A compartment-free digital plasmonic coupling assay via single-particle imaging and counting

Abstract

Plasmonic coupling-based biosensors have been conventionally quantified by bulk spectroscopic or color measurement methods. Such detection schemes limit assay sensitivity due to the high background noise of bulk measurement. In this work, we developed a digital plasmonic assay platform for sensitive biomarker detection by enumerating individual plasmon-shifted nanoclusters via single-nanoparticle darkfield scattering imaging. The assay is based on a pair of gold (Au) and silver (Ag) nanoparticles (NPs) that are functionalized with high-affinity surface ligands such as antibodies for target recognition. Molecular binding induces the formation of nanoparticle clusters with plasmonic features distinct from those of individual Au or Ag NPs, which can be counted digitally without the need for complex chip or droplet compartments. We systematically investigated nanoparticle–protein interactions during bioconjugation and revealed different effects of absorbed proteins on nanoparticle stability. Under optimized conditions, we achieved a limit of detection (LOD) of 107.5 fM for the detection of biotinylated gold nanoparticles with this assay. FDTD simulation was also performed to investigate different plasmonic coupling effects of various Au–Ag, Ag–Ag, and Au–Au combinations. Finally, a particle counting algorithm was developed to quantify individual nanoparticle clusters from darkfield scattering images with high accuracy. With a rational assay design and simple synthesis procedure, this simple digital assay platform could be engineered for high-sensitivity protein target detection for disease diagnostics.