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-gamma-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. (C) 2011 Elsevier B.V. All rights reserved.