Determination of Binding Constants on Microarrays with Confocal Fluorescence Detection

Confocal laser scanning microscopy was employed for the determination of binding constants of receptor-ligand interactions in a microarray format. Protocols for a localized immobilization of amine containing substances on glass via GOPTS (3glycidyloxypropyl)trimethoxysilane) were optimized with respect to the detection of ligand binding by fluorescence. Compatibility with miniaturization by nanopipetting devices was ensured during all steps. The interaction of the tripeptide L-Lys-D-Ala-D-Ala with vancomycin immobilized on glass served as a model. To minimize consumption of ligand, binding constants were determined by stepwise titration of binding sites. The binding constant of the unlabeled ligand was determined by competitive titration with a fluorescently labeled analogue. The determined binding constants agreed well with those determined by other techniques, previously. Labeled ligand bound stronger than the unlabeled one. This difference was dye-dependent. Still, binding was specific for the tripeptide moiety confirming that ligand and fluorescent analogue competed for the same binding sites these results validate the determination of binding constants by competitive titration. The protocols established for confocal fluorescence detection are applicable to axially resolved detection modalities and screening for unlabeled ligands by competitive titration in general.