Abstract: The present provides optical sensor based sensitive, label-free binding assay methods and kits for isothermal real-time detection of the binding of specific analytes (such as nucleic acids, proteins and low molecular weight antigenic or receptor binding ligands) present in low amount in different biological samples. In the binding assays of the invention, the analyte is captured at the specifically pretreated solid surface of optical biosensors in specific recognition reactions (such as hybridization, specific protein-protein interactions and receptor-ligand interactions). The specificity of the methods of the invention is further enhanced by a second specific recognition step using a padlock probe comprising an indicator sequence designed to keep the products of a subsequently performed isothermal nucleic acid amplification method (e.g.