Three-Dimensional DNA Nanomachine Biosensor by Integrating DNA Walker and Rolling Machine Cascade Amplification for Ultrasensitive Detection of Cancer-Related Gene

Stochastic DNA walkers capable of traversing on three-dimensional (3D) tracks have received great deal of attention. However, DNA walker-based biosensors exhibit limited amplification efficiency because of their slow walking kinetics and low processivity. Herein, by taking advantage of the high processivity of a DNA rolling machine, a sensitive ratiometric DNA nanomachine biosensor is designed. The biosensor is constructed with hairpin-loaded Au nanoparticles (NPs) (hpDNA@AuNPs) as a DNA walker and AgNCs-decorated magnetic NPs (AgNCs@MNPs) as a DNA rolling machine. In the presence of target DNA, exonuclease III (Exo HO-powered DNA walker is activated to accomplish first-stage amplification via a burnt-bridge mechanism, generating a great deal of toehold-loaded AuNPs (Toehold@AuNPs) to hybridize with magnetic nanoparticles loaded with silver-nanoclusters-labeled DNA (AgNCs@MNPs) with the assistance of Exo III. These trigger rapid rolling of AuNPs on the AgNCs@MNPs surface and release free AgNCs, converting the biological signal into a mass spectrometric signal ratio (Ag-107/Au-197) with detection by ICP-MS. A linear range of 0.5-500 fmol L-1 is achieved with a detection limit of 119 amol L-1 for the p53 gene. The practical applicability of the biosensor has been demonstrated in the accurate assay of the p53 gene in the human blood.