Abstract:
The quantification of L-malate in grapes and wine is an essential step in the winemaking process. In grapes, the amount of L-malate is correlated with ripeness, an indicator of the best harvest time. In wine production, L-malate is monitored throughout malolactic fermentation. The industry-standard method of L-malate determination is based on an enzymatic assay that requires the single use of enzymes for each sample. Several biosensors have been developed based on the immobilisation of one or more enzymes to determine L-malate concentrations, all of them allowing the re-use of the enzymes for multiple samples. In this work, the first bienzymatic biosensor, based upon the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) for L-malate determination has been developed.
First, different protocols of EDOT polymerization at glassy carbon electrodes have been tested and used to study the properties of the redox mediator Hexaammineruthenium (III) chloride (HAR(III)). Using cyclic voltammetry, it has been established that the nature of the counterion influences the adsorption of the redox mediator on the electrode surface. Robust counterions allow more significant adsorption phenomena, such as polystyrene sulfonate (PSS), enabled entrapment of the redox mediator into the PEDOT layer. The optimal approach developed involved polymerizing a PEDOT(PSS) layer and then entrapping HAR(III), producing a visible redox signal in HAR(III)-free solutions.
Afterwards, a kinetic study of the enzymes involved has been carried out, pinpointing an ideal ratio of 2 between diaphorase and L-malate dehydrogenase. The amperometric response using enzyme-bound electrodes yielded a significant calibration curve in the range of 0 to 3.0 mM L-malate, modelled with a non-linear equation derived from Michaelis-Menten kinetics. By developing novel statistical tools to assess and compare the goodness of fit of a calibration curve, the best conditions for the amperometric technique were obtained. These involved applying a potential of 0.02 V (Ag/AgCl) in a media enriched with 1 mM nicotinamide adenine dinucleotide (NAD) and 1 mM HAR(III). The inclusion of the conductive polymer within the biosensor construction, along with enzyme immobilization, yielded a biosensor with a more extensive dynamic range and current values than the system without enzyme immobilisation and PEDOT-modified electrodes.
Finally, the biosensors were tested with mock-up samples and compared with the results obtained with a conventional enzymatic kit. No significant differences were found between the two methods. While the biosensor calibration requires more analysis time, it allowed the re-use of enzymes and the testing of multiple samples.