Label-Free Electrochemiluminescent Aptasensor with Attomolar Mass Detection Limits Based on a Ru(phen)32+-Double-Strand DNA Composite Film Electrode

Precisely known ligand-induced conformation change and complex chemical labeling of the DNA sequence with probe molecules are often needed for the signal generation in most of the previous aptasensors. Herein, a solution to the above problems was reported by the use of the Ru(phen)(3)(2+) intercalated into double strand DNA (dsDNA) as an electrochemiluminescence (ECL) probe with thrombin as the target. After the antithrombin thiolated aptamer (27-mer) was attached to a gold electrode, ds-DNA structure was formed with its complementary 20-mer single strand DNA. Instead of the chemical modification of the aptamer or target with the probe molecule, Ru(phen)(3)(2+), as the probe, was intercalated into the ds-DNA structure. After thrombin hybridized with its aptamer, the ds-DNA dissociated and the intercalated Ru(phen)(3)(2+) released because of the higher stability of the aptamer-thrombin complex than that of the aptamer-complementary strand hybrid. The difference in ECL intensity with tripropylamine (TPA) as coreactant before and after the hybridization of thrombin and its aptamer was used to quantify thrombin. Besides the increase in the number of probe molecules over the single-site labeling, a ca. 80-fold improvement on the TPA oxidation at the dsDNA modified electrode was found over the bare gold electrode. With the two amplification factors, the mass detection limits of 0.2 attomolar for thrombin are obtained. Because of the independence of conformational changes, the present method is readily extended to the targets whose aptamers have no specific conformational changes or other DNA-related detection without the need for chemical labeling.