A reusable electrochemical biosensor for NF-κB p50: Precise detection of a critical biological macromolecule via CRISPR/Cas12a and pH-responsive signal regeneration

NF-κB p50 is a critical transcription factor that plays an essential role in regulating immune responses, inflammatory pathways, and tumor progression. Its aberrant expression is closely linked to various pathological conditions, making it a valuable biomarker for early disease diagnosis and therapeutic monitoring. In this work, we developed a highly sensitive and reusable electrochemical biosensor capable of detecting NF-κB p50 at ultralow concentrations. The biosensor integrates a pH-responsive regeneration mechanism and a multi-stage signal amplification cascade involving Exonuclease III (Exo III)-assisted recycling, CRISPR/Cas12a-mediated trans -cleavage, and DNA tetrahedron nanostructure-functionalized electrodes. This synergistic design significantly enhances detection sensitivity and stability. The platform offers a broad dynamic detection range from 5 aM to 100 fM with a remarkably low detection limit of 2.38 aM. More importantly, the biosensor demonstrates excellent reusability through efficient regeneration under alkaline conditions (pH 10.0), with minimal loss in signal output. Validation in tenfold-diluted human serum samples yielded high recovery rates (99.61 %–106.23 %) and low relative standard deviations, confirming its practical applicability. This study not only presents a robust approach for ultrasensitive protein detection but also introduces a generalizable strategy for the regenerative electrochemical analysis of protein-based biological macromolecules in clinical diagnostics.