Development of microbiochip for detection of metalloproteinase 7 using fluorescence resonance energy transfer

Abstract

A protease is any enzyme that catalyzes the hydrolysis of proteins into smaller peptide fragments and amino acids, a process known as proteolysis. They are involved in a multitude of normal biological processes as well as in diseases, including cancer, stroke and infections. Here we present a microfluidic-based assay system to detect proteolytic activity using fluorescence resonance energy transfer (FRET) by quantum dot (QD)-peptide conjugates immobilized on microbeads. As an energy donor, QD was immobilized on the microbead surface by the avidin-biotin interaction. As an energy acceptor, the fluorophore-labeled peptide was then associated with QD, thus quenching the photoluminescence (PL) of the QD. The functionalized microbeads were introduced into the microbiochip and captured by a micropillar in the reaction chamber. In the presence of matrix metalloprotease-7 (MMP-7) as a model protease, the PL of QD quenched by fluorophore was recovered due to the proteolytic activity of MMP-7 in the fabricated microbiochip. Moreover, the FRET efficiency induced by MMP-7 was linearly dependent on the logarithmic concentration of MMP-7. This technology is not limited to sensing MMP-7, but could be used to monitor other protease activities (Schematic diagram).