Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons

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.     

Enzymatic Reaction Coupled with Flow-Injection Analysis with Charged Aerosol,Coulometric, or Amperometric Detection for Estimation of Contamination of the Environment by Pesticides

Because excessive using of pesticides poses a threat to the environment and to human health, development of low-cost and sensitive methods for analysis of pesticides in the environment is needed. Several bacteria can release halide ions from the molecules of halogenated hydrocarbons. This can be used in a device for analysis of halogenated hydrocarbons in the environment by quantification of the halide anions. Here we directed our attention to selecting an instrument for detection of chloride anions. We tested three different detectors, amperometric, and coulometric, both coupled with flow-injection analysis and charged aerosol, coupled with high performance liquid chromatography. Detection limits (3 × S/N) for measurement of chloride anions by use of these detectors was 30 μM (charged aerosol), 100 nM (coulometric), and 1 nM (amperometric). Because of its lowest detection limit for chloride anions and the many technical possibilities of miniaturization, the amperometric detector was used to test of effect of different cations on the chloride signal under the optimized experimental conditions (working electrode potential −365 mV; “Current R” 5 μA; mobile phase 0.2 M phosphate buffer, pH 6; flow rate 0.5 mL min⁻¹). NaCl, SrCl₂, NH₄Cl, and CsCl were tested as sources of chloride anions. We then used the detector to detect chloride anions catalytically cleaved from 1-chlorohexane by the enzyme haloalkane dehalogenase LinB from the bacterium Sphingobium japonicum UT26. The activity of the enzyme increased with increasing reaction temperature until the maximum was observed at 39°C. The results obtained were in good agreement with data obtained by colorimetric detection.

     

Radioiodination and biodistribution of the monoclonal antibody TU-20 and its scFv fragment

The ability of the monoclonal antibody TU-20 and its scFv fragment to bind specifically to the C-end of the class III β-tubulin makes these substances useful as potential diagnostics for neurodegenerative diseases—especially peripheral neuropathies. TU-20 and its scFv were labeled with ¹²⁵I and ¹²³I by chloramine-T (with radiochemical yield 75 and 50%, respectively). Radiochemical purity and stability was revealed by gel filtration (decrease to 80 and 50% in 2 months, respectively). Immunoreactivity of the labeled TU-20 was determined by ELISA—the range of the preserved immunoreactivity varies from 60 to 95% in accordance to the used radiolabeling process. RIA and affinity coupling analytic methods were specifically designed with focusing on specifics of the antibody and its fragment. The results of RIA differ in depandance on the type of the reaction vessel (glass or polystyrene) and the affinity coupling results depend on the experimental arrangement—in the batch or on the column. Fragmentation of the labeled antibody and its fragment was estimated by bis–tris gel electrophoresis followed by silver staining and autoradiography (over 95% of radioactivity bound in the substances). The antibody binding in tissue slices was studied in vitro by immunohistochemistry. The Purkinje cells were observed conjugated with the radiolabeled substances, either TU-20 or its ScFv fragment in the area of the cerebellum. In vivo biodistribution of ¹²⁵I-TU-20, ¹²⁵I-scFv TU-20, ¹²³I-scFv TU-20 and Na¹²⁵I was proceeded in normal mice (wild type C57B/6/J). Both biomolecules labeled by ¹²³I were also proved in an imaging biodistribution study with use of the SPECT camera. Finally, a transgene population G93A1 Gur was used for comparative study to show the different behaviour of the substances in a normal mouse and in the modified organism with amyotrophic lateral sclerosis. The most part of differences is observed in the area of the muscles, rostal and caudal spinal cord. In summary, the monoclonal antibody TU-20 and its scFv were successfully radioiodinated and afterwards analysed by several quality control methods and biodistribution studies which confirmed their preserved or expected immunoanalytical characteristics in normal and genetically modified organism.