Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA.

Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a's target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.