A suitable immunosensor for chloramphenicol determination: study of two different competitive formats

Background: A deep analytical study was performed on two different formats based on a "competitive" ELISA-type assay to develop a suitable, sensitive and cheap immune device for chloramphenicol determination that could be advantageously applied to the analysis of real matrices (pharmaceutical, food and environmental). Methods: To this purpose peroxidase enzyme as a marker and an amperometric electrode for hydrogen peroxide, as a transducer, were used. Through the first competitive format, chloramphenicol determination was based on the competition between chloramphenicol and conjugated with biotin-avidinperoxidase chloramphenicol, both free in solution, for anti-chloramphenicol immobilized in the membrane, while the second competitive format was based on the competition between free in solution chloramphenicol and immobilized in membrane one, for anti-chloramphenicol biotin-avidin-peroxidase conjugated free in solution. Results: The immunosensor was optimized by comparing the two used different "competitive" working formats on the basis of respective K aff values, that were found to be about 10(5) and 10(4) (mol L-1)(-1). The developed immune device displayed good selectivity for Chloramphenicol and LOD (limit of detection) was of the order of 10(-9) mol L-1. The immunosensor was also used to test the presence of Chloramphenicol in real matrices such as cow milk, river wastewater and pharmaceutical formulations; recovery tests, using the standard addition method, gave satisfactory results. Conclusion: The results proved the validity of this immune device based on the competition between chloramphenicol and conjugated chloramphenicol obtained using biotin-avidin-peroxidase format, by which it is possible to carry out the analysis of chloramphenicol in milk and in river waste-waters with a % RSD <= 5 and with recovery values between 96% and 103%.