New bioanalytical platform based on the use of avidin for the successful exfoliation of multi-walled carbon nanotubes and the robust anchoring of biomolecules. Application for hydrogen peroxide biosensing

Abstract

We are reporting an innovative building-block for the development of biosensors based on the non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with avidin (MWCNTs-avidin). In this work, at variance with previous reports, avidin has the double role of simultaneously being the exfoliating agent of MWCNTs and the platform for anchoring different biotinylated biomolecules. The optimum dispersion was obtained by sonicating for 5.0 min 0.50 mgmL^-1 MWCNTs with 1.00 mgmL^-1 avidin solution prepared in 50:50 v/v ethanol/water. As proof-of-concept, we immobilized biotinylated horseradish peroxidase (b-HRP) at glassy carbon electrodes (GCE) modified with MWCNTs-avidin to develop a hydrogen peroxide biosensor using hydroquinone as redox mediator. Surface plasmon resonance, electrochemical impedance spectroscopy, cyclic voltammetry and amperometry demonstrated that, even after the partial denaturation of avidin due to the drastic conditions used to functionalize the MWCNTs, it preserves the biorecognition properties and efficiently interacts with biotinylated horseradish peroxidase (b-HRP). The analytical characteristics of the resulting hydrogen peroxide biosensor are the following: linear range between 1.0 × 10^-6 M and 1.4 × 10^-5 M, sensitivity of (1.37 ± 0.04) x 10⁵ μAM^-1, detection limit of 24 nM and reproducibility of 2.9%. The sensor was challenged with different samples, a mouthwash solution, human blood serum and milk, with very good performance.

Keywords: Avidin; Carbon nanotubes functionalization; Horseradish peroxidase; Hydrogen peroxide biosensor; Multi-walled carbon nanotubes.