Single-particle identification of encoded nanospheres.

Recently, it has been shown that 2-photon fluorescence correlation spectroscopy of single glycosylated 20-nm fluorescent spheres allows measurement of the relative carbohydrate binding affinities of unlabeled proteins and that these modified spheres can mimic the glycocalix of cell or virus surfaces. An especially useful extension would be the analysis of mixtures of nanospheres that each contain different fluorescent labels and are thus differentially "encoded." If the surfaces of these encoded nanospheres are modified with various receptors, many different biomolecule-surface interactions and concurrent reactions can be measured quickly and simultaneously in a single-reaction vessel. An essential prerequisite for this general assay principle is the ability to identify with an accuracy of nearly 100% any encoded nanosphere present in a mixture on a single-particle level. Here the authors present a method that indeed allows certain identification of differently encoded nanospheres during single transits through the focal volume of a microscope objective (ø approximately 200-500 nm) in aqueous solution. This opens the way for using the encoded nanospheres in 1-well measurements of a large variety of biomolecular receptor-ligand interactions, inhibition and concurrent reactions, and thus either for testing the behavior of ligands in a mimicked complex biomolecular environment or for a fast simultaneous measurement of a multitude of receptor-ligand interactions.