Cas12a-Programmed Modular CRISPR Cascade Reaction on Paper Supports for Dual-Mode Detection of Pathogenic Genomes

We developed an optical biosensing platform using cost-efficient and scalable paper support for the detection and identification of three major bacterial pathogens using fluorescent DNA-templated silver nanoclusters (FNPs) and an innovative CRISPR-Cas12a cascade reaction. The sensors were fabricated as ∼5 mm letter-shaped paper cutouts, with each letter representing a specific pathogen: “C” for Campylobacter jejuni, “E” for Shiga toxin-producing Escherichia coli, and “L” for Listeria monocytogenes. Detection was initially achieved via an ON-to-OFF mechanism, wherein target recognition by Cas12a led to FNP degradation and fluorescence loss using target strands identified from the conserved genomic regions from each pathogen. This platform successfully detected individual and multiple targets in all possible seven combinations. To enhance diagnostic clarity, we developed a two-step CRISPR-Cas12a cascade reaction enabling an ON signal output when the target is present, a more intuitive and desirable reporting format. In this design, the first Cas12a reaction detects the target and cleaves an activator strand, preventing activation of a second Cas12a reaction that would otherwise degrade FNPs. Consequently, fluorescence is retained in the presence of the target (ON-retention) and lost in its absence, providing a clear ON signal when the target is detected, and an OFF signal when it is not. Finally, we demonstrated both ON-to-OFF and ON-retention detection modes using the whole Listeria genome amplified by isothermal recombinase polymerase amplification, with reliable detection of as few as 40 full genomic copies using fluorescent images on paper substrates. This work represents a significant advancement in Cas12a-based biosensing, uniquely demonstrating multistep biochemical reactions directly on paper support, and offers a promising platform for low-cost, scalable pathogen detection in resource-limited settings.