Scientists at the University of Florida in Gainesville have demonstrated that single-wavelength excitation can be used to produce multiple emission signatures from silica nanoparticles labeled with dye. The approach may be suitable for in vivo multiplexed biological assays.As more insight is sought into the cellular and molecular signatures of disease, there is increasing interest in methods with which to perform highly multiplexed assays. Rather than testing for levels of a single compound or for cells with a specific membrane protein, bioassays may measure multiple analytes, perhaps from multiple sources. Doing so requires a means of easily and uniquely identifying each analyte.Silica nanoparticles impregnated with a Förster resonance energy transfer dye series are candidates for the spectral labeling of bioassays. By using different ratios of the dyes within the series, a single wavelength can excite a unique fluorescence spectrum from each type of nanoparticle. The image shows the discrimination possible with microbeads labeled with nanoparticles of five colors. Courtesy of Weihong Tan. ©2006, American Chemical Society.In the ideal method, many fluorescent markers would be excited with a single wavelength, and each marker would respond by emitting a unique spectrum. Quantum dots immediately leap to mind as such markers. However, they have a large hurdle to overcome for in vivo applications: The materials from which they are constructed are toxic at some level, and it is not clear how safely the body can process and excrete them.The new approach incorporates a three-dye Förster resonance energy transfer (FRET) complex of fluorescein isothiocyanate (FITC), rhodamine 6G (R6G) and ROX in 70 nm-diameter, biocompatible silica nanoparticles. If the dye molecules are near enough to one another and have the proper emission and excitation spectra, the energy emitted by one molecule will be absorbed by another.The FITC emission overlaps the R6G excitation, and the R6G emission overlaps the ROX excitation. So a particle featuring a mixture of the three dyes evenly distributed throughout it will absorb 488-nm light, exciting the FITC, and that energy will be transferred first to R6G and then to the ROX, creating a unique blend of emission colors.By regulating the levels of each dye molecule, an array of unique markers can be constructed. Although FITC, R6G and ROX were used in the initial particles, the researchers noted that the approach can be extended to more than three chromophores and that it can be applied to any energy-transfer dye series. The nanoparticles contain a high enough concentration of dye molecules so that the fluorescence is 2000 times higher than that of one fluorophore, but the concentration is not so high that self-quenching bcomes a problem. Because the particle size may be adjusted by changing the process parameters and is independent of the chromophores used, different labels can be the same size, eliminating the issues of size dependence associated with quantum dots. The silica nanoparticles do not show the blinking behavior seen with quantum dots, and the investigators contend that the preparation and surface modification is easier than with quantum dots.To demonstrate the applicability of the nanoparticles as biomarkers, the researchers biotinylated the silica structures and mixed them with streptavidin-coated microspheres at a ratio of 50,000:1. Confocal microscopy with 488-nm laser excitation clearly showed the simultaneous acquisition of microspheres with five unique colors. Nano Letters, January 2006, pp. 84-88.