Aptamer Nanoswitch-Mediated Lock-Expose Mechanism Enables Highly Sensitive In Vitro Detection and Precise In Situ Membrane Imaging of HER2 Protein.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer correlates with high aggressiveness, an increased recurrence rate, and poor survival, holding significant diagnostic value. In this work, a HER2-specific aptamer (Apt) was engineered into a Apt nanoswitch (hApt) and revealed the interaction of HER2-aptamers through molecular docking and quantified the binding energy and dynamic behavior through molecular dynamics simulations. A lock-expose mechanism was designed by combining the target-induced conformational switch of hApt with template-prefabricated rolling circle amplification (rRCA), forming a T-hApt-rRCA sandwich structure. Coupled with CRISPR/Cas12a and fluorescent probes, this sensor enabled highly sensitive detection, with a linear range of 10 fg/mL to 10 ng/mL and a limit of detection of 1.42 fg/mL. Using HUVEC, A549, MCF-7, and SK-BR-3 cell lines to model HER2 heterogeneity in circulating tumor cells (CTCs) enabled in situ imaging, differentiation, and quantitative detection of membrane HER2 expression, thereby providing direct visualization of expression levels and highlighting the translational promise of this approach. The accurate detection and clear differentiation between the eight healthy samples and the twenty-eight breast cancer patient samples further underscore the practical applicability of this sensing strategy.