Research On The Selection,Identification And Applification Of Aptamers Specific For Plasticiser Di(2-ethylhexyl) Phthalate

Di(2-ethylhexyl)phthalate(DEHP)is a kind of endocrine disrupting pollutant which is harmful to human health.The detection method based on aptamer is popular with its rapid and specificity.The aptamers were selected from an immobilized ssDNA library using the systematic evolution of ligands by exponential enrichment(SELEX).The real-time quantitative PCR(Q-PCR),high throughput sequencing(HTS),gold nanoparticles(Au NPs)biosensor and localized surface plasmon resonance(LSPR)were used to monitor enrichment and identify aptamers.After eight rounds enrichment,retention rate of the library was 14%.The HTS,Clustal X2 and Treeview analyses indicated that20000 sequences were enriched and 129 sequences with high homology rate were selected for bioactivity identification.The Au NPs biosensor and LSPR analysis indicated that four aptamers had higher binding activity and affinity constant were2.26±0.06 n M,5.33±0.01 n M,2.68±0.2 n M,43±0.7 n M,respectively.The aptamer 31 modified with sulfhydryl at its 5’ end was anchored on the surface of the gold electrode by Au-S bond.The electrochemical impedance spectroscopy(EIS)aptasensor was constructed after optimizing the incubation time between DEHP and aptamer 31.The results showed that the optimal incubation time between DEHP and aptamer 31 was 30 min.The EIS aptasensor had the limit of detection(LOD)of 0.103 pg/m L with no cross-reactivity to DEHP analogs and a mean recovery of 76.07 to 141.32% for detection of water samples.The aptamer 31 is novel(different from previously reported aptamers)with the highest affinity and sensitivity.