Cu(I)-Catalyzed Click Reaction-Triggered 3D DNA Walker for Constructing an "OFF-ON" Fluorescent Biosensor for Cu Detection.

Herein, a highly selective and sensitive "OFF-ON" fluorescent biosensor was designed for intracellular Cu detection. Compared to the fluorescent Cu biosensors reported so far, this work tackled the tricky issue of reliability of Cu, which mainly depends on the integration of the high selectivity of the Cu(I)-catalyzed click reaction with the ultrahigh sensitivity of a spherical nucleic acid-based 3D DNA walker. Typically, DNA track is carried out by coconjugating N-S1 and Cy3-HP onto gold nanoparticles (AuNPs). On this state, fluorophore (Cy3) was close to the surface of AuNPs (as a nanoquencher), generating a quenched fluorescence and thus causing the initial "OFF" state. In the presence of Cu and HC-swing arm, Cu was generated quickly from the reduction of Cu with the assistance of ascorbic acid, which could promptly and selectively trigger the Cu(I)-catalyzed click reaction-based 3D DNA walker between azide on N-S1 and alkyne on the HC-swing arm. Sequentially, the activated HC-swing arm was able to hybridize with adjacent Cy3-HP and the 3D DNA walker was automatically driven by N.BstNBI to produce multiple Cy3-labeled DNA fragments away from the AuNP surface for signal amplification, performing a recovered fluorescence response (turning into the "ON" state). Accordingly, the ingenious integration of an efficient click reaction and smart 3D DNA walker endows the constructed fluorescent biosensor with superior selectivity and ultrahigh sensitivity. We further apply this platform for Cu sensing in biological systems; this assay will provide a signal transduction strategy for evaluating intracellular Cu at picomolar levels.