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 Kaff values, that were found to be about 105 and 104 (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%.