The Reliable Detection of Homocysteine Using a Biosensor Based on Recombinant Cystathionine β-Synthase and Nanoporous Gold

Given the essential roles of homocysteine (Hcy) and the interference of cysteine in effectively monitoring human health, this study proposed a synergistic effect strategy that combines the unique structural and functional properties of nanoporous gold (NPG) with the selective recognition capability of a recombinant cystathionine β-synthase (CBS) for the sensitive and specific detection of Hcy. The CBS protein with specific catalytic activity for Hcy was successfully produced in recombinant <i>Escherichia coli</i> BL21 (pET-30a-<i>cbs</i>) using the <i>cbs</i> gene from <i>Pseudomonas aeruginosa</i> PAO1. The electrochemical mechanism demonstrated that the electrooxidation of H₂S, a catalytic product of the CBS, was an irreversibly surface-controlled process on the CBS/NPG/GCE electrode surface. The electrochemical detection of Hcy exhibited excellent linearity, with a high sensitivity reaching 10.43 µA mM^-1 cm^-2 and a low detection limit of 1.31 µM. Furthermore, the CBS/NPG/GCE biosensor was successfully used to detect Hcy in urine samples with strong anti-interference capability and high selectivity (relative standard deviation less than 2.81%), while effectively reducing the interference from cysteine. These results confirmed that the proposed CBS/NPG/GCE electrochemical sensor achieved specific, sensitive, and reliable rapid detection of homocysteine, making it highly promising for practical applications in clinical treatment and health assessment.