Construction Of G-Triplex-ThT Fluorescent Probes And Their Appl Ications In The Detection Of TNOS,Hg²⁺ And Kanamycin

Food safety is closely related to the national economy and the human's livelihood and has become an increasingly concerned problem.In order to ensure food safety,it is very important to sensitively and rapidly detect the harmful substances or products with potential hazards during the production and circulation of food and agricultural products.The traditional detection and analysis technologies for harmful substances in food and agricutural products often rely on chromatographic separation technology or antigen-antibody recognition,which have some disadvantages,like using sophisticated instruments,time-consuming,tedious operation,high cost and lack of accurate.Therefore,it is of great significance to develop a simple,rapid,sensitive and accurate detection method to detect harmful substances,as well as ensure food safety.In this work,three single strand DNA sequences which could recognize different target materials were designed and linked to G-triplex(G31)segment,therefore,three new label-free fluorescent probes were constructed by using thioflavin T(ThT)as fluorescent molecule,which were used to detect the terminator of nopaline synthase gene(TNOS),Hg²⁺and kanamycin.(1)The cDNA-G31 detection probe were designed by linking TNOS complementary DNA(cDNA)with G31,by using ThT as fluorescent molecule,a new label-free fluorescent probe was constructed,which was utilized for the quantitative detection of TNOS in transgenic soybean.ThT can be embedded into the groove of G-triplex formed by the G31 fragment in cDNA-G31,resulting in the single bond rotation of ThT between the two aromatic rings blocked,and the two aromatic rings kept in the same plane,as well as the ThT fluorescence signal enhanced.After the TNOS was added into the system,TNOS was hybridized with cDNA segment in cDNA-G31 and formed double stranded species.The newly formed double stranded structure could stabilize the binding of ThT and G-triplex,and further enhance the fluorescence signal of ThT.Through the optimization experiments,the optimal reaction conditions were obtained:the concentration of cDNA-G31 was 200 n M,the concentration of ThT was 6?M,the p H value of the solution was 7.3,the concentration of Na⁺was 40 m M,the concentration of Mg²⁺was 40 m M,and the reaction time between TNOS and cDNA-G31 was 60 min.Under the optimal reaction conditions,the detection limit of TNOS is 2.22 n M and the linear detection range is 2n M?200 n M.This label-free fluorescent probes has good selectivity,low cost,simple operation process and high sensitivity,and has been successfully used for the detection of TNOS in transgenic soybean samples,which provides the basis for the detection of other transgenic fragments and biomolecules,as well as the design of other probes.(2)The T-rich-G31 probe was designed by connecting the T-rich sequence with G31,and a label-free fluorescent probes was constructed by using ThT as fluorescent molecule to sensitively detect Hg²⁺in aqueous solution.Since Hg²⁺could specifically combine with T-base in T-rich-G31 to form T-Hg²⁺-T unit,therefore,induced several T-Hg²⁺-T intramolecular folded double chain structure.This double chain can stabilize the complex formed by ThT and G-triplex,and further enhance the fluorescence signal of ThT.Through the optimization experiments,the optimal reaction conditions were obtained:6T-G31 fragment containing 6 thymines was select,the concentration of 6T-G31 was 150 nm,the concentration of ThT was 4?M,the p H value of solution was 7.5,the reaction time between Hg²⁺and 6T-G31 was 10 min.Under the optimal detection conditions,the detection limit of Hg²⁺was 0.75 n M,and the detection range was 2 n M?300 n M.The label-free fluorescent probe has the advantages,like good selectivity,low cost,simple operation process,high sensitivity and less time consumption.It has been successfully applied for the detection of Hg²⁺in actual water samples with reasonable practicability and good accuracy.Gel electrophoresis analysis and circular dichroism spectroscopy were used to study the principle of the assay,the double chain DNA structure linked to the G-triplex could form new complex,which enhanced the conformation stability of G-triplex and ThT complex,and resulted in the further enhancement of ThT fluorescence signal.This assay is helpful for the construction of new probes,as well as the development of new method for food safety rapidly detection in future.(3)The Aptamer-G31 detection probe was designed by connecting kanamycin aptamer with G31,by using ThT as fluorescent molecule,a new label-free fluorescent probes was constructed for the sensitive and rapid detection of kanamycin in different meat samples.The aptamer unit of Aptamer-G31 contains several guanine(G)bases,which can form G-quadruplex analogous structure with G31.When ThT is embedded in this conformation,the fluorescence signal intensity of ThT increases significantly,which is much stronger than that of ThT combined with G-triplex alone.When kanamycin is added into the Aptamer-G31 system,since the highly specific interaction between kanamycin and aptamer segment in Aptamer-G31,the original G-quadruplex like structure of Aptamer-G31 was destructed,resulting in the decrease of ThT fluorescence signal.Because kanamycin mainly interacts with G bases in aptamer,when the concentration of kanamycin further increases to a certain extent,kanamycin will competitively bind to G-triplex,thus reducing the interaction between G31 and ThT,and further reducing the fluorescence signal intensity of the solution.Under the optimal reaction conditions,i.e.,the concentration of Aptamer-G31 was150 n M,the concentration of ThT was 4?M,the p H value of solution was 6.3,and the reaction time between kanamycin and Aptamer-G31 was 30 min,the detection limit of kanamycin was 1.05 n M,and the detection range was 50 n M?2000 n M.Through the determination of kanamycin in pork,chicken and beef by standard addition method,we could demonstrate this assay has good recovery.The label-free fluorescent probe has the advantages like,good selectivity,low cost,simple operation and short time-consuming.Our method extends the application of target-recognition DNA segments and G31 functional sequence,and has potential application in the sensitive detection of other biomolecules in food science?analysis and other fields.