In this work, we have developed and characterized a novel microfluidic immunoassay methodology for rapid and sensitive quantification of ethinylestradiol (EE2) in river water samples. The detection of EE2 was carried out using a competitive direct immunoassay method based on the use of anti-EE2 polyclonal antibodies immobilized on magnetic microspheres 3-aminopropyl-modified manipulated for an external removable magnet. The EE2 present in the water sample was allowed to compete with EE2-horseradish peroxidase (HPR) conjugated for the immobilized anti-EE2 antibody. The HPR, in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of catechol (Q) whose back electrochemical reduction was detected on gold electrode at 0.0 V. The response current obtained from the product of enzymatic reaction is inversely proportional to the amount of EE2 in the water sample. The electrochemical detection can be done within 1 min and total assay time was 30 min. The calculated detection limits for electrochemical detection and the ELISA procedure are 0.09 and 0.32 ng L(-1) respectively and the intra- and inter-assay coefficients of variation were below 5.8%. Our electrochemical immunosensor showed higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method, which shows the potential for assessment of EE2 in river water samples.
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