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

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.