rich single-stranded nucleic acid sequence and the metal terbium ion give higher fluorescence intensity at 290 nm excitation light.Then add a certain amount of melamine aptamers to construct melamine detection system.In the absence of melamine,the melamine aptamer binds to the complementary sequence,allowing fluorescence quenching.When melamine is present in the system,the aptamer binds to its target while releasing the complementary sequence,thus the fluorescence intensity is restored.Under the optimal conditions,the melamine was tested to prove the feasibility of this method.The linear range of melamine obtained in this experiment was from 0.1?g·mL^-1 to1.0?g·mL^-1 with the detection limit of 0.057?g·mL^-1.This method shows high selectivity to melamine against other structural analogues.In addition,the recovery results of this method were almost in accord with those from HPLC method in the real sample test,which indicates that this method can be used for the detection of melamine in milk.This new rare earth ion fluorescence sensor creates good conditions for on-site testing of melamine in milk.