Biomimetic co-immobilization of β-glucosidase, glucose oxidase, and horseradish peroxidase to construct a multi-enzyme biosensor for determination of amygdalin

Shuo Liu; Hongli Chao; Dajun He; Yi Wang; Yi Yang

doi:10.1016/j.ijbiomac.2025.139868

Biomimetic co-immobilization of β-glucosidase, glucose oxidase, and horseradish peroxidase to construct a multi-enzyme biosensor for determination of amygdalin

Int J Biol Macromol. 2025 Jan 13:139868. doi: 10.1016/j.ijbiomac.2025.139868. Online ahead of print.

Authors

Shuo Liu¹, Hongli Chao¹, Dajun He², Yi Wang³, Yi Yang⁴

Affiliations

¹ School of Light Industry and Chemical Engineering, Dalian Polytechnic University, Dalian 116034, China.
² Analysis and Testing Centre, Shihezi University, Shihezi 832003, China.
³ School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, China.
⁴ School of Light Industry and Chemical Engineering, Dalian Polytechnic University, Dalian 116034, China. Electronic address: yangyi105@mails.ucas.ac.cn.

PMID: 39814275
DOI: 10.1016/j.ijbiomac.2025.139868

Abstract

Accurate, specific, and cost-effective detection of toxic cyanogenic glycosides is crucial for ensuring biological health and food safety. In this study, a novel biosensor based on co-immobilized multi-enzyme system was constructed by artificial antibody-antigen-directed immobilization for the colorimetric detection of amygdalin through a cascade reaction catalyzed by β-glucosidase, glucose oxidase, and horseradish peroxidase. Artificial antibodies and antigens were prepared using catechol and 3,4-dihydroxybenzaldehyde, respectively, to generate mutual affinity recognition ability for enzyme immobilization. On this basis, the biosensing system showed a complete response to amygdalin within 4 min, with a linear range from 2 to 10 μM, a detection limit of 0.18 μM, and a quantification limit of 0.6 μM. In addition, this sensor had good precision, reproducibility, stability, and reusability. This study proposed a method for detecting cyanogenic glycosides, providing a successful case for the application of cascade biosensors in food safety detection.

Keywords: Artificial antigen-antibody; Immobilized multi-enzyme; Optical biosensor.