Direct monitoring of molecular recognition processes using fluorescence enhancement at colloid-coated microplates

Abstract

Direct monitoring of recognition processes at the molecular level is a valuable tool for studying reaction kinetics to assess affinity constants (e.g. drugs to receptors) and for designing rapid single step immunoassays. Methods currently used to gain information about binding processes predominantly depend on surface plasmon resonance. These systems use excitation with coherent light in attenuated total reflection geometry to obtain discrimination between surface-bound and free molecules in solution. Therefore labeling of the compounds is not necessary, but due to the complexity of the measuring setup the method is rather costly. In this contribution we present a simple method for performing kinetic single step biorecognition assays with fluorophore labeled compounds using the fluorescence enhancement properties of surface bound silver colloids. Silver colloids are bound to standard microplates via silanization of the plastic surface. Fluorophores close to this colloid coated surface show a significant gain in fluorescence compared to fluorophores farther away in the bulk solution. Therefore discrimination between surface bound and free fluorophores is possible and the binding of, for example, fluorophore labeled antibodies to antigens immobilized on the colloid surface results in increasing fluorescence intensity. Utilization of standard microplates makes this method fully compatible with conventional microplate processing and reading devices. Neither excitation with coherent laser light nor ATR geometry is required, the measurement is performed in a standard fluorescence microplate reader in front face geometry with a xenon flash lamp as excitation source. Methods for the preparation of colloid-coated microplates and fluorescence-enhanced biorecognition assays are presented. Additionally the dependence of the system performance on the structure and properties of the metal colloid coated surface is described. A two-component biorecognition model system shows a detection limit in the subnanomolar range. The ease of colloid-surface preparation and the high sensitivity makes fluorescence enhancement at colloid-coated microplates a valuable tool for studying reaction kinetics and performing rapid single-step immunoassays.