Novak Lara Marie, Hengge Elisabeth, Steyskal Eva-Maria, Würschum Roland, Nidetzky Bernd
Institute of Material Physics, Graz University of Technology, NAWI Graz, Petersgasse 16, 8010 Graz, Austria.
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria.
Langmuir. 2025 Mar 4;41(8):5136-5146. doi: 10.1021/acs.langmuir.4c04367. Epub 2025 Feb 20.
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 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) 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) was found with pores (diameter of ∼170 nm) supposedly large enough to facilitate enzyme diffusion into the porous structure during immobilization. Electrochemical sensing of HO 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.
将酶固定在（纳米）多孔金属载体上为适用于催化和传感的生物电极的先进设计奠定了基础。然而，吸附过程中的相互作用仍知之甚少，因此酶与电极表面的有效偶联仍然是主要挑战之一。在此，我们针对不同电荷的自组装单分子层（SAMs）对电极进行修饰，全面研究了L-乳酸氧化酶（LOx）在纳米多孔金（npAu）上的固定行为。对于磺酸酯封端的SAM，观察到最高活性（高达14 U/g）和电催化响应（灵敏度为3.9 μA mM）。这与酶在传统聚合物载体上的行为相反，因此，这种效应是金属电极所特有的。我们提出，带负电荷的LOx被捕获在紧邻强负电荷金表面的致密反离子层中。在带正电荷的胺封端的SAM上吸附导致类似的固定产率，但活性要低得多（低4倍）。重要的是，磺酸酯SAM的效应强烈依赖于npAu电极的孔径：在孔径（直径约170 nm）据推测足够大以促进固定过程中酶扩散到多孔结构中的情况下，发现了最高的LOx活性（以U/cm计）。对LOx反应产生的H₂O₂进行电化学传感显示，在带负电荷的表面上对L-乳酸的灵敏度高2.5倍。浸出研究支持了所提出的层捕获，并揭示了磺酸酯表面修饰后电极活性下降更快。总体而言，本研究揭示了LOx在带磺酸酯电荷的金表面上活性增强以及对孔径的强烈依赖性。这些发现加深了对LOx在带电纳米多孔金属上固定行为的理解，对酶电极的先进设计具有重要意义。
