Cu(I)-Catalyzed Click Reaction-Triggered 3D DNA Walker for Constructing an "OFF-ON" Fluorescent Biosensor for Cu²⁺...

Herein, a highly selective and sensitive "OFF-ON" fluorescent biosensor was designed for intracellular Cu²⁺ detection. Compared to the fluorescent Cu²⁺ biosensors reported so far, this work tackled the tricky issue of reliability of Cu²⁺, 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 N₃-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 Cu²⁺ and H₂C₂-swing arm, Cu⁺ was in situ generated quickly from the reduction of Cu²⁺ 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 N₃-S1 and alkyne on the H₂C₂-swing arm. Sequentially, the activated H₂C₂-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 Cu²⁺ sensing in biological systems; this assay will provide a signal transduction strategy for evaluating intracellular Cu²⁺ at picomolar levels.