Single-target molecule detection with nonbleaching multicolor optical immunolabels.
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.
Schultz
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Department of Physics 0319, University of California, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Smith
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Mock
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Schultz
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D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000906:Antibodies / Q000032:analysis; D001710:Biotin / Q000276:immunology; D002645:Chickens; D003102:Colloids; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development; D005136:Eye Proteins / Q000235:genetics; D006041:Goats; D017979:Gold Colloid; D007118:Immunoassay / Q000379:methods*; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D007202:Indicators and Reagents; D019476:Insect Proteins / Q000235:genetics; D007814:Larva; D008027:Light; D008853:Microscopy / Q000379:methods; D008863:Microspheres; D009124:Muscle Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis; D012542:Scattering, Radiation; D012834:Silver; D013053:Spectrophotometry; D020349:Surface Plasmon Resonance / Q000379:methods*; D014960:X Chromosome / Q000648:ultrastructure
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D003102:Colloids; D029721:Drosophila Proteins; D005136:Eye Proteins; D017979:Gold Colloid; D007202:Indicators and Reagents; D019476:Insect Proteins; D009124:Muscle Proteins; D011506:Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C059254:w protein, Drosophila; D012834:Silver; D001710:Biotin