Sensitive Assay of Protease Activity on a Micro/Nanofluidics Preconcentrator Fused with the Fluorescence Resonance Energy Transfer Detection Technique

Abstract

A fast and sensitive assay of protease activity on a micro/nanofluidics preconcentrator combining with fluorescence resonance energy transfer (FRET) detection technique has been developed in a homogeneous real-time format. First, the functionalized nanoprobes are formed by loading dye labeled protein onto gold nanoparticles (AuNPs), in which, the photoluminescence of donor dye was strongly quenched by AuNPs due to FRET mechanisms. For protease activity assay, the nanoprobes are enriched by a micro/nanofluidics preconcentrator. When the target protease is transported to the enriched nanoprobes, cleavage of protein occurs as a consequence of molecular recognition of enzyme to substrate. The release of cleavage fragments from AuNPs nanoprobes leads to the enhancement of fluorescence and enables the protease activity assay on the micro/nanofluidics chip. As a demonstration, digestion of fluorescein isothiocyanate labeled dog serum albumin (FITC-DSA) by trypsin was used as a model reaction. Because of the highly efficient preconcentration and space confinement effect, significantly increased protein cleavage rate and protease assay sensitivity can be achieved with enhanced enzyme activity. The present micro/nanofluidics platform fused with the FRET detection technique is promising for fast and sensitive bioanalysis such as immunoassay, DNA hybridization, drug discovery, and clinical diagnosis.