Analytical Capabilities of Fluorescence Polarization Immunoassay and Amperometric Tyrosinase Immunosensors in the Determination of Chloramphenicol

Abstract

A version of competitive fluorescence polarization immunoassay (FPIA) and an amperometric immunosensor with a tyrosinase tracer have been developed for the sensitive and selective determination of the chloramphenicol antibiotic. In the development of FPIA for the determination of chloramphenicol, a green fluorescent complex of terbium(III) was first used as a tracer, which changed emission in the green spectral region with a maximum at 545 nm. The competitive binding of the substance to be determined with a limited number of binding sites of specific antibodies (ABs) in using this tracer ensured the determination of chloramphenicol in the linear range of the calibration dependence of fluorescence polarization on the analyte concentration in the range 1 × 10^–11–1 × 10^–7 M, LOD = 9 × 10^–12 M. To ensure the maximum value of the analytical signal, tracer dilution was selected at 1 : 10 and antibody dilution, at 1 : 25. The optimal incubation time for the tracer–antibody immune complex was 5 min. Tyrosinase enzyme was used as a tracer in the development of an amperometric enzyme-linked immunosorbent assay (ELISA) sensor for the detection of chloramphenicol. The work of this sensor is based on a combination of immunochemical, enzymatic, and electrochemical reactions, leading to “quasi-inhibition” effects. The developed amperometric immunosensor for the determination of chloramphenicol ensured its determination in a narrower concentration range from 1 × 10^–9 to 1 × 10^–6 M, LOD = 8 × 10^–10 M. The conditions of analysis were as follows: AB dilution of 1 : 200 and the duration of an analysis 10 min. The developed versions for the determination of chloramphenicol were tested in analyses of food products (milk). The error of the determination dis not exceed 0.059.