Kai Yan^a,1, Ponnusamy Nandhakumar^a,1, Aman Bhatia^a, Nam-Sihk Lee^b, Young Ho Yoon^b, Haesik Yang^a,*

^aDepartment of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan, 46241, South Korea
^bEONE Laboratories, Incheon, 22014, South Korea

¹These two authors are contributed equally to this work.

^*Corresponding author.

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.

Keywords : Electrochemical immunoassay; Choline oxidase; Horseradish peroxidase; Enzymatic cascade; Parathyroid hormone detection