Self-assembled DNA nanosphere-powered confined-free self-circulating amplifier for high-fidelity microRNA imaging and...

The development of predictable and thermodynamically stable DNA nanomachines remains challenging for the monitoring of intracellular microRNAs (miRNAs) in complex biological matrices. In this study, a fluorescent biosensor utilizing a confined-free synergistic configuration is presented for the precise detection of miR-21. To mitigate the thermodynamic limitations of homogeneous catalytic hairpin assembly (CHA), a process in which unconstrained molecular diffusion induces background leakage, the amplification components were spatially partitioned. The sensing platform incorporates a rigid DNA nanosphere (NP), assembled via the annealing of three palindromic sequences, which serves as a sterically hindered scaffold to strictly confine hairpin probe 1 (HP1). Target miR-21 initiates the opening of the anchored HP1, subsequently recruiting freely diffusible auxiliary probes (HP2 and HP3) to form a localized Y-shaped complex. This reaction generates a surrogate miR-21 sequence that detaches and unlocks adjacent HP1 probes, establishing an inter-particle chemical communication network. By integrating nanoscale spatial confinement with this propagated catalytic cascade, the proposed confined-free self-circulating amplifier (CF-SCA) suppresses background leakage while maintaining rapid mass transfer, yielding a limit of detection of 100 pM. Additionally, the dense 3D nanoscaffold provides nuclease resistance. Following liposome-mediated cellular delivery, the confined-free mechanism counteracts macromolecular crowding, facilitating in situ dynamic imaging of miR-21 in living cancer cells. Evaluated through the stratification of clinical serum samples from multiple cancer cohorts, the CF-SCA nanoplatform offers an enzyme-free tool for clinical diagnostics and liquid biopsy applications.