Abstract Intensive agriculture associated with the use of large amounts of different pesticides, together with the growing concern about the potential contamination of ground water, have brought about the need for developing fast screening methods. This work presents the automa- tion of an enzyme-linked immunosorbent assay for atra- zine by means of a flow-through system. Three different solid supports for antibody immobilization were com- pared in a direct competitive assay format. Sensitivity reached in all cases was below the maximum level al- lowed in the EU (100 ng L –1 ). Cross-reactivity of atrazine- related compounds was also studied. The performance of the different supports is discussed regarding sensitivity and immunosurface regeneration. Introduction Atrazine and related s-triazine herbicides are used for the protection of crops from broadleaf weeds, as well as for soil sterilization and road maintenance. Due to the wide- spread application of atrazine, its persistence in the envi- ronment and its solubility in water, this pesticide has be- come a common pollutant. The determination of atrazine and other s-triazine herbicides is usually performed by liquid-liquid extraction with dichloromethane or solid- phase extraction using either reverse phase C 18 cartridges or Empore disks, followed by gas chromatography with nitrogen phosphorus or mass spectrometric detection [1]. These methods are sensitive, very robust and well estab- lished, but also time-consuming, expensive and require specialized instrumentation. Immunochemical techniques have become increas- ingly popular as screening methodologies for the analysis of environmental pollutants [2]. Very sensitive im- munoassays for triazines [3, 4] have been reported in the literature. Immunoassays are sensitive, fast, inexpensive, specific, portable, simple and easy to use as compared to chromatographic methods. However, they also suffer from disadvantages, such as interference by cross-reacting compounds, limitation to single analyte residue analyses, and matrix effects [5]. Immunoassays are usually performed on microtiter plates, enzyme-linked immunosorbent assay (ELISA) be- ing the mostly used variant of immunochemical analysis. This technique, although very sensitive, requires exten- sive pipetting steps, thus is not directly applicable to process control or alarm stations. These drawbacks can be resolved by the development of immunosensors, devices that can monitor analytes automatically in real time. An interesting approach to immunochemical sensing is the use of antigen-antibody reactions in flow systems [6] giv- ing rise to flow-through immunosensors. In direct flow-through immunosensors, an antibody raised against the analyte is immobilized on a solid sup- port, and all the reactions take place on this immunosor- bent. After the measurements, this immunosorbent can be replaced, or regenerated by the application of a solution (desorbent) that dissociates the antigen-antibody com- plexes. Immunoreactor regeneration is preferable because it reduces antibody consumption and partially circum- vents the present difficulties in producing reproducible immunoreactors [7]. In this work, the development of three flow-through immunosensors for atrazine is described and their perfor- mance comparatively discussed in terms of sensitivity, reusability, and cross-reactivity. Two of the reported sen- sors are based on an immunoreactor containing an ori- ented antibody covalently immobilized on two different supports. The third one is based on the capture of the im- munocomplexes formed in solution by an antibody-binder M. A. González-Martínez · S. Morais · R. Puchades · A. Maquieira · M. P. Marco · D. Barceló Automation of a heterogeneous enzyme immunoassay for atrazine. Comparison of three immobilization supports Fresenius J Anal Chem (1998) 361 : 179–184 © Springer-Verlag 1998 Received: 21 July 1997 / Revised: 7 November 1997 / Accepted: 11 November 1997 ORIGINAL PAPER M. A. González-Martínez · S. Morais · R. Puchades · A. Maquieira () Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, E-46071 Valencia, Spain M. P. Marco Departamento de Química Orgánica Biológica, Univ. Barcelona D. Barceló Departamento de Química Ambiental, C.I.D.-C.S.I.C., Jordi Girona, 18–26, E-08034 Barcelona, Spain