A tri-enzyme electrode probe for the sequential determination of fructose and glucose in the same sample

A new probe for the determination of fructose and glucose has been assembled using a platinum electrode and three enzymes co-immobilized on its surface: fructose dehydrogenase, glucose dehydrogenase and diaphorase. The mechanism of this probe is very simple: when the probe is immersed in a solution containing fructose and glucose and an electrochemical mediator, the current signal is due to the fructose. Then, by adding NAD(P)(+) a current response due to the glucose present in solution is obtained. Analytical parameters such as pH, buffer, temperature, concentration of mediator and ratio of the three enzymes co-immobilized have been optimised. Probe stability and reproducibility have been evaluated. Calibration curves for fructose and glucose have been constructed and all analytical parameters optimized. The linear range for both fructose and glucose was from 5x10(-6) to 2x10(-4) mol/L and the detection limit was 1x10(-7) mol/L. The probe has been used to determine the concentration of fructose and glucose in ten different samples of honey. The values obtained were compared with those obtained by UV-VIS spectrophotometry and the results were in good agreement.