Abstract
Despite the promising improvements made recently in fluorescence probes for enzymes and reactive small molecules detection, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag and avidin protein. The probes were constructed by following the current probe design for enzymes and reactive small molecules, but with an additional benzylguanine moiety for selective SNAP-tag conjugation or a short biotin linker for the conjugation to avidin protein. The SNAP-tag conjugated probes or avidin conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma and serum, whereas analyte concentrations were overestimated when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically as our probes displayed fluorescence enhancement upon SNAP-tag conjugation. In contrast to conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcome many key issues such as dye leakage, long preparation steps and limited application to detect only small metabolites.