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The use of the NIR spectral region (650-900 nm) for bioanalytical and biomedical analyses is advantageous due to the inherently lower background interference in biological matrices and the high molar absorptivities of NIR chromophores. There are several different groups of NIR fluorescing dye are available for bioanalytical applications. One of these groups, NIR carbocyanines are increasingly used in analytical, bioanalytical and medical applications. These dyes can be used as reporter labels for sensitive bioanalytical use, such as immunochemistry. Due to the spectroscopic sensitivity of NIR carbocyanines for polarity changes in the microenvironment fluorescence quantum yield can vary significantly dependent on the microenvironment. NIR dyes can have relatively low fluorescent quantum yields as compared to visible fluorophores, especially in aqueous buffers but the lower quantum yield is compensated for by a much higher molar absorptivity. The fluorescence intensity of NIR reporting labels can significantly be increased by enclosing several dye molecules in silica nanoparticles. Incorporation of NIR dyes in silica nanoparticles creates a unique challenge as these dyes can be unstable under certain chemical conditions present during silica nanoparticles syntheses. In addition, self quenching may also become a problem for carbocyanines at higher a concentrations that typically found inside of NIR dye loaded silica nanoparticles. Dyes possessing high Stokes’ shift can significantly reduce this problem. NIR carbocyanines are uniquely positioned for achieving this goal using a synthetic route that substitutes meso position halogens in NIR fluorescent carbocyanines with a linker containing amino moiety, which can also serve as a linker for covalently attaching the dye molecule to the nanoparticle backbone. The resulting silica nanoparticles can contain a large number of NIR dyes dependent on their size. For example some NIR fluorescent silica nanoparticle labels prepared that has an average radius around 15 nm, contains 16-20 covalently attached dye molecules inside of the nanoparticle. The primary applications of these particles are for bright fluorescent labels that can be used in applications such as immunochemistry, flow cytometry, and many other applications.
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