Plasmonic Single‐Molecule Affinity Detection at 10−20 Molar

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 10⁻2⁰ 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 102 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.