Target-silent self-driven DNA walker for small molecule detection with antibody immobilized on gold nanoparticles

A three-dimensional DNA walker offers an efficient strategy for sensitive detection of analytes with signal amplification. In this study, we report a target-silent, self-driven DNA walker for detecting small molecules (SMs). The DNA walker is composed of the Mg²⁺-dependent 8-17E DNAzyme walking strand conjugated with an SM (W-SM) and the three-dimensional walking track on gold nanoparticle (AuNP). The AuNP surface is functionalized with monoclonal antibody (mAb) and fluorescently labeled substrate of DNAzyme. Without target molecules, the W-SM is attached to the surface of AuNP via the antigen-antibody interaction. The DNAzyme catalytically cleaves the substrate, driving the W-SM autonomously moving along the walking track and generating high fluorescence. In the presence of SM target, the SM target competes with the W-SM in binding with the antibody on AuNP, and the DNA walker becomes inactive, causing fluorescence decline. This DNA walker enabled detection of digoxin and folic acid at concentrations as low as 0.2nM and 1 nM, respectively. It also performed well in diluted serum samples in responding to targets. This proposed strategy provides a new approach for constructing a DNA walker with a simple design for sensitive detection of small molecules in solution phase, showing promise in applications.