AND‑Gated Proximity‑Induced Assembly of Tandem G4 DNAzyme by Dual-aptamer Recognition Strategy for Integrated Enrichment and Colorimetric Detection of Circulating Tumor Cells

Accurate recognition and capture of circulating tumor cells (CTCs) remain challenging due to their extreme rarity and heterogeneity. Strategies based on logical analysis of multiple surface markers offer inherent advantages in specificity but often suffer from insufficient sensitivity. Herein, we report an intelligent colorimetric platform integrating enrichment and detection via dual aptamer recognition strategy-mediated proximity-induced in situ assembly of tandem G4 DNAzymes using MCF-7 breast cancer cells as a CTC model. The platform employs two aptamers that specifically recognize EpCAM and MUC1, two membrane-associated protein markers overexpressed on MCF-7 cells. Upon simultaneous recognition of two distinct membrane proteins on CTCs, the DNAzymes cooperatively assemble on the cell surface, enabling cascade signal amplification without exogenous enzymes and executing an AND logic gate. By coupling a micropore membrane-based enrichment device with a portable smartphone-assisted colorimetric readout system, the system achieves a detection limit as low as 2 cells/mL and a linear range of 10 - 5 × 10 4 cells/mL. Leveraging the dual-aptamer logic gate, it accurately distinguishes target cell subtypes from heterogeneous populations based on the presence or absence of specific biomarkers. Spike recovery in blood samples ranges from 95.42% to 107.26%, and visual quantitative analysis is completed within 10   min. This work provides a promising strategy for liquid biopsy by combining high sensitivity, intrinsic specificity, and operational simplicity in a compact, integrated format.