Metal-Enhanced Fluorescence by Bifunctional Au Nanoparticles for Highly Sensitive and Simple Detection of Proteolytic Enzyme.

Although fluorescence-based analytical methods have been used in intracellular analyses, their sensitivity is low for the precise analysis of intracellular proteolytic enzymes to observe cell apoptosis related to cancer and neurodegenerative diseases. In this study, a metal-enhanced-fluorescence (MEF)-based highly sensitive biosensor for the detection of proteolytic enzymes is proposed for the first time by using a bifunctional Au nanoparticle (AuNP), which is connected to the fluorophore by both single-stranded DNA (ssDNA) and a peptide. Once caspase-3, a proteolytic enzyme, cuts the peptide specifically, the fluorescence signal is drastically increased because the ssDNA maintains an optimal distance for the MEF. The proposed sensing method shows the highly sensitive detection of caspase-3 based on just a simple enzymatic cleavage reaction within 1 h, and caspase-3-related preapoptotic cell detection was successfully carried out with high sensitivity. The proposed sensing method is a rapid, simple, and one-step technique for the real-time monitoring of intracellular proteolytic enzymes and can be applied to the early diagnosis of cancer and neurodegenerative diseases.