Protease activity profiling in nanoliter-volume sample using the capillary microarrays

Keywords:
protease, fluorogenic substrate, microarray, capillary

Abstract:
Proteases play a key role in different (patho)physiological processes being at the same time important biomarkers and potential drug targets. As a result, specific, and sensitive assays to monitor the activity of proteases are very demanded. Here we describe the development of a capillary array for highly sensitive and fast fluorescent detection of proteolytic activity in biological samples. The fluorogenic polymers containing Rh110 substrates with specific protease cleavage sites were designed. These polymers were photochemically patterned onto the inner wall of fused silica capillary, each band corresponded to the specific protease substrate. The capillaries had an outer diameter of 363 mm and an internal diameter of 100 mm providing a filling capacity of about 76 nl per cm. For the assay, the samples containing various proteases were loaded into the capillary and the appearance of fluorescent bands was followed using laser scanner. We demonstrated that the capillary arrays could be used in different bioassays including detection of the specific protease, profiling of the complex proteolytic activity, and analysis of protease inhibitors. Having the sensitivity comparable to the fluorophore-based assays in a planar format, the capillary array possesses several advantages including nanoliter range sample a short assay time.

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Nanotech 2006 Conference Program Abstract