The successive steps of laccase immobilization on chemically modified Au electrodes were monitored using ATR-SEIRAS and in situ STM. Successful covalent immobilization of the enzyme on Au electrodes modified by a mixed aminophenyl-mercaptohexanol adlayer and on Au electrodes modified by a 4-aminothiophenyl SAM via a Schiff base reaction followed by the formation of amide bonds is revealed by the emergence of the corresponding bands in the ATR-SEIRA spectra, and an enzyme coverage on aminophenyl-mercaptohexanol-modified Au electrodes of about (7.27 ± 1.93) × 1011 laccase units per cm2 was calculated from STM images. The small differences between the ATR-SEIRA spectra of the enzyme immobilized on aminophenyl-mercaptohexanol-modified Au electrodes and the ATR-SEIRA spectra of the enzyme immobilized on 4-aminothiophenyl-modified Au electrodes are attributed to a different orientation of the immobilized enzyme due to the presence on the surface of aminophenyl-mercaptohexanol-modified Au electrodes of OH functional groups that favor an orientation of laccase with the Cu T1 center of the enzyme facing the electrode surface, thus, allowing a high activity for direct electrocatalysis of the ORR at low overpotentials.
Vaz-Dominguez, C., Pita, M., de Lacey, A. L., Shleev, S., & Cuesta, A. (2012). Combined ATR-SEIRAS and EC-STM study of the immobilization of laccase on chemically modified Au electrodes. The Journal of Physical Chemistry C, 116(31), 16532-16540. https://doi.org/10.1021/jp303818p