A highly sensitive ECL biosensor for NF-κB p50 detection based on entropy-driven amplification and CRISPR/Cas12a signal enhancement.

Transcription factors, particularly NF-κB p50, play crucial roles in regulating gene expression and are involved in several diseases such as cancer, autoimmune disorders, and chronic inflammation. The sensitive detection of NF-κB p50 is essential for clinical diagnostics and therapeutic monitoring. In this study, we present an electrochemiluminescence (ECL) biosensor designed for the highly sensitive and specific detection of NF-κB p50. The biosensor integrates entropy-driven amplification and CRISPR/Cas12a-based signal enhancement to detect trace amounts of NF-κB p50. Upon detection of NF-κB p50, a ternary complex forms with a double-stranded DNA (dsDNA) probe, which prevents subsequent cleavage by exonuclease III (Exo III) and inhibits the CRISPR/Cas12a system. In the absence of NF-κB p50, Exo III digestion triggers entropy-driven amplification, which activates CRISPR/Cas12a, leading to enhanced electrochemical signals. The ECL biosensor demonstrated a detection limit of 0.56 pM, high selectivity, and excellent reproducibility. Furthermore, the biosensor successfully detected NF-κB p50 in complex biological samples, such as HeLa cell lysates, showcasing its potential for clinical applications in disease diagnostics.