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A sensitive and rapid magnetic-nanoparticles-based fluorescent immunoassay (MNPs-FIA) for individual and simultaneous determination of dichlorvos (DDVP) and paraoxon in untreated raw milk was developed. The conjugates DDVP-cBSA (cationized bovine serum albumin) and paraoxon-BSA (bovine serum albumin) were labeled by fluorescent dyes. The optimal amount of immobilized polyclonal anti-DDVP and anti-paraoxon antibodies on MNPs (0.375 mg Ab-MNPs) and optimal concentrations of DDVP-cBSA-FITC (18 µg/mL) and paraoxon-BSA-FITC (22 µg/mL) for MNPs-FIA were determined. The calibration curves of individual and simultaneous immunoassays of DDVP and paraoxon were investigated. The IC 50 value of the individual paraoxon assay in raw cow's milk was 60 ng/mL and linear range 2-300 ng/mL. The IC 50 value of the individual DDVP assay was 70 ng/mL and linear range 5-300 ng/mL. The IC 50 values of the paraoxon and DDVP (100 and 120 ng/mL respectively) in simultaneous assay were higher than those of the individual ones, and their linear ranges were 10-400 ng/mL.

The sensitive competitive immunofluorescence method for simultaneous determination of paraoxon and dichlorvos with immobilized multi-polyclonal antibody on magnetic nanoparticles was developed. The multi-polyclonal antibody was obtained after the immunization of mixture of two prepared immunogens dichlorvos-cBSA and paraoxon-BSA (1:1). The immunogens dichlorvos-cBSA and paraoxon-BSA were synthesized preliminary. Multi-polyclonal antibody against dichlorvos and paraoxon was covalently coupled on magnetic nanoparticles. The competitive fluorescence conjugates dichlorvos-cBSA-FITC and paraoxon-BSA-ATTO 620 were synthesized. Two typical calibration curves of immunofluorescence assay for determination of dichlorvos and paraoxon in buffer solutions were obtained. The linear interval from 2 to 200 ng/mL for these two pesticides was determined. Then the calibration curves for dichlorvos and paraoxon were obtained in cow milk solutions. The linear range of pesticides in cow milk was determined (from 5 to 300 ng/L) and the detection limit for paraoxon (3.5 ng/mL) and dichlorvos (4 ng/mL) was found. The obtained results for cow milk samples were compare with results in UHT, pasteurized cow milk, sheep and goal milk. Quite different are the results when analyzing paraoxon and dichlorvos in standard solutions prepared in sheep's milk. The linear working range for the two pesticides is between 7 and 300 ng/mL it is obviously, that there is a shift of all analytical characteristics up to higher values. The reason for this is the high fat content of sheep's milk. The obtained results were showed that the developed method was 2 time more sensitive than method with the results obtained with mixture (1:1) of two separated antibodies – anti-paraoxon and anti-dichlorvos. These results confirmed the potential of the immunoassay for quantitative simultaneous screening of both dichlorvos and paraoxon. Practical applications:The use of immunofluorescence assay based on immobilized multi-polyclonal antibody on magnetic nanoparticles allows us to detect simultaneous paraoxon and dichlorovos in raw milk and ather diary products.

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