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Homogenous immunoassay technique based on functionalized nanoparticles. Application to detection of pesticide contaminant 2,4-dichlorophenoxyacetic acid from alimentary and environmental samples
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Acronym: HINANODET
Contracting Authority: Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI)
Number / Date of the contract: 98 / 2012
Parteneriate in domeniile prioritare
Project Manager: Ioan Dorobantu
Partners: Alexandru Ioan Cuza University of Iasi (UAIC); National Institute for Research and Development of Isotopic and Molecular Technologies Cluj-Napoca (INCDTIM)
Starting date / finishing date: 2012-07-02 / 2016-12-31
Project value: 2000000 RON
Abstract: Immunological methods such as enzyme linked immunosorbent assay (ELISA) are increasingly becoming important for pesticides residual analysis due to the high specificity of detecting molecules like antibodies. These immunoassay methods are highly specific, sensitive (nanogram or picogram) and accuracy for the detection of low molecular weight contaminants presents in our environment. ELISA is a technique based on the ability of non-labeled antigen (e.g. pesticide) in a specific volume of standard solution or in an unknown sample to compete with a fixed of amount of enzymatic labeled antigen for a limited number of binding sites of a specific binding antibody protein. The objective of the project is to develop a new and innovative immunochemical technique based on functionalized nanoparticles, homogenous enzyme linked immunosorbent assay (HnELISA) technique for detection of pesticide contaminant 2,4-dichlorophenoxyacetic acid (2,4D) from alimentary and environmental samples. 2,4D is one of the most used herbicide in agriculture to control and destroy of the weeds that can affect agricultural crops. The remanence of this organochlorurate compound in alimentary products, transfer and contamination of ground water in the areas where this pesticide is used require the analysis of this chemical in order to establish the contamination level of the alimentary products and the environmental factors (water, soil). The objective of the project is to develop an immunoassay technique with improved qualities in comparison with traditionally ELISA technique existed on the market for detection of pesticide contaminants from alimentary and environmental factors. Qualitative characteristics as sensitivity, accuracy, stability of the nanoimmunosorbent and low cost per assay are finally taken in account. Development of HnELISA technique would have practical application in monitoring of the pesticide contaminant 2,4D from alimentary and environmental samples.

Objectives: 1. To obtain the immunogenic conjugate 2,4-Dichlorophenoxyacetic acid-protein. 2. To obtain the anti 2,4-Dichlorophenoxyacetic acid antibody by immunization, preparation and purification of antisera. 3. To obtain the enzymatic markers 2,4-Dichlorophenoxyacetic acid-peroxidase and 2,4-Dichlorophenoxyacetic acid-alkaline phosphatase for ELISA technique and anti 2,4-Dichlorophenoxyacetic acid antibody-peroxidase and anti 2,4-Dichlorophenoxyacetic acid antibody-alkaline phosphatase. 4. To study kinetics of the antibody-antigen system and to evaluate the association and dissociation rate constants, the affinity constant of the immune components. 5. To obtain the nanoimmunosorbent (anti2,4-Dichlorophenoxyacetic acid antibody covalently coupled on the surface of the nanoparticles) and physico-chemical and structural characteristics of the nanoimmunosorbents. 6. To study kinetics and thermodynamics of nanoimmunosorbent-antigen system and to evaluate the kinetics and thermodynamic parameters as: the adsorbtion and desorbtion rate constants, the equilibrium constant, free energy, enthalpy and entrophy of the system. 7. To obtain the auxiliary reagents necessary for HnELISA technique. 8. To prepare biological sample, to extract 2,4-Dichlorophenoxyacetic acid from alimentary and environmental samples with different solvents and to evaluate the recovery coefficient. 9. To elaborate the HnELISA technique for 2,4-Dichlorophenoxyacetic acid and to prepare auxiliary reagents necessary in this technique. 10. To obtain the validation criteria of HnELISA technique: specificity, sensitivity, accuracy and the precision and quality control to be achieved by comparison with other method used in assays of pesticide HPLC (High Performance Liquid Chromatography) and GC-MS (Gas Chromatography-Mass Spectrometry)

THE STAGES OF THE PROJECT AND DELIVERY DATES
1. Obtainment of immunogenic conjugates pesticide-protein and antipesticide antibodies (2012-12-31) Results
2. Obtainment of enzymatic labels: pesticide-enzyme and antipesticide antibody-enzyme and of nanoimmunosorbents (2013-12-31) Results
3. Obtainment of non magnetic and magnetic nanoimmunosorbents antipesticide-antibody coupled to nanoparticles and physico-chemical and structural characterization of the nanoimmunosorbents (2014-12-31) Results
4. Kynetic and thermodynamic properties of the systems nanoimmunosorbents- analyte in the presence of enzymatic label. Obtainment of the auxiliary reagents for HnELISA and preliminary preparation of the biological and environmental samples (2015-12-31) Results
5. Elaboration of HnELISA technique for assay of 2,4-Dichlorophenoxyacetic acid from alimentary and the environmentally samples (2016-12-31) Results

RESULTS
PUBLISHED ARTICLES
RESEARCH TEAM

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