available at a reasonable price. Project Rationale The overall goal of this project is to develop a simple, inexpensive immuno-biosensor, which will be used in the form of a portable analyser for rapid field and on-line determination of organophosphorous and organochlorine pesticides present in water at ng/l concentrations. To achieve these goals, the following tasks will have to be tackled: Development of two basic sample handling techniques, one for microreactor and one for SPR. Optimization of two detection techniques: fluorescence and SPR. Research Plan SUMMARY OF THE ACHIEVMENTS OF THE FIRST PROGRAM PHASE adapted from the summary provided by the project partners
Synthetic pesticides and herbicides are widely used in agriculture. Many of these are organochlorine and organophosphorous derivatives. Due to their recalcitrant nature, such toxins enter the food chain, causing serious risk to humans and animals. Therefore, it is essential to monitor their contamination in food, soil and water. The conventional analytical methods are not suitable for quick and specific field analyses. They are also expensive and laborious to apply. Biosensor based methods present an attractive alternative. In this concluded project, the development of biosensors useful for monitoring pesticides and herbicides was the prime objective. Biosensors based on enzymatic and immunological principles and modern detection systems not only present an effective solution to this vexing problem, but also a value-added product with good potential for commercialization and gainful job creation. Manpower training, scientific exchange between India and Switzerland and proliferation of knowledge between developed and developing countries were other important benefits expected from this project. A joint project comprising four different labs from India and Switzerland (IMTECH, Chandigarh; CFTRI, Mysore; <strong>EPFL</strong>, Lausanne, institutes LPI and LCPPM; IMT, Neuchatel), having interdisciplinary and complimentary expertise, was formed in order to develop sensitive, analyte specific, easy to use and cost-effective biosensor devices for monitoring pesticide samples in aqueous media. LPI-<strong>EPFL</strong> functioned as the overall project coordinator. The partners chose 5 pesticides (insecticides and herbicides) for the biosensor development. These were methyl and ethyl parathions, 2,4-dichlorophenoxy acetic acid (2,4-D), atrazine and monochrotophos. The primary task was to develop good quality polyclonal antibodies against these molecules. Secondly, appropriate assay techniques to individually detect up to nano (ppb) or sub-nanogram (ppt) levels of these molecules in water were required. CFTRI accepted the responsibility of developing polyclonal antibodies suitable for the 2 types of parathions. Monochrotophos was kept as a molecule to be studied after completing the work with parathions, since parathions are still widely used in India with serious harmful consequences. IMTECH selected atrazine and 2,4-D as their target molecules, particularly since the manufacture and use of these two compounds are rapidly increasing in India . Apart from the development and characterization of antibodies, the Indian labs were also involved in assay and transducer development. <strong>EPFL</strong> was involved in identifying appropriate detection methods, antibody-antigen interactions and pre-concentration methods. The preselected detection methods were surface plasmon resonance (SPR) and fluorescence. IMT , NE was involved in developing a Si (silicon) based micro-machined flow-device platform for repeated multiple use. In order to select the best option, the efficacy of the two detection methods was reviewed towards the end of the first two years, based on the actual experience and performance data of the developed antibodies. SPR was abandoned at that point, as this method was less sensitive with small molecules in comparison to fluorescence techniques. However, the work in CFTRI and <strong>EPFL</strong> also showed that chemiluminescence could be an equally competitive method for detecting pesticides in low concentration levels. Therefore, fluorescence and chemiluminescence were finally chosen as the suitable detection methods. Neverthless, at <strong>EPFL</strong>, SPR functioned throughout as an excellent workhorse in characterising the generated antibodies and evaluating their purity and specificity. IMTECH produced both polyclonal and monoclonal antibodies, which reached ppm limits of detection. CFTRI clearly showed that large quantities of polyclonal antibodies against parathions could be produced through egg-yolks of poultry. This method avoids the traditional production of antibodies in rabbit blood. Both CFTRI and IMTECH used appropriate conjugation chemistry for making haptens and optimized the conditions for obtaining best quality antibodies. Hapten-protein conjugates with different hapten density were made by chemical modification of selected organic toxins and subsequently linking covalently with suitable carrier proteins (BSA and KLH). The developed conjugates were thoroughly characterized by various methods such as UV, fluorescence, SDS-PAGE, MALDI etc., in order to optimize the hapten-protein conjugation density. The suitable conjugates were used as immunogens for the purpose of antibody generation. Specific antibodies were screened out using various affinity purification techniques. Hapten columns were designed for getting specific antibodies from the serum. Antibodies of different binding characteristics were eluted using suitable elution buffers and characterized by ELISA, SPR and Western blot techniques. These antibodies were used for different immunoassay formats.
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