Preparation, characterization and application of alkanethiol self-assembled monolayers modified with tetrathiafulvalene and glucose oxidase at a gold disk electrode

Abstract

A critical study of the different variables affecting the preparation of enzyme electrochemical biosensors using self-assembled monolayer (SAM)-modified electrodes is reported. Regarding variables affecting the obtention of SAMs on a gold disk electrode (AuE), the type of alkanethiol employed, its concentration, the time and temperature of formation and the composition of the medium were evaluated. Concerning a glucose biosensor based on SAM-modified AuEs, the performances of several redox mediators for the enzymatic oxidation of glucose were compared. Two glucose oxidase (GOx) immobilization methods on the SAM-modified AuE were tested: a covalent binding using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide (NHSS), and a cross-linking with glutaraldehyde. The cross-linking method produced slopes of the substrate calibration graphs at a mercaptopropionic acid (MPA)-modified electrode two orders of magnitude higher than those obtained with the covalent binding. Moreover, the mediator tetrathiafulvalene (TTF) was co-immobilized atop the SAM together with GOx. A heterogeneous electron transfer constant between TTF and the MPA–GOx bioelectrode of 1.25 s −1 was calculated. The enzyme loading, the amount of TTF on the electrode, the applied potential and the pH were also optimized. A good repeatability of the measurements with the TTF–GOx–MPA–AuE biosensor was demonstrated, with no need of pretreatment of the modified electrode. No significant changes in the slope value for the glucose calibration graph were found after 5 days when working with the same biosensor. An apparent Michaelis–Menten constant of (13.9±0.5) mM, and a limit of detection for glucose of 3.5×10 −6 M were obtained. Moreover, the TTF–GOx–MPA–AuE also performed well in the flow-injection mode and in the analysis of glucose in real samples.