Single-target molecule detection with nonbleaching multicolor optical immunolabels

TLDR

PRPs are ultrabright, nanosized optical scatterers that can be tuned to any visible color and scatter light elastically. The study introduces colloidal silver PRPs as optical reporters and proposes they can replace or complement conventional labels in biological assays. The authors demonstrate that surface‑coated PRPs serve as target‑specific labels in in‑situ hybridization and immunocytology assays. PRPs can be visualized individually under dark‑field microscopy, are nonbleaching and bright enough for rapid counting, enabling ultrasensitive single‑molecule detection as demonstrated in a model sandwich immunoassay for goat anti‑biotin antibody.

Abstract

We introduce and demonstrate the use of colloidal silver plasmon-resonant particles (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 spectrum. 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, as target-specific labels in an in situ hybridization and an immunocytology assay. We propose that PRPs can replace or complement established labels, such as those based on radioactivity, fluorescence, chemiluminescence, or enzymatic/colorimetric detection that are used routinely in biochemistry, cell biology, and medical diagnostic applications. Moreover, because PRP labels are nonbleaching and bright enough to be rapidly identified and counted, an ultrasensitive assay format based on single-target molecule detection is now practical. We also present the results of a model sandwich immunoassay for goat anti-biotin antibody, in which the number of PRP labels counted in an image constitutes the measured signal.