Abstract
Immobilization of enzymes on (nano)porous metal carriers provides the foundation for an advanced design of bioelectrodes suitable for catalysis and sensing. However, interactions upon adsorption are still poorly understood, and so the efficient coupling of the enzymes to the electrode surface remains one of the major challenges. Here, we present a comprehensive study of the immobilization behavior of Aerococcus viridans l-lactate oxidase (LOx) on nanoporous gold (npAu) in dependence of electrode modification with differently charged self-assembled monolayers (SAMs). The highest activity (up to 14 U/g) and electrocatalytic response (sensitivity of 3.9 μA mM(-1)) were observed for a sulfonate-terminated SAM. This is contrary to enzyme behavior on conventional polymer carriers, and thus, the effect is specific to the metal electrodes. We propose the capture of the negatively charged LOx in a dense counterion layer in close proximity to the strongly negatively charged gold surface. Adsorption on positively charged amine-terminated SAMs resulted in a similar immobilization yield but gave much lower activity (4-fold). Importantly, the effect of the sulfonate SAM was strongly dependent on the npAu electrode pore size: the highest LOx activity (in U/cm(2)) was found with pores (diameter of ∼170 nm) supposedly large enough to facilitate enzyme diffusion into the porous structure during immobilization. Electrochemical sensing of H(2)O(2) produced by the LOx reaction showed a 2.5-fold higher sensitivity for l-lactate on the negatively charged surface. Lixiviation studies supported the proposed layer capture and revealed a faster decline in the electrode activity with sulfonate surface modification. Collectively, the present study reveals enhanced activity of LOx on sulfonate-charged gold surfaces and a strong pore size dependence. These findings deepen the understanding of the immobilization behavior of LOx on charged nanoporous metals and have importance for the advanced design of enzyme electrodes.