Procedure 36 Genosensor on gold thin-films with enzymatic electrochemical detection of a SARS virus sequence

摘要

This chapter presents a procedure for the construction of a hybridization-based genosensor for a SARS (severe acute respiratory syndrome) virus sequence on a 100nm sputtered gold film, which works as immobilization and transduction surface. The chapter tests the sensitivity and the selectivity of the SARS genosensor using complementary strands of SARS virus and three-base mismatch strands. Genosensor construction include following steps: a drop of 5 mL of 1.02 mM thiolated probe deposited on the gold film and maintain at 37°C for 20 min or at 41°C for 12 h; it is cleaned with 0.1M Tris-HCl buffer; further a15 mL drop of a 2% 1-hexanethiol solution is deposited on the gold film and maintained for 10 min and again cleaned with a 2×SSC buffer solution pH 7. From the results of hybridization assay and recording of the analytical signal no significant difference found between the analytical signal obtained for a 3.03nM solution of the complementary target strand and three-base mismatch strand. The limit of detection, calculated as the concentration corresponding to a signal which is three times the standard deviation of the intercept, results to be 5 pM. This means an improvement of various orders of magnitude when compared with limits of detection reported in the bibliography for DNA assays.