The way of immobilization of the monoclonal antibody (type IgG) on the electrode surface has a significant effect on the amount of the immobilized protein and in consequence on current signal of protein. Herein, we demonstrate that the application of appropriately functionalized phenyl film allowed us to control the orientation of the antibody (Ab) molecules on the electrode surface. The influence of Ab orientation on the efficiency of antigen-antibody interaction was tested with an example blood plasma protein (ferritin; Ft). To control the orientation of Ab molecules the phenyl films containing -COOH or -NH2 groups were applied. Contrary to aminoethylophenyl layer, the carboxyphenyl film guaranteed the shortest distance between the redox center of the protein and the electrode surface. Additionally, the application of an external magnetic field together with magnetic nanoparticles allowed achieving the best orientation to observe well-defined ferritin current signals. The proposed method of ferritin detection can be successfully used in the concentration range of Ft between 0.1 and 30 µg dL-1. The detection limit for a carboxyphenyl film was estimated as 0.40 ± 0.04 and 0.13 ± 0.04 µg dL-1 for impedance and voltammetric measurements, respectively. In turn, for an aminoethylophenyl film the detection limit was 0.03 ± 0.002 (electrochemical impedance spectroscopy; EIS) and 0.02 ± 0.002 µg dL-1 (differential pulse voltammetry, DPV). The interday precision (reproducibility) was calculated (4.10 ÷ 9.10% RSD) together with the intraday precision / repeatability (3.20 ÷ 8.0% RSD) for the studied samples. The functionality of the sensor has been tested on rat blood samples. Based on the performed investigations it can be stated that the developed sensor was characterized by high selectivity and good sensitivity.
Keywords: Diazonium salt; Ferritin; Immunosensor; Magnetic field; Voltammetric detection.
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