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Reaction Monitoring with FTIR Spectroscopy

ReactIR 4000

Experimental setup with ReactIR 4000

A new publication in the journal Analytical Biochemistry describes the use of in-situ FTIR spectroscopy to monitor the degradation of toxic organophosphorus componds by the enzyme DFPase. The use of Attenuated Total Reflexion (ATR) allows direct meassurements in the reaction vessel without the need for cuvettes. In comparison to established methods the total reaction volume can be significantly reduced, which also leads to a substantial reduction in the required ammount of toxic substrate and therefore to an increase in work safety. Detection is based on the different IR absorption bands of the organophosphorus compound and its hydrolysis product. We were able to show that changes in the IR bands are directly proportional to changes of substrate and product concentrations in solution. The Lambert-Beer law therefore holds. The limit of quantitation (LOQ) of the methods is around 1 mM. This is a significant progress compared to older work in the field of in-situ FTIR spectroscopy. Dadd and co-workers used FTIR spectroscopy to monitor the reaction of nitriles (Dadd et al., J. Microbiol. Methods 2000; 41:69-75), but the required substrate concentration were in the range of 250 mM. For the experiments we used a ReactIR 4000 made by Mettler-Toledo.

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy.
Gäb J, Melzer M, Kehe K, Richardt A, Blum MM.
Anal Biochem. 2009; 385(2):187-193.

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P–O–R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert–Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).


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