Development of MNPs Based Enzyme Immuno-Sorbent Analysis for the Determination of Organophosphorus Pesticides in Milk

Abstract

Objectives: High toxicity of pesticides requires accurate and reliable methods to monitor their levels for food, water and soil safety. Methods: Magnetic nanoparticles were synthesized and functionalized with (3-aminopropyl)triethoxysilane. Sheep polyclonal-anti-paraoxon and anti-dichlorvos antibodies were produced. Dichlorvos-cationized ovalbumin-HPR and paraoxon-ovalbumin-HPR conjugates were prepared and characterized. The optimal conditions for antibody immobilization were determined. Enzyme immunosorbent assay based on Magnetic Nanoparticles (MNPs) for the determination of pesticides was developed. A comparison of the developed modified MNPs-based ELISA with the conventional ELISA was performed. The effect of protein, pH, and fats in milk samples on the analytical characteristics of the MNPs-based ELISA was investigated. In order to validate the results obtained from the MNPs-based ELISA method, experiments with HPLC were performed. Results: The linear range of the standard curve for the determination of paraoxon in buffer with anti-paraoxon antibody was 0.125-5 ng/mL, for the dichlorvos with anti-dichlorvos antibody was 0.0625-5 ng/mL, and for the mixture of paraoxon and dichlorvos in ratio (1:1) with multi-antibody was 0.125-5 ng/mL. A comparison of the developed modified MNPs-based ELISA with the conventional ELISA was performed. The recovery of the obtained results for the determination of pesticides in milk samples in both methods was from 104 to 110%. Conclusion: The stated advantages of magnetic nanoparticles provide good sensitivity and rapidity of immunoassay and a simple procedure for separating of antibody-magnetic nanoparticles with the bound antigen from the non-bound antigen in the sample.