Molecular Engineering of Sarcosine Oxidase and Horseradish Peroxidase: Impact of Lysine Accessibility on Covalent Bioconjugation and Bioelectrocatalysis

Sarcosine has emerged as a critical biomarker for the early diagnosis of prostate cancer, necessitating the development of highly sensitive and simplified electrochemical detection methods. While the bioconjugation of enzymes offers a pathway toward enhanced bioelectronic performance, biosensors based on bioconjugated sarcosine oxidase remain largely unexplored. In this study, we used a synthesized redox mediator, N-(ferrocenylmethyl)-6-aminohexanoic acid, featuring an extended spacer arm designed to decrease the tunneling distance between the enzyme cofactor and the electrode surface. Sarcosine oxidase and horseradish peroxidase were subjected to bioconjugation via carbodiimide-mediated coupling. The resulting bioconjugates were characterized by cyclic voltammetry, diffusion-kinetic analysis, and high-resolution mass spectrometry following tryptic and peptic digestion. Furthermore, an in silico structural analysis was employed to correlate lysine solvent-accessible surface area with conjugation efficiency. Based on these bioconjugates, a sarcosine biosensor was constructed and tested using human serum as real samples.