Ultrasensitive detection of NF-κB p50 using DNA tile–mediated electrochemical biosensing and Exo III amplification

Nuclear factor kappa B (NF-κB) p50 is a critical transcription factor that regulates immune responses, inflammation, and cell proliferation. Precise detection of NF-κB p50 at ultralow concentrations is of paramount importance for early disease diagnosis and mechanistic studies. Here, we report an electrochemical biosensor integrating double-crossover DNA tiles and entropy-driven strand displacement, amplified by Exonuclease III (Exo III) digestion. By designing a self-assembled DNA tile scaffold on a gold electrode and employing a DNA tetrahedron architecture, the sensor achieves a highly ordered surface configuration, minimizing background noise and enhancing specificity. In the absence of NF-κB p50, Exo III selectively digests a double-stranded DNA substrate, releasing single-stranded triggers that undergo an entropy-driven reaction to form a Y-shaped scaffold on the electrode. This assembly effectively hinders redox probe access, lowering the differential pulse voltammetry (DPV) signal. Conversely, NF-κB p50 binding protects the DNA substrate from digestion, preserving a higher current output. Under optimized conditions—30 nM DNA tiles, 2 U μL −1 Exo III, 30 min cleavage, and 100 nM DNA3/DNA4/DNA5—the biosensor exhibits an ultralow detection limit of 2.65 aM and excellent specificity, as confirmed by negligible cross-reactivity with other proteins. Furthermore, the method shows robust anti-interference performance in complex matrices (e.g., diluted cell lysates). These findings underscore the potential of this DNA tile-based biosensor for clinical diagnostics and fundamental research on NF-κB p50–mediated pathways.