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 microfluidicbased 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 fluorophorelabeled 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).