When using screen printed electrodes (SPE), the sensor results very cheap, thus even disposable, which constitutes an advantage in order to overcome the problem of the lifetime of enzymes when fixed on conducting substrates.Different enzymes were used for the construction of amperometric biosensors for glycerol determination, including glycerol dehydrogenase (GDH) [8�C12], glycerol kinase/glycerol-3-phosphate oxidase [10,12�C14], pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase [15�C17], glycerol kinase/creatine kinase/creatinase/sarcosine oxidase/peroxidase [18], glycerol kinase/pyruvate kinase/pyruvate oxidase [19] and glycerol oxidase [20].

Among the others, GDH is commercially available and, in the presence of Nicotinamide Adenine Dinucleotide (NAD+), leads to the formation of the redox active NADH cofactor, according with the reaction:glycerol+NAD+��dihydroxyacetone+NADH+H+(1)Due to the high overpotential affecting the oxidation of the NADH and the severe passivation of the electrode surface, redox mediators are generally added to the electrochemical system in order to catalyse the oxidation of NADH [21]:NADH+Mox��NAD++Mred(2)The analytical data finally consist of the current values registered when the reduced form of the redox mediator produced by the enzymatic reaction is newly oxidised at the electrode surface. Reaction (2) can be in turn catalysed by diaphorase (DP) that can be also added to the catalytic system; in this case, it is also anchored at the electrode surface [10,12].

Amperometric biosensors consisting of GDH/DP bi-enzymatic system have been already reported in the literature for the quantification of glycerol in wines [12,22]. Since this analyte is massively produced during alcoholic fermentation, its concentration is particularly high in this matrix (from 4 to 20 g/L [12]) and high dilutions of the sample are necessary; thus, matrix effects are not particularly meaningful and the analysis can be finally carried out through an external calibration registered in a simple buffered solution [12].To the best of our knowledge, no attempts have been made to determine the concentration of glycerol in grapes by means of amperometric biosensors. In this matrix the concentration of this chemical species is significantly lower (generally <1 g/L) with respect to wine samples, thus requiring much lower dilution factors.

Moreover, the chemical composition of the sample is very different from the winery product at the end of alcoholic fermentation process.In this GSK-3 paper we report the development of a fully automated instrument for the determination of glycerol in grapes. The analysis of samples coming from the same vine but containing increasing amount of grapes affected by Botrytis allowed us to verify that this analyte can be considered a good benchmark of the sanitary quality of the grapes.