Endogenous Protease-Activatable Nanosensor Based on PNA–Peptide–DNA Engineering for AND-Gated and Dual-Model Detection of MicroRNA in Single Living Tumor Cells

The in situ detection of low-content cancer biomarkers by an endogenous activator instead of an exogenous initiator in vitro remains a great challenge, leaving a gap in the development of a tumor-specific nanosensor with an endogenous protease-activatable manner. Herein, we proposed an endogenous protease-activatable nanosensor (PA-NS) guided by peptide nucleic acid-peptide-DNA copolymers to realize AND-gated and dual-model sensing of miRNA-21 (miR-21) by combining electrochemical detection with optical imaging in living tumor cells, without an additional introduction of an exogenous activator or nanomaterials. Moreover, the PA-NS can only be activated by "dual keys" (overexpressed miR-21 and cathepsin B protease in tumor cells) simultaneously, which enables effective improvement of the tumor-to-healthy cells ratio. The fluorescence intensity measured in single tumor cells was ∼3.5-fold higher than that in single healthy cells, and the electrochemical response decreased ∼30% in the presence of target miRNA. Furthermore, studies on regulation of the protease activity and miR-21 fluctuation under external stimulation have contributed to our understanding of the biological processes and drug screenings underlying disease development. This specific endogenous protease-mediated manner for dual-model detection of miRNA guarantees excellent tumor-selective capability, which offers new opportunities to study cell heterogeneity and provides more reliable fundamentals for the diagnosis and treatment of cancer down to the single-cell level.