Exonuclease III-assisted CRISPR/Cas12a electrochemical biosensor for detection of transcription factor binding catalytic hairpin assembly signal amplification technology

Transcription factors (TFs) play a critical role in regulating gene expression, influencing essential cellular processes such as development, growth, and differentiation. Dysregulation of TFs, such as NF-kappa B p50, is linked to a range of diseases, including cancer and inflammatory disorders, highlighting the need for sensitive and accurate detection methods. This study presents a novel electrochemical biosensor that utilizes exonuclease III (Exo III)assisted CRISPR/Cas12a combined with catalytic hairpin assembly (CHA) signal amplification for the quantitative detection of NF-kappa B p50. The detection strategy is based on the specific binding of NF-kappa B p50 to doublestranded DNA (dsDNA), which inhibits Exo III digestion, preserving a higher electrochemical signal. In the absence of NF-kappa B p50, Exo III cleaves dsDNA, triggering CHA and activating Cas12a's trans-cleavage activity, resulting in a weakened signal. Under optimized conditions, the biosensor demonstrated exceptional sensitivity with a detection limit of 0.24 pM. The system also showed excellent specificity and stability, with minimal interference from other proteins. Recovery tests performed in spiked nuclear extracts demonstrated high accuracy, confirming the biosensor's suitability for real sample analysis. This innovative biosensor offers a rapid, costeffective, and ultrasensitive approach for detecting transcription factors like NF-kappa B p50, with significant potential for early clinical diagnostics and biomedical research applications.