Development of an immobilized-antigen immunofluorescence glass slide system that exploits fluorescent silica nanoparticles

The aim of the study was to develop an immobilized-antigen immunofluorescence glass slide system using fluorescent silica nanoparticles (FSNPs) for the detection of biomarkers to evaluate food functionality. A plain glass slide was first cleaned by treatment with piranha solution, after which C-reactive protein (CRP), a model antigen, was immobilized on the slide via procedures that exploited activation of the slide surface with 3-aminopropyltrimethoxysilane (APTMS) and glutaraldehyde, or 3-mercaptopropyltrimethoxysilane (MPTMS) and N-γ-maleimidobutyryloxysuccinimide ester (GMBS). Streptavidin-modified FSNPs that contained a fluorescent dye, dichlorotris(1,10-phenanthroline)ruthenium(II) hydrate, were conjugated with biotinylated monoclonal anti-rat CRP antibody to produce FSNP–antibody conjugates. The bioconjugate bound more effectively to the surface of slides that had been activated with APTMS before immobilization of the antigen than to those activated with MPTMS–GMBS. Specific binding of the coating antigen and bioconjugate to the slide surface was confirmed by fluorescence and atomic force microscopy. The fluorescence intensity due to formation of a complex between the antigen and bioconjugate increased gradually with increasing bioconjugate concentration up to 0.250 mg/mL.