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

Abstract

Biosensors for organophosphates in solution may be constructed by monitoring 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 to construct microsensors with increased surface area for enzyme immobilization, we used a sol–gel process to create highly porous and stable silica matrices. 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 analysis, porous and non-porous silica beads were used as model substrates of sol–gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-γ-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded 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 activity. Enzyme was also immobilized to sol–gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.