Organophosphorus acid anhydrolase: Secondary structure analysis in solution and Langmuir-Blodgett film and the direct CDS quantum dots conjugation for detection of diisopropylfluorophosphate

Organophosphorus acid anhydrolase (OPAA) can catalyze the hydrolysis of P-F or PCN bonds of organophosphorus (OP) compounds. My main objective is to develop the OPAA-based biosensor to detect and detoxify OP compounds by the conjugation of CdS quantum dots (QDs) and OPAA.First, the surface chemistry and secondary structure of enzyme OPAA was studied. The pH (from 4.6 to 10.5) and temperature (from 20 to 56°C) effects on the secondary structure of OPAA enzyme in solution, LB film and dry film were studied by circular dichroism using CDPro software package. The secondary structure of OPAA is well defined as alpha-helix structure at the isoelectric point (pH = 6.8). The thermal stability of OPAA LB and dry films indicates that OPAA secondary structure could be preserved by being immobilized in LB and dry films.Second, the bioconjugation between CdS QDs with the enzyme OPAA was synthesized and the CdS/OPAA bioconjugate was characterized. The OPAA was coupled to the CdS QDs directly without any stabilizer. This direct conjugation necessitates no nanoscale templating agents such as surfactants, which are commonly used to prepare bioconjugate of inorganic nanoparticles. Direct conjugation is expected to minimize enzymatic structural changes and loss of activity. The characterization of CdS/OPAA QDs by surface chemistry and spectroscopic techniques showed that no significant changes in secondary structure, which suggests the activity of the enzyme may be preserved.Third, the interactions between OPAA with paraoxon or DFP in aqueous solution and at air-water interface were investigated utilizing different spectroscopic techniques, that is CD, UV-vis and fluorescence. The characterization of OPAA and its secondary structure in aqueous solution and as a Langmuir monolayer at the air-water interface in the absence and presence of its substrates dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive feature.Last, the interactions between CdS/OPAA QDs with DFP in aqueous solution and air-water interface were also investigated utilizing CD and fluorescence spectroscopies. The enzyme-QDs system was used to detect DFP at the nanomolar level through photoluminescence (PL) quenching. Percent PL quenching as a function of DFP concentration had a hyperbolic dependence. The OPAA-based QDs not only detected DFP but also hydrolyzed DFP into less toxic products.This research project demonstrated that the direct conjugated CdS/OPAA QDs is a good enzyme-based biosensor for OP compound detection