Abstract

"PIWI-interacting RNAs (piRNAs) are well-recognized as promising diagnostic biomarkers for cancer, yet their quantitative detection remains a great challenge owing to their short sequences, low cellular abundance, high degradation susceptibility, and significant sequence homology among family members. Herein, we developed an ultrasensitive and highly specific biosensor for the detection of piRNA-54265—a colorectal cancer (CRC)-associated piRNA—by integrating strand displacement amplification (SDA) with the CRISPR/Cas12a system. After systematic optimization, the biosensor exhibited remarkably enhanced amplification efficiency and target specificity, achieving an ultra-low limit of detection (LOD) of 57.54 aM for piRNA-54265. Notably, this CRISPR-SDA platform enabled accurate discrimination of CRC cells from other cancer cells via high-fidelity intracellular imaging of piRNA-54265 and also realized reliable detection of the target in complex biological matrices with favorable recovery. Benefiting from its simple sequence design, user-friendly operation, and isothermal reaction conditions, the developed biosensor not only overcomes the inherent technical bottlenecks in piRNA detection but also shows great potential for applications in cellular imaging and early clinical diagnosis of CRC. Graphical abstract