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Fluorescence spectroscopy has become a widely accepted method in many applications in chemistry, biology, biochemistry and biophysics just to mention a few areas because of its practicality and sensitivity. Major trend for these applications during the recent years is the use of longer wavelength fluorophores and encapsulation of fluorophores by utilizing nanoparticles. Longer fluorescence wavelengths have several advantages such as lower background interference that is especially important in complex biological samples. While many different types of nanoparticles can be utilized for encapsulation of these dyes, silica nanoparticles have significant advantages in biological system. The synthesis of silica nanoparticles is relatively straightforward and covalent copolymerization of fluorescent dyes during the silica nanoparticle synthesis is a easily controllable process by using modified TEOS reactive analogues that are widely available. Adding layers on silica nanoparticles different chemical or sensor functionality can be added to the silica nanoparticle surface. The fluorescence intensity of a fluorescent silica nanoparticle can significantly be increased by enclosing larger number of dye molecules in silica nanoparticles. Dependent on the size of the silica nanoparticle 20-50 or more dye molecules can be included using this copolymerization process. For high dye concentrations in the silica nanoparticle self quenching can be significant but it can be minimized by synthesizing large Stokes’ shift dyes. Using NIR dyes excellent sensitivity can be achieved but one major disadvantage is that most biological fluorescence instruments are designed for shorter excitation wavelengths frequently matching the optical properties of fluorescein, a widely used fluorophore. One approach to make these new fluorescent silica nanoparticles more compatible with widely used optical wavelength windows is the development of fluorescent silica nanoparticles containing copolymerized dyes that are good candidates for fluorescence energy transfer. For example if the donor is fluorescein the excitation wavelength is compatible with most instruments on the market. Systems that contain two or more dyes can be used to achieve long fluorescence wavelengths. This presentation discusses the facile synthesis and practical applications of silica nanoparticles containing copolymerized multiple fluorophores that are suitable for fluorescence energy transfer. Optimization of these systems requires the evaluation of individual dye concentrations (donors, acceptors and intermediators), concentration ratios to achieve high fluorescence. During these studies copolymerized energy transfer fluorescence silica nanoparticles were evaluated for their stability, fluorescence intensity and utility. The surface properties of fluorescence silica nanoparticles were modified by adding hydrophobic or hydrophilic molecules on the surface to achieve biocompatibility. Biocompatibility was evaluated by hemolytic experiments. Typical applications of these particles are for immunochemistry, flow cytometry, CE, forensic applications, biomolecule characterizations, etc.
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