Label-Free Detection Of Thrombin Based On AP Site

DNA is the main component of chromosome, and also is the carrier of genetic information. It was attacked unceasingly resulted from external or internal factors in all cells. In the general physical condition or the action of the outside, however, about 10,000 apurinic sites and 500 apyrimidinic sites, referred to as abasic site (AP site), were produced in a cell one day. At present, the detection of AP sites is under going and has been applied to the analysis field. Aptamers, short nucleic acid-binding species isolated from systematic evolution of ligands by exponential enrichment, have emerged as promising candidates for molecular recognition events due to their high sensitivity and specificity to bind large numbers of ligands.Thrombin, as an important measure in the body’s blood coagulation system, with the double function of coagulation and anticoagulation, is a kind of very vital enzyme. It plays an important role in thrombosis regulation and tissue repair. Therefore, rapid and accurate detection of thrombin is of great importance in clinical diagnosis.An AP site was introuduced in the single-stranded DNA which was partly matched with the DNA-containing thrombin aptamer composed of the reported 15-mer thrombin aptamer and an additional 6 nucleoside bases at the 3’-terminal. With silver nanoclusters (Ag NCs) and 2-Amino-6,7-dimethyl-4-hydroxypteridine (diMe-pteridine), two kinds of label-free thrombin sensors were constructed by double-stranded DNA-containing AP site (AP-dsDNA) based on fluorescence quenching and recovery mechanism.The study found that the length of AP-DNA has important influence on the sensitivity of the system and the suitable length of AP-DNA can improve the sensitivity of detection. At the same time, the buffer solution, the incubation time, temperature and other factors of the detection system were also optimized. Under optimal conditions, the fluorescence intensity had a good linearity with the concentration of thrombin in the range from 9.09×10-9 to 9.2×10-8mol/L and 2.98×10-8 to 1.00*10"7mol/L with a detection limit of 7.06×10-9mol/L and 1.69×10-8 mol/L (S/N=3) for diMe-pteridine and Ag NCs, respectively. Finally, the selectivity of systems was detected by equal concentrations of glucose, glutathione, lysozyme and thrombin. It concludes that results are in accord with expectation, and anticipative purpose is obtained.