Chip-Based Protease Assay Using Fluorescence Resonance Energy Transfer Between Quantum Dots and Fluorophores

Abstract

The analysis of proteolytic activity is essential for understanding the biological functions of living systems as well as for developing therapeutics and diagnosis. We eveloped a chip-based assay system for proteases that measures the fluorescence resonance energy transfer (FRET) between quantum dots (QD) and fluorophore-labeled peptides. In this system, while the photoluminescence (PL) of donor QD immobilized on a surface was quenched due to the presence of an energy acceptor (fluorophore) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the highly sensitive assay. Unlike solution-based analyses, our chip-based format enables a more reliable analysis with no aggregation of QD, and it is able to function using a much smaller reaction volume. The system presented here will be useful for the detection of various proteases with high selectivity and nsitivity in a high-throughput manner.