Single-target molecule detection with nonbleaching multicolor optical immunolabels

Abstract

We introduce and demonstrate the use of colloidal silver plasmonresonant 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 enzymatic͞colorimetric 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. R obust optical reporters for diagnostic detection and͞or labeling are used extensively in areas of biomedical and clinical chemistry research, for instance in immunology, microbiology, molecular biology, pharmacology, pathology, virology, or drug testing. Current methods of detection use colorimetric, fluorometric, or chemiluminescent (1) reporter molecules either as enzyme substrates or as direct labels. The measured optical signal in such assays typically results from the accumulated sum of all reporter labels present in the target region, including contributions from both specific and nonspecific binding events. Alternative optical assay formats based on detecting and counting individual binding events are possible, but have not yet been demonstrated to be feasible. Although single fluorescent molecules, upconverting phosphors (2), and the recently introduced quantum dots (3, 4) can be individually detected, such systems have very low light yield and often exhibit time-dependent blinking and irreversible photodestruction. Thus, to indicate reliably the presence of a target, a population of such labels is still required, potentially limiting both the minimum quantity of target detected and the spatial localization of the labeled region.We introduce here a new assay platform (both probe and instrumentation) capable of individual target molecule detection that uses plasmon-resonant particles (PRPs) as optical reporters. PRPs are metallic nanoparticles, typically 40-100 nm in diameter, which scatter light elastically with remarkable efficiency because of a collective resonance of the conduction electrons in the metal (i.e., the surface plasmon resonance; ref. 5). The magnitude, peak wavelength, and spectral bandwidth of the plasmon resonance associated with a nanoparticle are dependent on the ...

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