In winemaking
gluconic acid is an important marker for quantitative evaluation of grape
infection by Botrytis cinerea. A screen-printed amperometric bienzymatic sensor for the determination of
gluconic acid based on
gluconate kinase (GK) and 6-phospho-D-gluconate
dehydrogenase (6PGDH) coimmobilized onto
polyaniline/poly (2-acrylamido-2-methyl-1-propanesulfonic
acid; PANI-PAAMPSA) is reported in this study. The conductive
polymer electrodeposed on the working
electrode surface allowed the detection of
NADH at low potential (0.1 V) with a linear range from 4 × 10(-3) to 1 mM (R2 = 0.99) and a sensitivity of 419.44 nA∙mM(-1). The bienzymatic sensor has been optimized with regard to GK/6PGDH enzymatic unit ratio and
ATP/NADP+ molar ratio which resulted equal to 0.33 and 1.2, respectively. Under these conditions a sensitivity of 255.2 nA∙mM(-1), a limit of detection of 5 μM and a Relative Standard Deviation (RSD) of 4.2% (n = 5) have been observed. Finally, the biosensor has been applied for
gluconic acid measurements in must grape samples and the matrix effect has been taken into consideration. The results have been compared with those obtained on the same samples with a commercial kit based on a spectrophotometric
enzyme assay and were in good agreement, showing the capability of the bienzymatic PANI-PAAMPSA biosensor for
gluconic acid measurements and thus for the evaluation of Botrytis cinerea
infection in grapes.