A peroxidase-tetrathiafulvalene biosensor based on self-assembled monolayer modified Au electrodes for the flow-injection determination of hydrogen peroxide

Abstract

The construction and performance under flow-injection conditions of an integrated amperometric biosensor for hydrogen peroxide is reported. The design of the bioelectrode is based on a mercaptopropionic acid (MPA) self-assembled monolayer (SAM) modified gold disk electrode on which horseradish peroxidase (HRP, 24.3 U) was immobilized by cross-linking with glutaraldehyde together with the mediator tetrathiafulvalene (TTF, 1 μmol), which was entrapped in the three-dimensional aggregate formed. The amperometric biosensor allows the obtention of reproducible flow injection amperometric responses at an applied potential of 0.00 V in 0.05 mol L −1 phosphate buffer, pH 7.0 (flow rate: 1.40 mL min −1, injection volume: 150 μL), with a range of linearity for hydrogen peroxide within the 2.0 × 10 −7–1.0 × 10 −4 mol L −1 concentration range (slope: (2.33 ± 0.02) × 10 −2 A mol −1 L, r = 0.999). A detection limit of 6.9 × 10 −8 mol L −1 was obtained together with a R.S.D. ( n = 50) of 2.7% for a hydrogen peroxide concentration level of 5.0 × 10 −5 mol L −1. The immobilization method showed a good reproducibility with a R.S.D. of 5.3% for five different electrodes. Moreover, the useful lifetime of one single biosensor was estimated in 13 days. The SAM-based biosensor was applied for the determination of hydrogen peroxide in rainwater and in a hair dye. The results obtained were validated by comparison with those obtained with a spectrophotometric reference method. In addition, the recovery of hydrogen peroxide in sterilised milk was tested.