Assessing Surface Coverage of Aminophenyl Bonding Sites on Diazotised Glassy Carbon Electrodes for Optimised Electrochemical Biosensor Performance

Abstract

Electrochemical biosensors using carbon-based electrodes are being widely developed for the detection of a range of different diseases. Since their sensitivity depends on the surface coverage of bioreceptor moieties, it necessarily depends on the surface coverage of amine precursors. Electrochemical techniques, using ferrocene carboxylic acid as a rapid and cheap assay, were used to assess the surface coverage of amino-phenyl groups attached to the carbon electrode. While the number of electrons transferred in the first step of diazotisation indicated a surface coverage of 8.02 ± 0.2 × l0−10 (mol/cm2), and those transferred in the second step, a reduction of nitrophenyl to amino-phenyl, indicated an amine surface coverage of 4–5 × l0−10 (mol/cm2), the number of electrons transferred during attachment of the amine coupling assay compound, ferrocene carboxylic acid, indicated a much lower available amine coverage of only 2.2 × l0−11 (mol/cm2). Furthermore, the available amine coverage was critically dependent upon the number of cyclic voltammetry cycles used in the reduction, and thus the procedures used in this step influenced the sensitivity of any subsequent sensor. Amine coupling of a carboxyl terminated anti-beta amyloid antibody specific to Aβ(1-42) peptide, a potential marker for Alzheimer’s disease, followed the same pattern of coverage as that observed with ferrocene carboxylic acid, and at optimum amine coverage, the sensitivity of the differential pulse voltammetry sensor was in the range 0–200 ng/mL with the slope of 5.07 µA/ng.mL−1 and R2 = 0.98.

Electrochemical Immunosensors Developed for Amyloid-Beta and Tau Proteins, Leading Biomarkers of Alzheimer’s Disease

“…Tehrani et al, 2021, reported another immunosensor for detecting Aβ (1-42) using a diazotized GCE [74]. The surface of the GCE was electrochemically functionalized with an amino group (-NH 2 ) using cyclic voltammetry (CV) in a mixture solution of diazonium salt in an electrolyte of BU 4 NBF 4 and acetonitrile.…”

Antigen-Mimic Nanoparticles in Ultrasensitive on-Chip Integrated Anti-p53 Antibody Quantification

“…14 Typically, these assays utilize electrodeconfined p53 antigens. 15 However, on planar two-dimensional interfaces, the combined effects of moderately low epitope surface density (∼pmol/cm 2 ), 16 efficacy of large target (e.g., antibody) capture. Additionally, in protein-rich real samples (e.g., serum), recruitment against a large excess of background remains challenging.…”

“… 14 Typically, these assays utilize electrode-confined p53 antigens. 15 However, on planar two-dimensional interfaces, the combined effects of moderately low epitope surface density (∼pmol/cm 2 ), 16 restricted target accessibility, and sluggish (planar) target diffusion serve to reduce the efficacy of large target (e.g., antibody) capture. Additionally, in protein-rich real samples (e.g.…”