Reagent-Free Molecular Pendulum Biosensor with Antibody–Aptamer Dual Recognition for Protein Analysis

The development of reagent-free biosensors that can continuously monitor proteins in complex biological fluids is still a critical unmet need. Molecular pendulum sensors have recently emerged as a promising reagent-free platform, enabling direct biomolecule detection without interference from surrounding matrices. However, conventional molecular pendulums employ only a single recognition element, which constrains their tunability and regulatory range. To overcome this limitation, a molecular pendulum design incorporating dual recognition elements was introduced here. By simultaneously engaging two nonoverlapping epitopes on human serum albumin (HSA) or Aβ 42 monomers, the dual recognition pendulum underwent a marked conformational re-orientation that amplified the intrinsic signal gain. Replacing one aptamer with a high-affinity antibody further increased target binding efficiency, boosting charge transfer efficiency and delivering substantially larger electrical read-out than the corresponding dual-aptamer architecture. Besides, cryogenic surface modification promoted rapid self-assembly of the probes, further lowering the detection limit. This design also enabled rapid regeneration and reuse of the sensor by allowing reset completion within 30 s through adjustment of applied voltage and time. The sensor demonstrated excellent specificity even in the presence of multiple interferents and exhibited high sensitivity in biological samples (serum and artificial cerebrospinal fluid).