Ultrasensitive Solution-phase Electrochemical Molecular Beacon-based DNA Detection with Signal Amplification by Exonuclease III-assisted Target Recycling

Taking advantage of the preferential exodeoxyribonuclease activity of exonuclease III in combination with the difference in diffusivity between an oligonucleotide and a mononucleotide toward a negatively charged ITO electrode, a highly sensitive and selective electrochemical molecular beacon (eMB)-based DNA sensor has been developed. This sensor realizes electrochemical detection of DNA in a homogeneous solution, with sensing signals amplified by an exonuclease III-based target recycling strategy. A hairpin-shaped oligonucleotide containing the target DNA recognition sequence, with a methylene blue tag close to the 3' terminus, is designed as the signaling probe. Hybridization with the target DNA transforms the probe's exonuclease III-inactive protruding 3' terminus into an exonuclease III-active blunt end, triggering the digestion of the probe into mononucleotides including a methylene blue-labeled electro-active mononucleotide (eNT). The released eNT, due to its less negative charge and small size, diffuses easily to the negative ITO electrode, resulting in an increased electrochemical signal. Meanwhile, the intact target DNA returns freely to the solution and hybridizes with other probes, releasing multiple eNTs and thereby further amplifies the electrochemical signal. This new immobilization-free, signal-amplified electrochemical DNA detection strategy shows great potential to be integrated in portable and cost-effective DNA sensing devices.