The present project is aimed at developping immunochemical detection techniques which allow the fast screening of pesticides and cross-reactive transformation products in aquatic environmental matrices. All methods to be developed will be based on commercially available antibodies against pesticides.
An immunobiosensor has been developed which is based on the kinetically fast association of fluorescein labelled antibodies with antigenic analytes eluting from the high performance liquid chromatography (HPLC) column or injected via a flow injection system. In a first step, the antibodies are added to the liquid chromatography (LC) effluent via a mixing union. Antigenic analytes react with the antibodies to form strongly fluorescent immunocomplexes in concentrations proportional to the analyte concentration. In a second step the excess of free antibodies is removed via the passage through a small column packed with an antigen bound support. The reporter molecules covalently attached to the polyclonal fab fragments used as immunoreagent are detected by fluorescence.
This detection system is entirely based on fast association reactions between antigens and antibodies providing short reaction times and compatibility with continuous flow systems. The detection sensitivity is not limited by the binding constant of the antibody antigen complex and the precision and accuracy is higher than in competitive assays. Due to the high capacity of the antigen support used to trap free antibodies a large number (over 2500) of samples can be measured before regeneration of the antigen support is required. The method has proven to be equally sensitive as batch fluoroimmunoassays.
The determination of atrazine in the presence of simazine in water samples has been analyzed by direct rapid magnetic particle based solid phase enzyme linked immunosorbent assay (ELISA), by dichloroethane liquid liquid extraction (LE) and Florisil clean up followed by ELISA and by LLE Florisol followed by gas chromatography-nitrogen phosphorus detection (GC-NPD).
Results on the use of a rapid atrazine ELISA indicated that when analysing freeze dried water samples coming from river water with various pesticides and glycine, a prior clean up step using Florisil was needed. Good correlation was observed between the values obtained using Florisil clean up followed by ELISA and by GC-NPD. Both techniques allow similar limits of detection for atrazine in water samples. Higher deviations (26%) have been encountered in one of the samples were the proportion of simzine was about 3 times higher than atrazine and it has been attributed to certain cross reactivity between atrazine and simazine. The present techniques permit the rapid determination of atrazine in drinking water samples at the levels required by the European Community Drinking Water Directive and achieve also the goals of avoiding false positive determinations.
The use of the rapid ELISA assay is ideal for adaptation to on site monitoring of atrazine since the analytical test can be performed in less than one hour, can be used to perform real time analysis and it can be used as a potent alarm monitoring system.
Further research will include the performance of this rapid ELISA test for the determination of atrazine and simazine in combination with various transformation products and using different water matrices. Other clean up steps will be investigated and comparison made with other analytical techniques.
Due to the fact that currently no continuous-flow on-line immunobiosensors are used in environmental monitoring, three different strategies will be pursued to set up screening methods. The methods differ in the types of antibodies to be used, detection principles, sensitivity to be expected, ease of use and requirements with respect to regeneration. All immunobiosensors will be based only on association reactions between analytes and free antibodies. The fast kinetics of this type of reactions which will guarantee analysis times lower than 10 minutes.
Three immunobiosensor techniques will be developed representing the range of immunoassays technology used in clinical and biochemistry:
1. surface plasmon resonance detection using non labelled antibodies
2. fluorescence detection using fluorescein-labelled antibodies
3. Amperometric detection using enzyme-labelled antibodies
The immunobiosensors will be implemented in flow-injection analysis systems allowing the continuous, automated processing of samples. On-site sampling devices will be developed and coupled to the immunobiosensor detection system without manual transfer of samples. The detection devices will be linked to data acquisition systems which will automatically identify positive samples. Non-biological instrumental analysis methods (LC-MS, GC-MS, LC-DAD) will be used to validate the methods developed in this project. The continous-flow immunobiosensors will be compared with commercially available immunoassay kits (batch immunoassays) with respect to sensitivity, reproducibility, reliability and sample throughput.
Funding SchemeCSC - Cost-sharing contracts
221 00 Lund
3700 AJ Zeist
28801 Alcalá De Henares - Madrid