AND-Gated Intramolecular Confined CHA Converts Forked DNA Probes into Brushlike Assemblies for Tumor-Cell Imaging

Sensitive and selective imaging of low-abundance intracellular biomarkers is important for cellular analysis and disease-related biomarker evaluation, but it is often hampered by insufficient sensitivity and false-positive signals from single-target analysis. Herein, we developed a dual-locked forked DNA fluorescent probe (DL-forked probe) that integrated intramolecular confined catalytic hairpin assembly (intra-CHA) with AND-logic gating. The probe facilitated cellular uptake in the tested tumor cells via the AS1411 aptamer, without transfection reagents. APE1 and miRNA-21, two biomarkers overexpressed in tumor cells, served as the dual inputs. APE1 first cleaved the AP site in H2 to expose a toehold for miRNA-21, which then triggered a rapid intra-CHA cycle, converting the forked structure into brushlike DNA assemblies with fluorescence recovery. The spatial confinement effect restricted the reactants within ∼13 nm, with the geometric local concentration approximately 9.2 × 103-fold higher than the nominal concentration of the free-CHA system, thereby drastically accelerating reaction kinetics. The DL-forked probe exhibited a detection limit of 29 pM for miRNA-21 and 7.0 × 10−3 U/mL for APE1, with outstanding selectivity. In live-cell imaging, the probe generated 2.2-fold stronger fluorescence than the free-CHA control and produced distinct fluorescence responses among the tested tumor cells (MDA-MB-231, MCF-7, A549) and normal AML12 cells, with signal enhancements of 14.7- to 33.0-fold. These results agreed well with the independent reference methods of ELISA and RT-qPCR. This AND-gated, confinement-accelerated CHA platform provides a potential strategy for dual-biomarker-gated intracellular imaging and warrants further evaluation in clinically relevant models.