A novel class of micro-electrodes was fabricated by synthesizing high density carbon nanotube islands on lithographically defined, passivated titanium nitride conductors on a silicon dioxide substrate. Electrochemical characterization in phosphate buffered saline of these new electrodes reveals superb electrochemical properties marked by featureless rectangular cyclic voltammetry curves corresponding to a DC surface specific capacitance and a volume specific capacitance as high as 10 mF cm(-2) and 10 F cm(-3), respectively. These electrodes are also characterized by a slowly varying impedance magnitude over the range of 1 Hz to 20 kHz. High fidelity extracellular recordings from cultured neurons were performed and analysed to validate the effectiveness of the fabricated electrodes. The enhanced electrochemical properties of the electrodes, their flexible and simple micro-fabrication preparation procedure as well as their bio-compatibility and durability suggest that carbon nanotube electrodes are a promising platform for high resolution capacitive electrochemical applications.
Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
Serum
paraoxonase 1 (PON1) is a native lactonase capable of promiscuously
hydrolyzing a broad range of substrates, including organophosphates,
esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller
with a flexible loop (residues 70–81) covering the active site.
This loop contains a functionally critical Tyr at position 71. We
have performed detailed experimental and computational analyses of
the role of selected Y71 variants in the active site stability and
catalytic activity in order to probe the role of Y71 in PON1’s
lactonase and organophosphatase activities. We demonstrate that the
impact of Y71 substitutions on PON1’s lactonase activity is
minimal, whereas the kcat for the paraoxonase
activity is negatively perturbed by up to 100-fold, suggesting greater
mutational robustness of the native activity. Additionally, while
these substitutions modulate PON1’s active site shape, volume,
and loop flexibility, their largest effect is in altering the solvent
accessibility of the active site by expanding the active site volume,
allowing additional water molecules to enter. This effect is markedly
more pronounced in the organophosphatase activity than the lactonase
activity. Finally, a detailed comparison of PON1 to other organophosphatases
demonstrates that either a similar “gating loop” or
a highly buried solvent-excluding active site is a common feature
of these enzymes. We therefore posit that modulating the active site
hydrophobicity is a key element in facilitating the evolution of organophosphatase
activity. This provides a concrete feature that can be utilized in
the rational design of next-generation organophosphate hydrolases
that are capable of selecting a specific reaction from a pool of viable
substrates.
A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 10(7) M(-1) min(-1). Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 × 10(-4) in favor of the R(P) isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S(P) isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.
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