Carbon nanotubes functionalized by click chemistry as scaffolds for the preparation of electrochemical immunosensors. Application to the determination of TGF-beta 1 cytokine

Abstract

An electrochemical immunosensor for the determination of the multifunctional Transforming Growth Factor β1 (TGF-β1) cytokine has been prepared using multi-walled carbon nanotube (MWCNT)-modified screen-printed carbon electrodes. MWCNTs were functionalized by means of copper(I) catalyzed azide–alkyne cycloaddition (“click” chemistry) as an efficient strategy for the covalent immobilization of immunoreagents without altering their configurations and preserving their biological activity. Alkyne-functionalized IgGs were also prepared and used to assemble IgG-alkyne–azide–MWCNT conjugates used as scaffolds for the immunosensor preparation. After a blocking step with casein, anti-TGF was immobilized and the target cytokine was sandwiched with biotinylated anti-TGF labeled with poly-HRP-labeled streptavidin. The affinity reaction was monitored amperometrically at −0.20 V using the hydroquinone (HQ)/H₂O₂ system. The calibration plot for TGF-β1 exhibited a range of linearity (r² = 0.995) extending between 5 and 200 pg mL⁻¹ which is suitable for the determination of the target cytokine in human serum. A limit of detection of 1.3 pg mL⁻¹ was achieved. The analytical performance of the immunosensor can be advantageously compared with that claimed for ELISA kits. The immunosensor was applied to the analysis of spiked human serum samples at different concentration levels with excellent recoveries.