Dual Steric-Hindrance-Modulated Switchable DNAzyme for Sensitive Antibody Detection, Logic Circuits, and Dynamic Monitoring

Antibodies play a pivotal role in clinical diagnostics and biomedical research, yet conventional detection platforms are often constrained by limited signal tunability and an inability to perform complex signal processing. Herein, we report a fluorescence-based sensing platform that leverages switchable DNAzymes based on a dual steric-hindrance effect on the gold nanoparticle surface for multifunctional antibody detection. This research suggests that the steric-hindrance effect can be flexibly tuned by regulating the valency of antigen labeling on the DNAzyme and the length of the flexible spacer on the substrate probe. The optimized system exhibits excellent analytical performance for the detection of anti-biotin (Anti-Biotin) and anti-digoxigenin antibody (Anti-Dig). Beyond single-antibody detection, advanced molecular logic circuits were engineered. NOR and NOT logic circuits were constructed in the heterobivalent antigen-modified DNAzyme system, with Anti-Biotin and Anti-Dig serving as input. Additionally, a streamlined fluorescence workflow was established for sequential monitoring of antibody binding events, leveraging distinct kinetic responses of Anti-Biotin and Anti-Dig. Collectively, this work developed a general design principle for engineering dual-steric-hindrance-modulated DNAzyme sensors, which transcends the limitations of conventional static antibody detection methods. Notably, the platform combines sensitive detection, complex logic operation, and dynamic sequential monitoring, which highlight its substantial potential for applications in clinical diagnostics, especially for diseases with distinct antibody-expression signatures.