Protease biosensing on novel high surface area organosilicate nanoporous films

We report on the development of a protease biosensing platform using novel high surface area nanoporous organosilicate (NPO) thin film supports. NPO films, with their three dimensional interconnected pores are constituted of organosilicate nanoparticles and are characterized by their large and accessible surface areas, ideal for high density binding of biological probes. Here, we demonstrate enhanced protease sensing of a serine protease, trypsin using a Forster resonance energy transfer (FRET) assay performed on the NPO films. Dual fluorescent dye labeled peptide sequences specific to proteolytic activity of trypsin were used as the sensor probes and immobilized with high density on the NPO surfaces. Enzymatic activity of trypsin was monitored by measuring the donor to acceptor fluorescence ratio changes as a function of both reaction time (time response) and trypsin concentration (dose response). Performance of the same assay on planar glass substrates yielded inconclusive results owing to the inherently low signal/noise ratio from these substrates and potential unfavorable orientation of peptides toward trypsin. Using NPO films, a detection limit of 2.45 μg/ml was achieved in 14 min. The use of solid supports for the performance of protease assays is expected to significantly miniaturize the assays and aid in multiplexing akin to antibody based microarrays.