CRISPR/Cas12a-functionalized silicon nanowires field-effect transistor sensor for ultra-sensitive detection of...

Rapid, sensitive, and accurate detection of pathogen nucleic acids is critical for ensuring public safety and health. Nevertheless, current methods still encounter significant challenges. Field-effect transistor (FET) biosensors are renowned for high sensitivity, rapid response, and label-free detection. However, when employed for the direct detection of long-chain DNA extracted from pathogens, these sensors exhibit low recognition efficiency, poor accuracy, and prolonged reaction times. To address these limitations, we propose a novel silicon nanowires FET sensing strategy functionalized with the CRISPR/Cas12a system. The Cas12a/crRNA complex rapidly scans and precisely cleaves target sequences within long double-stranded DNA (dsDNA). This mechanism effectively mitigates detection performance degradation caused by nucleic acid folding and entanglement, thereby significantly enhancing both sensitivity and accuracy. Additionally, Cas12a/crRNA cleaves long dsDNA into specific-length fragments, thereby ensuring their distribution within the Debye length and enhancing signal consistency. Using this approach, we successfully achieved quantitative detection of Bacillus anthracis dsDNA within 10 min, with a detection limit at the attomolar (aM) level. Furthermore, the correlation coefficient between detection results of real whole-genome samples and digital PCR reached 0.912, validating the reliability of this strategy. In summary, this strategy provides a highly valuable reference for the direct detection of pathogen nucleic acids.