This Article addresses the important issue of the characterization of surface functional groups for optical bioassay applications. We use a model system consisting of spherical dye-doped silica nanoparticles (NPs) that have been functionalized with amine groups whereby the encapsulated cyanine-based near-infrared dye fluorescence acts as a probe of the NP surface environment. This facilitates the identification of the optimum deposition parameters for the formation of a stable ordered amine monolayer and also elucidates the functionalization profile of the amine-silanization process. Specifically, we use a novel approach where the techniques of fluorescence correlation spectroscopy (FCS) and fluorescence lifetime measurement (FL) are used in conjunction with the more conventional analytical techniques of zeta potential measurement and Fourier transfer infrared spectroscopy (FTIR). The dynamics of the ordering of the amine layer in different stages of the reaction have been characterized by FTIR, FL, and FCS. The results indicate an optimum reaction time for the formation of a stable amine layer, which is optimized for further biomolecular conjugation, whereas extended reaction times lead to a disordered cross-linked layer. The results have been validated using an immunoglobulin (IgG) plate-based direct binding assay where the maximum number of IgG-conjugated aminated NPs were captured by immobilized anti-IgG antibodies for the NP sample corresponding to the optimized amine-silanization condition. Importantly, these results point to the potential of FCS and FL as useful analytical tools in diverse fields such as characterization of surface functionalization.
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