Amperometric bienzymatic biosensor for l-lactate analysis in wine and beer samples

Abstract

A novel amperometric bienzymatic biosensor has been developed based on the incorporation of Lactate Oxidase (LOx) and Horseradish Peroxidase (HRP) into a carbon nanotube/polysulfone membrane by the phase inversion technique onto screen-printed electrodes (SPEs). In order to improve the sensitivity and reduce the working potential, experimental conditions have been optimized and ferrocene has also been incorporated into the membrane as a redox mediator of the enzymatic reactions, which allows the reduction of H₂O₂ at −100 mV. Measurements were carried out in phosphate buffer solution at pH 7.5 and under batch conditions. The biosensor response time to L-lactate was only 20 s and showed a good reproducibility (RSD 2.7%). Moreover, the detection limit was 0.05 mg L⁻¹ of L-lactate with a linear interval range from 0.1 mg L⁻¹ to 5 mg L⁻¹. Finally, the biosensor has been applied to the determination of L-lactic acid in different wine and beer samples. Then, the results obtained with the biosensor were compared with the ones obtained using, as a reference method, a commercial kit based on spectrophotometric measurements, obtaining an excellent agreement between the results, validating our approach.