Lactate biosensing based on covalent immobilization of lactate oxidase onto chevron-like graphene nanoribbons via diazotization-coupling reaction

We have designed and prepared an electrochemical biosensor for lactate determination. Through a diazotation process, the enzyme lactate oxidase (LOx) is anchored onto chevron-like graphene nanoribbons (GNR), previously synthesized by a solution-based chemical route, and used as modifiers of glassy carbon electrodes. In a first step, we have performed the grafting of a 4-carboxyphenyl film, by electrochemical reduction of the corresponding 4-carboxyphenyl diazonium salt, on the GNR-modified electrode surface. In this way, the carboxylic groups are exposed to the solution, enabling the covalent immobilization of the enzyme through the formation of an amide bond between these carboxylic groups and the amine groups of the enzyme. The biosensor design was optimized through the morphological and electrochemical characterization of each construction step by atomic force microscopy, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy.The cyclic voltammetric response of the biosensor in a solution of hydroxymethylferrocene in presence of Llactate evidenced a clear electrocatalytic effect powered by the specific design of the biosensing platform with LOx covalently attached to the GNR layer. From the calibration procedures employed for L-lactate determination, a linear concentration range of 3.4 center dot 10 5- 2.8 center dot 10 4 M and a detection limit of 11 mu M were obtained, with relative errors and relative standard deviations less than 6.0% and 8.4%, respectively. The applicability of the biosensor was tested by determining lactate in apple juices, leading to results that are in good agreement with those obtained with a well-established enzymatic spectrophotometric assay kit.

Automated summary

We have developed an electrochemical biosensor for lactate determination by immobilizing lactate oxidase onto chevron-like graphene nanoribbons and using them as modifiers of glassy carbon electrodes. The biosensor showed a wide linear concentration range and low detection limit, and demonstrated good agreement with a well-established enzymatic spectrophotometric assay kit.