Bienzyme self-assembled monolayer on gold electrode: An amperometric biosensor for carbaryl determination

•Two enzymes covalently immobilized in a self-assembled monolayer nanoenvironment.•Simple, and fast fabrication of the economically biosensor is proposed for pesticide detection.•Two redox mediators behavior were compared by cyclic voltammetry and EIS.•Acetylcholine was used as substrate rather than acetylthiocholine.

A new electrochemical biosensor based on the covalent immobilization of two enzymes on self-assembled monolayer attached to a polycrystalline gold electrode is proposed, experimentally. The acetylcholinesterase (AChE) and cholineoxidase (ChO) are two enzymes that covalently co-immobilized on the mercaptopropionic acid self-assembled monolayer on polycrystalline gold electrode (Au-MPA-AChE/ChO SAM). Fabrication steps and electrochemical interaction of the Au-MPA-AChE/ChO SAM with carbaryl were monitored by general electrochemical methods like cyclic voltammetry (CV) and chronoamperometry (CA), and by a more advanced method, electrochemical impedance spectroscopy (EIS) in the presence of parabenzoquinone (PBQ) or K3[Fe(CN)6] redox probes. The close distances between two immobilized enzymes on Au-MPA SAM result in preconcentration of choline, the product of acetylcholine hydrolysis by AChE, in ChO nano-environment. Enzyme activity was measured by chronoamperometric tracing of hydroquinone from the reduction of PBQ mediator which in turn is oxidized at Au electrode in diffusion layer. Carbaryl was chosen as a model toxin and its inhibition characteristics were utilized for the toxin detection. The linear range for the determination of Carbaryl was 10–1000 nmol l−1. A limit of detection 5.96 nmol l−1 was obtained.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide