Electrochemiluminescent biosensor of ATP using tetrahedron structured DNA and a functional oligonucleotide for Ru(phen)3(2+) intercalation and target identification.

Restricted target accessibility and surface-induced perturbation of the aptamer structure are the main limitations in single-stranded DNA aptamer-based electrochemical sensors. Chemical labeling of the aptamer with a probe at the end of aptamer is inefficient and time-consuming. In this work, tetrahedron-structured DNA (ts-DNA) and a functionalized oligonucleotide (FO) were used to develop an electrochemiluminescence (ECL) aptasensor with adenosine triphosphate (ATP) as a model target. The ts-DNA was formed with three thiolated oligonucleotides and one oligonucleotide containing anti-ATP aptamer. The FO contained a complementary strand to the anti-ATP aptamer and an intermolecular duplex for Ru(phen)3(2+) intercalation. After the ts-DNA was immobilized on the electrode surface through gold-thiol interactions, hybridization between the anti-ATP aptamer and its complementary strand introduced the intercalated Ru(phen)3(2+) to the electrode. ECL emission from Ru(phen)3(2+) was observed with tripropylamine as a co-reactant. Once ATP reacted with its aptamer, the aptamer-complimentary strand duplex dissociated and the intermolecular duplex containing Ru(phen)3(2+) was released. The difference in emission before and after reaction with ATP was used to quantify ATP with a detection limit of 0.2nM. The ts-DNA increased the sensitivity compared to conventional methods, and the intercalation strategy avoided a complex chemical labeling procedure.