A rapid method for determination of l-lactic acid in real samples by amperometric biosensor utilizing nanocomposite

Amperometric biosensors based on gold planar electrode and on two types of nanocomposite electrodes consisting of multi-walled carbon nanotubes for determination of l-lactic acid designed for wine-makers were developed and used for an analysis of real food samples and beverages. The biosensors were constructed by immobilization of l-lactate oxidase and peroxidase within chitosan layers on the surface of the electrodes. As a mediator, ferrocyanide was used. The current resulted from re-oxidation of produced ferricyanide was measured at working potential of −50 mV against Ag/AgCl reference electrode. The biosensor based on gold electrode showed linearity over the range 5-244 μM with the detection limit of 0.96 μM. Calibration curves for biosensors utilizing two types of nanocomposites consisting of N-eikosane and multi-waled carbon nanotubes (d = 60-100 nm, L = 5-15 μm, 95+% purity or d = 9.5 nm, L = 1.5 μm, 90+% purity) were obtained both with the linear range of 5-340 μM. The detection limits were 1.62 and 1.66 μM, respectively. All types of biosensors had response time of 65s. The biosensors showed satisfactory operational stability (no loss of sensitivity after 60 consecutive measurements) and an excellent storage stability (90% of the initial sensitivity after 15 months of storage at room temperature). Finally the results obtained from measurements of food and wine samples were compared with those obtained with an enzymatic-spectrophotometric and standard HPLC method. The biosensors based on 3 different configurations were able to determine low concentration (0.02-0.12 g L−1) of lactic acid level, what was not possible by reference analytical methods and a comparison between results obtained by the biosensors and reference methods agreed very well at concentration of lactic acid above 1.4 g L−1.

► The lactate biosensor based on gold and nanocomposite electrodes was developed. ► Enzymes were immobilized between chitosan “sandwich”. ► Biosensor has improved storage stability compared to accepted commercial ones. ► The results of measurements of real samples correlated with reference methods. ► Nanocomposite electrodes are capable to replace the gold electrode surface.