Karl Wally

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 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 mm2) 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-gamma-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.

Development of sensors for direct detection of organophosphates. Part II : Sol-gel modified field effect transistor with immobilized organophosphate hydrolase

Abstract pH-sensitive field effect transistors (FET) were modified with organophosphate hydrolase (OPH) and used for direct detection of organophosphate compounds. OPH is the organophosphate degrading gene product isolated from Pseudomonas diminuta . OPH was selected as an alternative to acetylcholinesterase, which requires inhibition mode sensor operation, enzyme regeneration before reuse, long sample incubation times, and a constant source of acetylcholine substrate. OPH was covalently immobilized directly to the exposed silicon nitride gate insulator of the FET. Alternatively, silica microspheres of 20 or 200 nm were formed via a base catalyzed sol–gel process and were dip-coated onto the gate surface; enzyme was then covalently immobilized to this modified surface. All sensors were tested with paraoxon and displayed rapid response ( −6 molar. The 200 nm sol–gel gate modification enhanced the signal of enzyme-modified devices without effecting device pH sensitivity. Sensors were stored at 4°C in buffer and tested multiple times. Devices coated with 200 nm silica microspheres maintained significant enzymatic activity over a period of 10 weeks while uncoated devices lost all enzyme activity during the same period. The 20 nm sol–gel modification did not enhance device response or enzyme stability. Successful reuse of sensor chips was demonstrated after stripping inactive enzyme with an RF oxygen plasma system and reimmobilizing active enzyme.

Hydrogen production in a compact supercritical water reformer

Abstract Experiments were conducted to investigate the reforming of organic compounds (primarily methanol) in supercritical water at 550–700°C and 27.6 MPa in a tubular Inconel 625 reactor. The results show that methanol can be completely converted to a product stream that is low in methane and near the equilibrium composition of hydrogen, carbon monoxide, and carbon dioxide. The effect of reactor temperature, feed concentration of methanol, and residence time on both conversion and product gas composition was investigated and the results are presented. Reaction pathways and potential applications of this technology are discussed. Ethanol and ethylene glycol resulted in less desirable effluent gas, with high concentrations of methane and carbon monoxide. Acetone and diesel fuel both resulted in the reactor becoming plugged.

Electrokinetically Pumped Liquid Propellant Microthrusters for Orbital Station Keeping

For most orbital maneuvers, small satellites in the sub-10 kg range require thrusters capable of spanning the micro-Newton to milli-Newton force range. At this scale, electrokinetic (EK) pumping offers precise metering of monergolic or hypergolic liquid propellants under purely electrical control at pressures and flow rates well-suited to microthruster applications. We have demonstrated direct and indirect EK pumping for delivery of anhydrous hydrazine and hydrogen peroxide monopropellants, respectively, into capillary-based microthrusters with integrated in-line catalyst beds. Catalytic decomposition generates gases which accelerate through a plasma-formed converging-diverging nozzle, producing thrust. Specific impulses up to 190 s have been shown for hydrazine in non-optimized nozzles.

Immobilization, stabilization and patterning techniques for enzyme based sensor systems.

Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore, Calif. to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of our biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused upon covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. We are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces are presented.