Construction of ratiometric nano-converter based on DNA-templated silver nanocluster for piRNA sensing

PiRNAs have emerged as promising biomarkers for cancer diagnosis, however, their sensitive detection remains challenging due to the reliance on enzymatic amplification, complex labeling procedures. Herein, we report an integrated sensing strategy that combines a structure-switching ratiometric nano-converter with an enzyme-free entropy-driven catalytic (EDC) DNA circuit for highly sensitive and reliable piRNA detection. A hairpin DNA templated silver nanocluster (HP-DNA-AgNCs) that functions as a programmable nano-converter. In its closed hairpin state, the probe exhibits strong green fluorescence (530   nm). Upon hybridization with the target piRNA, the hairpin undergoes a structural transformation, triggering AgNC redistribution and a pronounced fluorescence shift to red emission (620   nm), thereby generating a ratiometric signal (F 620 nm / F 530 nm ). Notably, this structural transition is rationally coupled to an EDC amplification circuit, enabling each target piRNA to catalytically activate multiple nano-converters under isothermal conditions. Current method allows ultrasensitive piRNA detection with a limit of detection as low as 0.146 pM and a wide dynamic range. The clinical applicability of the platform was further validated using breast cancer tissue samples, yielding an area under the ROC curve (AUC) of 0.951, in excellent agreement with qRT-PCR analysis. By unifying ratiometric self-calibration, enzyme-free amplification, and label-free design, this work establishes a robust and versatile sensing paradigm with strong potential for clinical diagnostics and point-of-care testing.