Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons |
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Authors: | Sarka Bidmanova Radka Chaloupkova Jiri Damborsky Zbynek Prokop |
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Institution: | (1) Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic; |
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Abstract: | An enzyme-based biosensor was developed by co-immobilization of purified enzyme haloalkane dehalogenase (EC 3.8.1.5) and a
fluorescence pH indicator on the tip of an optical fiber. Haloalkane dehalogenase catalyzes hydrolytic dehalogenation of halogenated
aliphatic hydrocarbons, which is accompanied by a pH change influencing the fluorescence of the indicator. The pH sensitivity
of several fluorescent dyes was evaluated. The selected indicator 5(6)-carboxyfluorescein was conjugated with bovine serum
albumin and its reaction was tested under different immobilization conditions. The biosensor was prepared by cross-linking
of the conjugate in tandem with haloalkane dehalogenase using glutaraldehyde vapor. The biosensor, stored for 24 h in 50 mM
phosphate buffer (pH 7.5) prior to measurement, was used after 15 min of equilibration, the halogenated compound was added,
and the response was monitored for 30 min. Calibration of the biosensor with 1,2-dibromoethane and 3-chloro-2-(chloromethyl)-1-propene
showed an excellent linear dependence, with detection limits of 0.133 and 0.014 mM, respectively. This biosensor provides
a new tool for continuous in situ monitoring of halogenated environmental pollutants. |
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