Localized DNA Logic Circuit Equipped with Cascade Amplifiers for Precise Identification of Cancer Cells

Precise and highly sensitive identification of cancer cells plays a pivotal role in early cancer detection, diagnosis, and effective treatment. While DNA logic circuits have shown great promise as diagnostic tools, their practical application has been hindered by inadequate sensitivity arising from limited signal amplification capabilities in complex biological matrices. To address this issue, we constructed a localized DNA circuit (LDC) equipped with cascaded amplifiers by introducing a Y-shaped AND-gate circuit module and three hairpin amplifier modules into a DNA tetrahedron. The Y-shaped logic gate is activated only in the simultaneous presence of two cancer-specific biomarkers: intracellular microRNA-21 (miR-21) and flap endonuclease 1 (FEN1). Upon activation, the logic gate releases output strands that trigger the assembly of hairpin amplifiers, initiating a localized strand displacement amplification cascade that generates a significantly enhanced fluorescent signal. The LDC exhibits remarkable sensitivity with detection limits of 82.5 pM for miR-21 and 0.015 U/mL for FEN1. Fluorescence assays demonstrate that the LDC achieves a 15.5-fold improvement over circuits without amplifiers and a 5.2-fold enhanced sensitivity compared to nonlocalized circuits. The LDC enables simultaneous detection of the dual biomarkers, generating significantly amplified fluorescent signals exclusively in tumor cells expressing both miR-21 and FEN1, thus allowing precise discrimination between cancerous and healthy cells. Furthermore, we demonstrated that the LDC system enables in vivo tumor imaging, effectively differentiating between normal and tumor tissues. This work highlights the potential of the proposed localized cascade-amplification DNA circuit strategy for tumor-specific imaging, paving the way for precise cancer diagnosis and treatment.