Highly selective impedimetric determination of Haemophilus influenzae protein D using maze-like boron-doped carbon nanowall electrodes

Abstract

This study reports a novel impedimetric immunosensor for protein D detection in purified and bacterial (Haemophilus influenzae, Hi) samples. The detection was based on antigen recognition by anti-protein D antibodies (apD) immobilised at the maze-like boron-doped carbon nanowall electrodes (B:CNW). The B:CNW electrodes were synthesised, and their surface was characterised by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) methods. The sensor was prepared in a two-step procedure: apD were covalently linked on the previously modified B:CNW electrodes using diazonium salt. Modification steps were controlled by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements. The immunosensor exhibited excellent electrochemical performance, stability, satisfactory sensitivities, and linear ranges for antigen detection. Protein D was detected down to 2.39 × 10²fg/mL with a linear range extending from 3.37 × 10^-11to 3.37 × 10^-3μg/mL (in purified sample). Next, Hi's LOD was 5.20 × 10²CFU/mL with a linear range of 8.39 × 10¹-8.39 × 10³CFU/mL. Selectivity studies showed no reaction with negative samples as Streptococcus pyogenes, Streptococcus pneumoniae or Bordetella parapertussis bacteria. Therefore, the new approach is suitable for rapid and quantitative detection of Hi, and is a good candidate for further tests on clinical samples.

Keywords: Carbon nanowalls; Electrochemical impedance; Haemophilus influenzae; Pathogen detection; Protein D.