Molecular interaction between organophosphorus acid anhydrolase and diisopropylfluorophosphate.

Organophosphorus acid anhydrolases (OPAA; E.C.3.1.8.2) are a class of enzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus (OP) compounds, including pesticides and fluorine-containing chemical nerve agents. In this paper, subphase conditions have been optimized to obtain stable OPAA Langmuir films, and the diisopropylfluorophosphate (DFP) hydrolysis reaction catalyzed by OPAA in aqueous solution and at the air-water interface was studied. OPAA-DFP interactions were investigated utilizing different spectroscopic techniques, that is, circular dichroism and fluorescence in aqueous solution and infrared reflection absorption spectroscopies at the air-water interface. The characterization of OPAA and its secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and in the presence of DFP dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive features. The research described herein demonstrated that OPAA can be used in an enzyme-based biosensor for DFP detection.

Hydrolysis of acetylcholinesterase inhibitors--organophosphorus acid anhydrolase enzyme immobilization on photoluminescent porous silicon platforms.

We report on the immobilization of an OPAA enzyme on luminescent porous silicon devices, and on the utilization of this new platform to hydrolyze p-nitrophenyl-soman.

The interaction between OPH and paraoxon at the air-water interface studied by AFM and epifluorescence microscopies.

The paraoxon hydrolysis reaction catalyzed by organophosphorus hydrolase (OPH) monolayer at the air-water interface was studied. OPH-paraoxon interactions, occurring at the two-dimensional interface, by close-packed, highly orientated OPH monolayer, were investigated by several different surface chemistry techniques; e.g. surface pressure area isotherms, atomic force microscopy (AFM), and in situ epifluorescence microscopy. The characterization of OPH Langmuir and Langmuir-Blodgett films prepared in both the presence and absence of paraoxon, demonstrated significantly distinctive feature when compared with one another. Continuous growth of the OPH aggregates is a distinct phenomenon associated with hydrolysis, in addition to the pH changes in the local environment of the enzyme macromolecules.

(CdSe)ZnS quantum dots and organophosphorus hydrolase bioconjugate as biosensors for detection of paraoxon.

In this paper, we first report a novel biosensor for the detection of paraoxon based on (CdSe)ZnS core-shell quantum dots (QDs) and an organophosphorus hydrolase (OPH) bioconjugate. The OPH was coupled to (CdSe)ZnS core-shell QDs through electrostatic interaction between negatively charged QDs surfaces and the positively charged protein side chain and ending groups (-NH2). Circular dichroism (CD) spectroscopy showed no significant change in the secondary structure of OPH after the bioconjugation, which indicates that the activity of OPH was preserved. Detectable secondary structure changes were observed by CD spectroscopy when the OPH/QDs bioconjugate was exposed to organophosphorus compounds such as paraoxon. Photoluminescence (PL) spectroscopic study showed that the PL intensity of the OPH/QDs bioconjugate was quenched in the presence of paraoxon. The overall quenching percentage as a function of paraoxon concentration matched very well with the Michaelis-Menten equation. This result indicated that the quenching of PL intensity was caused by the conformational change in the enzyme, which is confirmed by CD measurements. The detection limit of paraoxon concentration using OPH/QDs bioconjugate was about 10(-8) M. Although increasing the OPH molar ratio in the bioconjugates will slightly increase the sensitivity of biosensor, no further increase of sensitivity was achieved when the molar ratio of OPH to QDs was greater than 20 because the surface of QDs was saturated by OPH. These properties make the OPH/QDs bioconjugate a promising biosensor for the detection of organophosphorus compounds.

Langmuir and Langmuir-Blodgett films of organophosphorus acid anhydrolase.

In this paper, we describe the preparation and characterization of Langmuir and Langmuir-Blodgett (LB) monolayers of the enzyme organophosphorus acid anhydrolase (OPAA). Langmuir films of OPAA were characterized on different subphases, such as phosphate, ammonium carbonate, and bis-tris-propane buffers. Monolayers at the air-water interface were characterized by measuring the surface pressure and surface potential-area isotherms. In situ UV-vis absorption spectra were also recorded from the Langmuir monolayers. The enzyme activity at the air-water interface was tested by the addition of diisopropylfluorophosphate (DFP) to the subphase. LB films of OPAA were transferred to mica substrates to be studied by atomic force microscopy. Finally, a one-layer LB film of OPAA labeled with a fluorescent probe, fluorescein isothiocyanate (FITC), was deposited onto a quartz slide to be tested as sensor for DFP. The clear, pronounced response and the stability of the LB film as a DFP sensor show the potential of this system as a biosensor.

Layer-by-layer self-assembled chitosan/poly(thiophene-3-acetic acid) and organophosphorus hydrolase multilayers.

The aim of this study is to immobilize an enzyme, namely, organophosphorus hydrolase (OPH), and to detect the presence of paraoxon, which is an organophosphorus compound, using the layer-by-layer (LbL) deposition technique. To lift the OPH from the solid substrate, a pair of polyelectrolytes (positively charged chitosan (CS) and negatively charged poly(thiophene-3-acetic acid) (PTAA)) were combined. These species were made charged by altering the pH of the solutions. LbL involved alternate adsorption of the oppositely charged polyions from dilute aqueous solutions onto a hydrophilic quartz slide. This polyion cushion was held together by the electrostatic attraction between CS and PTAA. The growing process was monitored by fluorescence spectroscopy. OPH was then adsorbed onto the five-bilayer CS/PTAA system. This five-bilayer macromolecular structure compared to the solid substrate rendered stability to the enzyme by giving functional integrity in addition to the ability to react with paraoxon solutions. The ultimate goal is to use such a system to detect the presence of organophosphorus compounds with speed and sensitivity using the absorption and fluorescence detection methodologies.