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

ABSTRACT 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.