Chapter 12
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Optical Immunosensor and ELISA for the Analysis of Pyrethroids and DDT in Environmental Samples 1
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Petra M. Kramer , Cristina M. Weber , Elisabeth Kremmer , Christina Räuber , Dieter Martens , Stephan Forster , Larry H. Stanker , Peter Rauch , Paul M. Shiundu , and Francis J. Mulaa 1
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GSF-National Research Centre for Environment and Health, Institute of Ecological Chemistry, 85764 Neuherberg Oberschleissheim, Germany GSF-National Research Centre for Environment and Health, Institute of Molecular Immunology, 81377 München, Germany Department of Ecological Chemistry and Environmental Analysis, Technische Universitat München, 85350 Freising-Weihenstephan, Germany Agricultural Research Service, U.S. Department of Agriculture, Albany, C A 94710 Candor Bioscience GmbH, 48149 Münster, Germany Departments of Chemistry and Biochemistry, University of Nairobi, Nairobi, Kenya Current address: LUFA Speyer, Obere Langgasse 40, 67346 Speyer, Germany 2
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An optical immunosensor (AQUA-OPTOSENSOR) and ELISA (enzyme-linked immunosorbent assay) for the analysis of pyrethroids and D D T in river water and/or sediment, are described. The optical immunosensor consists of a bench-top optical read-out-device and disposable single-use sensor chips. ELISA was carried out in the coating antigen format. As examples, phenothrin (pyrethroid) and p,p'-DDT were chosen. Herein we describe the overall strategy, the set-up and principle of the immunosensor platform, and show represent -tative results for immunosensor and ELISA analysis. The immunosensor employs fluorophore (Oyster®-645)-labeled
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© 2007 American Chemical Society
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monoclonal antibodies (mouse mAb Py-1 and rat mAb D D T 7C12), and makes use of the evanescent field, thus operating without washing steps. ELISA in the coating antigen format uses a second antibody labeled with peroxidase. Both, phenothrin and p,p'-DDT can be analyzed with these immunochemical techniques in the low ppb levels. Advantages and drawbacks of both immunochemical platforms are discussed.
Introduction Contamination of different bodies of water with pesticide residues occur during application and runoffs. Some pesticide residues are also persistent and can be found throughout the year. Especially in developing countries, where most communities living in slum areas use untreated water for drinking and for domestic activities, there is an urgent need to provide cost-effective tools for monitoring and control of these contaminations. In addition, new E U directives, such as the Water Framework Directive (WFD; (/)), ask for monitoring programs, which will not be affordable, when conventional analytical methods (e.g., L C , GC) will be used. Immunochemical methods, either as immunoassays or as immunosensors, are seen as analytical techniques, which can be established for screening and monitoring scenarios, especially when many samples should be monitored for a limited number of analytes, and when a fast result is needed. In a recent review, the increasing significance of antibody based methods for environmental and food analysis is evident (2). In comparison to immunoassays, which still need a lot of pipetting and which are therefore prone to manual mistakes, the advantage of immunosensors is the higher degree of automation. For this purpose, a new optical immunosensor platform ( A Q U A OPTOSENSOR) was employed, which uses monoclonal antibodies (mAbs) labeled with a fluorophore (Oyster®-645). Here we describe and demonstrate this new technique together with conventional ELISA (coating antigen format) for the analysis of pyrethroids (5) and D D T [ 1,1,1-trichloro-2,2-bis(4chlorophenyl) ethane] (4) in different environmental samples. Although D D T has been banned in many countries since 1972 (5), it is still present in the environment, especially in tropical countries. Advantages and limitations of this immunosensor in comparison to ELISA will be discussed.
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Materials and Methods
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Chemicals, Reagents, Instruments Pestanal® standard phenothrin (mixture of isomers, 94.9%) was purchased from Riedel de Haen (Seelze, Germany; now Sigma-Aldrich, Taufkirchen, Germany). D D T isomers and metabolite standards were purchased from the Institute of Organic Industrial Chemistry (Warsaw, Poland). Stock solutions were prepared either in methanol or in ethanol with concentrations of 1 mg/mL and stored at 4 °C. 3-Phenoxybenzoic acid (3-Pba ( C H , o 0 , M 214.2), Fluka), dimethylformamide (DMF, Fluka), Af-hydroxysuccinimide (NHS, Aldrich), 1,3dicyclohexylcarbodiimide (DCC, Aldrich), bovine serum albumin (BSA, fraction V powder), ovalbumin (OVA), keyhole limpet hemocyanine (KLH), and 3,3',5,5'-tetramethylbenzidine ( T M B ) were purchased from Sigma-Aldrich, Taufkirchen, Germany. The derivative of D D T (4-{4-[l-(4-chloro-phenyl)2,2,2-trichloroethyl]phenyl}butanoic acid ( C j g H ^ Q ^ ; M 406.14)), was synthesized by Solvias A G , Basel, Switzerland. A l l buffer salts, hydrogen peroxide (30% w/w), and solvents were purchased from Merck (Darmstadt, Germany). Pierce Slide-A-Lyzer® dialysis cassettes ( M cut-off 10,000) and ImmunoPure goat anti-mouse IgG, (Fc), peroxidase (HRP) conjugated, were purchased from Perbio Science Deutschland GmbH, Bonn, Germany. Goat antirat-HRP was from Dianova GmbH, Hamburg, Germany. I3
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Ultra pure water was prepared by purifying demineralized water in a Milli-Q (MQ) filtration system (Millipore, Eschborn, Germany) and used for preparation of all buffers. Washing steps were carried out with the automated microtiter plate washer ELX405R (Bio-Tek Instruments, Bad Friedrichshall, Germany). Absorbance of microtiter plates was read with a ThermoMax microtiter plate reader (Molecular Devices, Palo Alto, U S A ) at 450 nm (reference 650 nm). Data evaluation was carried out with Softmax Pro (Molecular Devices, Palo Alto, USA).
Preparation of Hapten-Protein Conjugates for Coating Antigens The 3-Pba hapten and the DDT-derivative (DDT-hapten) were conjugated to different proteins. 3-Pba was conjugated to B S A and O V A as described by Karu et al. (
