Quantifying the Capture of Dye-Loaded Silica Nanoparticles on Magnetic Beads

The current standard method for detecting proteins and antibodies is an enzyme-linked immunosorbent assay, or ELISA. Even though this method proves to be successful, the procedure is very complex and time consuming. An alternative method that is demonstrated in this study is to use dye-loaded silica nanoparticles coupled with a sandwiching immunoassay approach to detect proteins. We hypothesize that high surface area, dye-loaded silica nanoparticles will provide a rapid and robust detection of proteins and antibodies with high signal to noise ratio. The binding of biotinylated fluorescently labeled silica nanoparticles to streptavidin (protein)-functionalized magnetic beads served as a model system to investigate the variables relevant to signal detection from dye-loaded nanoparticles, where the strong binding of biotin-streptavidin binding is commonly used to link biomolecules to surfaces. Biotin was covalently attached to the surface of amine-functionalized nanoparticles. Two types of dye-loaded biotinylated mesoporous silica were investigated (fluorescein isothiocyanate (FITC) dye bound through covalent attachment to surface amines or sulforhodamine (SR)-101 dye bound through surface charge to surface amines). To amplify the signal of these fluorescently labeled nanoparticles after capture on the magnetic beads, a high pH buffer was introduced to the solution. The fluorescence intensity of the dye released from the bound particles was measured via spectroscopy as a function of time and particle concentration. The biotinylated nanoparticles produced more fluorescence than the non- biotinylated (amine-functionalized only) nanoparticles, when captured by the streptavidin coated magnetic beads, meaning that biotin-streptavidin binding was taking place on the functionalized surfaces. Detection using dye-loaded nanoparticles was further improved by adding bovine serum albumin (to reduce nonspecific binding) or alginate (to reduce the surface charge of the amines and trap the dye). The SR-101 dye-loaded particles released the fluorescent dye much faster than the FITC labeled particles after the high pH buffer solution was added. Overall, the simple real-time detection assay using fluorescently labeled silica nanoparticles was successful.