DNA Tetrahedron-enhanced single-particle counting integrated with cascaded CRISPR Program for ultrasensitive dual RNAs logic sensing

CRISPR-Cas-based technology, emerging as a leading platform for molecular assays, has been extensively researched and applied in bioanalysis. However, achieving simultaneous and highly sensitive detection of multiple nucleic acid targets remains a significant challenge for most current CRISPR-Cas systems. Herein, a CRISPR Cas12a based calibratable single particle counting-mediated biosensor was constructed for dual RNAs logic and ultra-sensitive detection in one tube based on DNA Tetrahedron (DTN)-interface supported fluorescent particle probes coupled with a novel synergistic cascaded strategy between CRISPR Cas13a system and strand displacement amplification (SDA). As expected, our platform enables dual RNA molecules intelligent detection using only one crRNA of Cas13a, achieving a sensitivity enhancement of three orders of magnitude assisted with multiple signal amplification and accurate fluorescence particle counting with DTN mediated nano-biointerface enhancement, compared to traditional bulk Cas13a assays. Moreover, the effectiveness and universality of our strategy are experimentally investigated and demonstrated through the detection of mRNAs (cervical cancer swab clinical samples and cultured cancer cells) and bacterial 16s rRNAs. This work not only proposes a highly promising avenue for designing CRISPR-based multiplex detection systems that excel in ultra-sensitivity, specificity, and clinical molecular diagnostics, but also provide new insights into the potential applications of nanotechnology in molecular diagnostics, functional surface engineering, and interface-mediated bioreactions.