Use of Hydrophobin Roda Protein for Modification of Gold Electrodes as Part of Glucose Biosensor

Abstract

Hydrophobins are proteins, consisting of approximately 70–130 amino acids and containing eight cysteines, linked by four disulfide bonds, which are characteristic of the entire hydrophobin family. The main advantage of hydrophobins is their ability to form amphiphatic layers on surfaces and thus to change their properties from hydrophilic to hydrophobic and vice versa. It is for this reason that hydrophobins can be widely used in a variety of applications to improve the properties of materials, such as hydrophilicity, activity and stability of immobilized molecules. In this work, the hydrophobin RodA of Aspergillus fumigatus and its properties were investigated. The gene responsible for the synthesis of the RodA protein was identified by molecular biology methods and used to design an expression system. The purified recombinant RodA protein was used to modify the surface of a gold electrode in order to investigate the effect of this hydrophobin as a matrix on the performance of the engineered glucose biosensor. The engineered biosensor with the RodA matrix was compared with a biosensor without the RodA matrix. The data obtained were fitted to Michaelis–Menten and linear models to calculate the KM and the maximum current generated (Imax). In the case of Au/GOx, the KM value was 6.99 mM and the Imax was 34.8 μA·cm−2; in the case of the Au/RodA/GOx biosensor, the KM value was 2.37 mM and the Imax was 0.432 μA·cm−2. The lower Imax value for the Au/RodA/GOx biosensor could be explained by the possible formation of an excessively thick monolayer of RodA protein or by possible conformations of the protein that blocked the glucose oxidase molecules. However, the KM value obtained for Au/RodA/GOx showed that for this biosensor, the immobilized glucose oxidase has a significantly higher affinity for the substrate, indicating that such a protein may be suitable for electrode modifications.

Keywords

• hydrophobins
• RodA
• biosensors
• surface modification