Fluorescent silica nanospheres for digital counting bioassay of the breast cancer marker HER2/nue

This paper describes the use of fluorescent silica nanospheres as luminescent signal amplifiers in biological assays based on digital counting of individual particles instead of measuring averaged fluorescence intensity. We recently described a simple method to prepare highly fluorescent mono-dispersed silica nanospheres that avoids microemulsion formulations and the use of surfactants. Modification of the Stöber method was used successfully to prepare fluorescent silica spheres with the inorganic dye dichlorotris(1,10-phenanathroline)ruthenium (II) hydrate encapsulated during the condensation of tetraethylorthosilicate in ethanol and dye aqueous mixtures. Modifications in the ammonia and water content in the reaction mixture resulted in mono-dispersed silica spheres of 65, 440 and 800 nm in diameter. The dye-encapsulating particles emit intense red luminescence when excited at 460 nm. We observed an increased photostability and longer fluorescence lifetime in our particles that we attributed to increased protection of the encapsulated dye molecules from molecular oxygen. The newly prepared fluorescent silica particles were easily modified using trialkoxysilane reagents for covalent conjugation of anti-HER2/neu. We demonstrated the utility of the fluorescent nanospheres to detect the cancer marker HER2/neu in a glass slide based assay. The assay was shown to be simple but highly sensitive with a limit of detection approaching 1 ng/mL and a linear range between 1 ng/mL and 10 μg/mL of HER2/neu.