Surface immobilization of peptides on SiO/sub 2/ nanobeads for a protease biosensor

We have been investigating the development of a biosensor that detects medically relevant enzymes. Silica nanobeads were utilized as an optical platform for the development of the protease biosensor. The biosensor uses the chemical transduction method, fluorescence resonance energy transfer, FRET. FRET requires the utilization of special molecular groups termed donor and acceptor, whose combined purpose is to elicit a change in fluorescence in the presence of a given analyte. Donor (AMCA) and acceptor (FITC) fluorophores were covalently attached at either ends of peptide constructs that were synthesized using Fmoc (9-fluorenylmethoxycarbonyl) strategy, a method for solid-phase synthesis of peptides. Two peptide sequences were constructed: CF6 (a positive substrate) and 051-4 (a negative substrate), and were investigated as a prototype trypsin substrate. The peptides were immobilized onto silica nanobeads (15 nm in diameter) using silanization and simple adsorption and then exposed to various concentrations of trypsin. Only the peptides that were immobilized via silanization gave a significant response. When these peptides were cleaved by trypsin, the donor and acceptor fluorophores were separated, resulting in an increase in donor fluorescence. The response time of the sensor was less than 5 minutes and the limit of detection was 13 ng/ml of trypsin in solution