Enhancing electrochemical xanthine detection: A two-step incubation strategy to minimize interference from ascorbic acid

Enzyme-based biosensors with mediated electrochemical detection offer a straightforward and cost-effective approach for detecting xanthine. However, electro-active interfering species such as ascorbic acid (AA) complicate the achievement of sensitive and selective detection in biological fluids. Direct and mediated oxidation of AA elevates electrochemical background levels. While ascorbate oxidase (AOx) is employed to oxidize AA into an electro-inactive product, incomplete removal of AA allows it to reduce the electron mediator, resulting in still considerable background levels. Additionally, excess AOx can oxidize the signaling species, the reduced form of the electron mediator, albeit slowly, leading to decreased signal levels. To address these challenges, a two-step incubation process and the use of appropriate AOx concentration are implemented. Once AA is fully oxidized by AOx, an electron mediator is added to the solution. To enhance the electrochemical signal-to-background ratio, an optimal pairing of a xanthine-oxidizing enzyme and an electron mediator is selected from two xanthine-oxidizing enzymes [xanthine dehydrogenase (XDH) and xanthine oxidase] and three electron mediators [Os(bpy)₂Cl₂⁺, Ru(NH₃)₆³⁺, and Fe(CN)₆³⁻]. The combination of XDH and Os(bpy)₂Cl₂⁺ provides high signal and low background levels. When these conditions are applied to xanthine detection in artificial serum, a detection limit of approximately 500 nM is achieved, making it applicable in various clinical and research fields.