Illuminating NF-KB p50 detection: a cascaded electrochemiluminescent biosensor powered by T7 RNA polymerase transcription and DSN-mediated cleavage on an MXene interface.

Early and sensitive detection of transcription factors such as NF-κB p50 is essential for accurate disease diagnosis and monitoring. We present a highly sensitive electrochemiluminescence (ECL) biosensor for the detection of NF-κB p50, integrating a multifunctional nanocomposite and a three-enzyme signal amplification cascade. The sensing platform is constructed by modifying a glassy carbon electrode with a nanocomposite composed of gold nanoparticles (AuNPs), Ti₃C₂Tₓ MXene, and a Ru(II)-polyethylenimine (PEI) complex, which enables strong and stable ECL emission and efficient probe immobilization. The detection mechanism combines Exonuclease III (Exo III)-mediated target recycling, T7 RNA polymerase-driven transcription, and duplex-specific nuclease (DSN)-facilitated signal amplification. Upon binding of NF-κB p50 to the DNA probe, the T7 promoter is exposed, initiating transcription of abundant RNA strands. These RNAs subsequently act as templates for DSN to cleave Fc-labeled DNA on the electrode surface, releasing quenching tags and restoring the ECL signal. The proposed biosensor exhibits excellent analytical performance, including a wide dynamic range (1 aM-10, pM), a low detection Limit of 0.273 aM, and high specificity against interfering proteins. It also demonstrates strong anti-interference capability and satisfactory recovery in diluted cell lysate samples. This work provides a versatile and scalable biosensing strategy with great potential for clinical diagnostics, real-time disease monitoring, and broader biomarker detection in complex biological matrices.