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. 2014 Jun 23;14(6):11097-109.
doi: 10.3390/s140611097.

A doped polyaniline modified electrode amperometric biosensor for gluconic acid determination in grapes

Donatella Albanese  1 , Francesca Malvano  2 , Adriana Sannini  3 , Roberto Pilloton  4 , Marisa Di Matteo  5
Affiliations

A doped polyaniline modified electrode amperometric biosensor for gluconic acid determination in grapes

Donatella Albanese et al. Sensors (Basel). .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Enzymatic reactions involved for gluconic acid chronoamperometric determination by GK and 6PGDH.
Figure 2.
Figure 2.
Electrochemical synthesis of PANI-PAAMPSA on SPCE surface.
Figure 3.
Figure 3.
CV of PANI-PAAMPSA/SPCE in PBS, pH 7 without NADPH (a) and with 0.4 mM NADPH (b).
Figure 4.
Figure 4.
Characterization of NADPH oxidation in PBS pH 7. Calibration curves of PANI-PAAMPSA modified electrodes after electropolymerization with 10 cycles, 20 cycles, 5 cycles.
Figure 5.
Figure 5.
Effect of ATP/NADP+ ratio on the amperometric response obtained for 0.1 mM gluconic acid solution.
Figure 6.
Figure 6.
Effect of ATP/NADP+ at different concentrations (mM), for their concentration ratio of 1.2 on the amperometric response obtained injecting 0.1 mM gluconic acid solutions into the FIA system.
Figure 7.
Figure 7.
Calibration curve of GK-6PGDH/PEDGE/PANI-PAAMPSA/SPCE biosensor in optimized condition. Insert: amperometric biosensor response obtained injecting gluconic acid solutions into the FIA system.
Figure 8.
Figure 8.
Operational stability of gluconic acid biosensors during 6 h working. Amperometric response to gluconic acid 0.1 mM (70 injections) in PBS, applied potential of 0.1 V vs. Ag/AgCl.
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References

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