Accelerated CRISPR/Cas12a-Based Point-of-Care Diagnostics Through Critical Coupling Distance Control.

Prompt pathogen detection in resource-limited settings remains constrained by energy-intensive instrumentation and a shortage of trained personnel. The CRISPR/Cas12a-based diagnostic technology, despite its robustness as a promising tool, is constrained by suboptimal detection speed and sensitivity. Here we designed triblock DNA-mediated spherical nucleic acids (tSNA) that acts as a spatially confined reporter with critical coupling distances between substrates, enabling Cas12a protein to rapidly identify concentrated and stretched single-stranded substrates with size-matching intervals. Precise control of distances on tSNA of varying sizes revealed a direct correlation between trans-cleavage efficiency and coupling distance, indicating that only when the distance exceeds the protein size can it offer an appropriate reaction space. It demonstrates a rapid "scooting" reaction model on tSNA, resulting in a trans-cleavage rate of 10 nm A30-tSNA 12 times faster and a sensitivity that is two orders of magnitude higher than that in bulk solution. Furthermore, tSNA can serve as a novel recognition and colorimetric element in lateral-flow strips, thereby reducing the detection time for pathogen nucleic acids to just 3 min. This "size-matching" model of tSNA offers a new perspective on the regulation of Cas12a enzymatic activity, establishing a versatile platform to advance diagnostic development through ultrafast, CRISPR-powered POC systems.