A two-step hybridization-capture aptasensor with dithiol-DNA-AuNPs interface for rapid, label-free detection...

The global control of tuberculosis (TB) urgently demands diagnostic tools that are rapid, specific, and amenable to resource-limited settings. Here, we report a novel, label-free aptasensor for Mycobacterium tuberculosis (MTB) H37Rv, which operates on an innovative two-step "pre-incubation & hybridization-capture" strategy, fundamentally departing from conventional competitive displacement designs. First, a high-affinity, in-house selected aptamer specifically complexes with target bacteria in solution. This pre-formed complex is then efficiently captured on a gold interdigitated electrode via hybridization with a short, dithiol-anchored DNA probe, which is pre-assembled with conductive gold nanoparticles (AuNPs) to form a robust sensing interface. This architecture decouples target recognition from signal transduction, enhancing assay robustness. The captured bulky and negatively charged bacterial complex synergistically creates pronounced steric and electrostatic barriers at the interface, drastically impeding charge transfer and generating a measurable frequency shift in a multichannel piezoelectric quartz crystal (MSPQC) system. The sensor demonstrates exceptional specificity, clearly distinguishing H37Rv (ΔF = 116 Hz) from non-target bacteria, including Bacillus Calmette-Guérin (BCG) (ΔF < 30 Hz). It exhibits a wide linear response from 10³ to 10⁶ CFU/mL (R² = 0.9666) and a low experimental detection limit of 100 CFU/mL within a total assay time of 65 min. With excellent reproducibility (RSD 2.1-4.6%) and stability, this work establishes a new paradigm for rapid, label-free and equipment-simplified whole-cell detection, holding significant promise for point-of-need TB diagnosis.