Cytokines are small proteins and form complicated cytokine networks to report the status of our health. Thus, accurate profiling and sensitive quantification of multiple cytokines is essential to have a comprehensive and accurate understanding of the complex physiological and pathological conditions in the body. In this study, we demonstrated a robust electrochemical immunosensor for the simultaneous detection of three cytokines IL-6, IL-1β, and TNF-α. First, graphene oxides (GO) were loaded with redox probes nile blue (NB), methyl blue (MB), and ferrocene (Fc), followed by covalent attachment of anti-cytokine antibodies for IL-6, IL-1β, and TNF-α, respectively, to obtain Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc, acting as the signal reporters. The sensing interface was fabricated by attachment of mixed layers of 4-carboxylic phenyl and 4-aminophenyl phosphorylcholine (PPC) to glassy carbon surfaces. After that, the capture monoclonal antibody for IL-6, IL-1β, and TNF-α was modified to the carboxylic acid terminated sensing interface. And finally a sandwich assay was developed. The quantitative detection of three cytokines was achieved by observing the change in electrochemical signal from signal reporters Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc. The designed system has been successfully used for detection of three cytokines (IL-6, IL-1β, and TNF-α) simultaneously with desirable performance in sensitivity, selectivity, and stability, and recovery of 93.6%-105.5% was achieved for determining cytokines spiked in the whole mouse serum.
Keywords: cytokines; electrochemistry; graphene oxides; immunosensors; multiplex detection; redox probes.