Laccase biosensor based on N-succinimidyl-3-thiopropionate-functionalized gold electrodes

A laccase biosensor, in which the enzyme was immobilized oil N-succinimidyl-3-thiopropionate (NSTP)-modified gold electrodes, is reported. Two different approaches for the preparation of N-Succinimidyl-terminated monolayers were evaluated: a) activation of a preformed 3-mercaptopropionic acid (MPA) SAM by reaction with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydrosulfosuccinimide (NHS); b) assembling of dithiobisuccinimidyl propionate (DTSP). NSTP-modified electrodes were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Biosensors prepared by covalent binding of the enzyme and by cross-linking with glutaraldehyde atop NSTP-modified electrodes were compared in terms of sensitivity and operational range for caffeic acid. A much better analytical performance was found using the latter approach. Variables affecting the amperometric detection (enzyme loading pH and applied potential) were optimized. The operational stability and characteristics of functioning of the laccase biosensor in terms of repeatability of the amperometric measurements, reproducibility with different biosensors and useful lifetime, were evaluated. The kinetic parameters of the enzyme reactions and the analytical characteristics of the corresponding calibration plots were calculated for eight phenolic compounds. Limits of detection of 0.07 mu M, 0.05 mu M and 0.09 mu M were obtained for caffeic acid, catechol and 3,4-dihydroxyphenylacetic acid (DOPAC), respectively. The practical usefulness of the developed biosensor was evaluated by estimating the "Pool" Of Phenolic compounds ill olive oil mill wastewaters (OMW).