Plasmonic Single‐Molecule Affinity Detection at 10−20 Molar

Abstract DNA can be readily amplified through replication, enabling the detection of a single‐target copy. A comparable performance for proteins in immunoassays has yet to be fully assessed. Surface‐plasmon‐resonance (SPR) serves as a probe capable of performing assays at concentrations typically around 10⁻⁹ molar. In this study, plasmonic single‐molecule assays for both proteins and DNA are demonstrated, achieving limits‐of‐detections (LODs) as low as 10⁻ 2 ⁰ molar (1 ± 1 molecule in 0.1 mL), even in human serum, in 1 h. This represents an improvement in typical SPR LODs by eleven orders‐of‐magnitude. The single‐molecule SPR assay is achieved with a millimeter‐wide surface functionalized with a physisorbed biolayer comprising trillions of recognition‐elements (antibodies or protein–probe complexes) which undergo an acidic or alkaline pH‐conditioning. Potentiometric and surface‐probing imaging experiments reveal the phenomenon underlying this extraordinary performance enhancement. The data suggest an unexplored amplification process within the biomaterial, where pH‐conditioning, driving the biolayer in a metastable state, induces a self‐propagating aggregation of partially misfolded proteins, following single‐affinity binding. This process triggers an electrostatic rearrangement, resulting in the displacement of a charge equivalent to 1.5e per 10 2 recognition elements. Such findings open new opportunities for reliable SPR‐based biosensing at the physical detection limits, with promising applications in point‐of‐care plasmonic systems.