Single-target molecule detection with nonbleaching multicolor optical immunolabels

We introduce and demonstrate the use of colloidal silver plasmon-resonant p articles (PRPs) as optical reporters in typical biological assays. PRPs are ultrabright, nanosized optical scatterers, which scatter light elastically and can be prepared with a scattering peak at any color in the visible spe ctrum. PRPs are readily observed individually with a microscope configured for dark-field microscopy, with white-light illumination of typical power. Here we illustrate the use of PRPs, surface coated with standard ligands, a s target-specific labels in an in situ hybridization and an immunocytology assay. We propose that PRPs can replace or complement established labels, s uch as those based on radioactivity, fluorescence, chemiluminescence, or en zymatic/colorimetric detection that are used routinely in biochemistry, cel l biology, and medical diagnostic applications. Moreover, because PRP label s are non-bleaching and bright enough to be rapidly identified and counted, an ultrasensitive assay format based on single-target molecule detection i s now practical. We also present the results of a model sandwich immunoassa y for goat anti-biotin antibody, in which the number of PRP labels counted in an image constitutes the measured signal.