Development of sensors for direct detection of organophosphates. Part I: immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitorin g the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (<1 mm(2)) for microsensors. In order t o construct microsensors with increased surface area for enzyme immobilizat ion, we used a sol-gel process to create highly porous and stable silica ma trices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this a nalysis, porous and non-porous silica beads were used as model substrates o f sol-gel coated and uncoated sensor surfaces. Two different covalent chemi stries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followe d by enzyme attachment using the homobifunctional linker glutaraldehyde. Th e second chemistry used sulfhydryl-silanization followed by enzyme attachme nt using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinim ide ester (GMBS). Surfaces were characterized in terms of total enzyme immo bilized, total and specific enzyme activity, and long term stability of enz yme activity. Amine derivitization followed by glutaraldehyde linking yield ed supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme ac tivity. Enzyme was also immobilized to sol-gel coated glass slides. The mas s of immobilized enzyme increased linearly with thickness of coating. Howev er, immobilized enzyme activity saturated at a porous silica thickness of a pproximately 800 nm. Published by Elsevier Science S.A.