Electrochemical immunoassay based on choline oxidase-peroxidase enzymatic cascade

Horseradish peroxidase (HRP)-based electrochemical immunoassays are considered promising techniques for point-of-care clinical diagnostics, but the necessary addition of unstable H₂O₂ in the enzymatic system may hinder their practical application. Although glucose oxidase (GOx) has been widely explored for in situ generation of H₂O₂ in HRP-based immunoassay, the GOx-catalyzed reduction of oxidized peroxidase substrate may limit the immunosensing performance. Here, we report a sensitive electrochemical immunosensor based on a choline oxidase (ChOx)-HRP cascade reaction. In this design, ChOx catalyzes the oxidation of choline, during which H₂O₂ is generated in situ and thus oxidizes acetaminophen (APAP) in the presence of HRP. The electrochemical behavior of APAP in the ChOx-HRP cascade was compared with that of the commonly used GOx-HRP cascade, which confirmed that ChOx could be a superior preceding enzyme for sensitive immunoassay based on the bienzymatic cascade. The developed ChOx-HRP cascade was also further explored for a sandwich-type electrochemical immunoassay of parathyroid hormone in artificial and clinical serum. The calculated detection limit was ~3 pg/mL, indicating that the ChOx-HRP cascade is especially promising for highly sensitive electrochemical immunoassays when APAP is used as the peroxidase substrate.