A new amperometric biosensor for fructose determination based on epoxy-graphite-TTF-TCNQ-FDH-biocomposite

In this work a novel amperometric biosensor for fructose determination in solutions was developed. The device was constructed by the incorporation of a tetrathiofulvalene-tetracyanoquinodimethane organic conducting salt and fructose dehydrogenase enzyme, include in a polymeric matrix of epoxy resin and graphite powder. Because of the electrocatalytic function of the salt, the direct transfer of the electron between the reduced prosthetic group (PQQH₂) of the enzyme and the transducing material, was verified at a low working potential (150 mV vs. Ag/AgCl), where the interfering reactions were minimized. The response time at 90% of the steady state value was less than 20 s. The current response was directly proportional to the D-fructose concentration from 0.01 to 0.3 mmol/l with a detection limit of 0.005 mmol/l (signal/noise of 3) and a sensitivity of 1.9985 μA/mmol. The biosensor sensitivity diminishes when its surface is not polished between successive determinations, and remains constant (rsd=1.85, n=10) when the surface is polished between determinations. The effects of temperature and pH on the biosensor response were studied and analyzed; also the properties of the enzyme (Km _ap, I _max, Q₁₀) were determinate in this work. The biosensor was used to determine fructose in high fructose syrups and there were not significant differences between these results and those obtained by HPLC (p≤0.05). During 4 months, in intermittent determinations the biosensor kept 100% of its original sensitivity and after 18 months stored at 4°C, it only lost 32% of its sensitivity. The simplicity, low working potential, high stability and good performance of this biosensor shows a great potential for its use in the fructose determination.