Electrochemical and morphological layer-by-layer characterization of electrode interfaces during a label-free impedimetric immunosensor build-up: The case of ochratoxin A

In this paper, we provide an in-depth electrochemical characterization of a label-free impedimetric immunosensor for rapid detection of ochratoxin A. The sensor was based on a carbodiimide-mediated amide coupling reaction to immobilize a specific ochratoxin A antibody onto 4-mercaptobenzoic acid-modified commercial screen-printed gold electrode. Different variables affecting the performance of the developed sensor were optimized. Cyclic voltammetry and electrochemical impedance spectroscopy were used to analyse modifications of the interfacial properties occurring at each step of the biosensor assembly. The free electrode surface area, the diffusion coefficient, the peak-to-peak separation, the heterogeneous electron transfer constant, and charge transfer resistance have been calculated and compared. The decrease of charge transfer resistance values was linearly proportional to the ochratoxin A concentration in the range of 0.37- 2.86 ng/mL, with a detection limit of 0.19 ng/mL, a limit of quantification of 0.40 ng/mL, very good selectivity, reproducibility, and storage stability in the absence of antifouling agents. Surface morphology and topographic data at each step of the immunosensor assembly were studied by Atomic Force Microscopy, which also provided information on the specific binding of ochratoxin A. Finally, contact angle measurements revealed the hydrophilicity evolution of the surface during sensor assembly enabling OTA binding.