Characterization of an amperometric laccase electrode covalently immobilized on platinum surface

Laccase from DeniLite™ was covalently assembled on a silane-modified platinum surface and the electrode is characterized with respect to response time, sensitivity, linear range, detection limit, pH dependence, and long-term stability. 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), diammonium salt (ABTS), p-phenylenediamine (PPD), and p-aminophenol (PAP) were selected as the test substrates and detected based on the principle of enzymatic oxidation of substrate and following electrochemical regeneration. The sensitivities of the electrode are 75, 330, and 385 nA/μM with linear ranges of 0.6–15, 0.14–29, and 0.12–22 μM for ABTS, PPD, and PAP, respectively. The detection limits (S/N=3) are 45 and 40 nM for PPD and PAP, respectively, with around 5 nA background noise. The response time (t90%) is less than 2 s for PPD and PAP. The long-term stability of the electrode is over 2 months (retaining 80% of initial activity). The immobilized laccase shows high affinity to PPD and PAP with Km,app values of 55 and 85 μM, respectively. The pH, temperature, and inhibition effects on the activity of the immobilized laccase show that characteristics of the immobilized enzyme are similar to those of the free one. The very stable and fast response, and the remarkable long-term stability of the sensor are the principal advantages over those of reported.