Surface property effects in the immobilization of L-lactate oxidase on gold electrodes by self-assembled monolayers

The performance of enzyme electrodes depends critically on the interactions between the enzyme and the electrode surface. In this study, L-lactate oxidase with an N-terminal His-tag (His-LOx) was immobilized on planar gold electrode modified with self-assembled monolayers (SAMs) engineered to provide distinct surface characteristics: hydrophilic, hydrophobic, or Ni²⁺-chelating for His-tag affinity binding. Electrochemical measurements revealed that the hydrophilic 1-thioglycerol (TG) SAM supported the highest initial activity (10.5 µA/cm²), whereas the hydrophobic 1-octanethiol led to a substantially lowered activity. Ni²⁺-complexed nitrilotriacetic acid (Ni-NTA) SAM markedly enhanced the operational stability of His-LOx, retaining over 65% of activity after 20 h compared to less than 20% for TG. A mixed SAM combining TG and Ni-NTA enabled the simultaneous achievement of high activity and improved stability, demonstrating a synergistic effect of hydrophilic and affinity-based interactions. These results provide mechanistic insight into how SAM surface properties and enzyme-surface interactions govern both activity and stability of LOx, offering practical design principles for the development of robust and sensitive LOx electrodes for biosensing applications.