Immobilized Fluorescent Probes for Simultaneous Multiple Protease Detection

A new concept for simultaneous detection of two model proteases based on immobilized fluorescently labelled peptides was developed and evaluated. Each probe was composed of a carrier modified by poly(ethylene glycol) (PEG) chains, a specifically cleavable linker, and a fluorescent dye incorporated in the peptide tail. Based on a single excitation–double emission fluorescence response of liberated fluorophores caused by enzymatic cleavage, the presence of a single or both proteases in a mixture was unambiguously determined in an experimentally established concentration range. Among the tested solid supports, Rink Amide PEGA resin was recognized as the most suitable choice from the perspective of on-resin enzyme assays.