Development of biosensors for determination of the total antioxidant capacity
In this study, an amperometric laccase biosensor was developed for determination of the oleuropein concentration that is the biological active component of olive leaf and contributes dominantly to the total antioxidant capacity. The biosensor was prepared by immobilization of laccase from Trametes versicolor by addition of cross-linking agent, glutaraldehyde, into the carbon paste electrode. Different biosensors were prepared by changing the amount of crosslinking agent and concentration of the enzyme solution. So, effect of these parameters on biosensor performance was investigated. The best biosensor performance was determined for the biosensor having glutaraldehyde amount of 12.03 % vol. of the biosensor bottom part and 5 mg/ml of laccase enzyme. The effect of scan rate and temperature on the biosensor performance was also investigated in this study. The scan rate of 10 mV/s was decided to be the optimum for the amperometric detection of oleuropein considering the fastest response and maximum reduction current. 250C was chosen as an optimum temperature value due to the maximum laccase activity and capability of oleuropein acting as an antioxidant. Extraction of phenolics from olive leaf was also an important part of this study. The extract was divided into fractions varying in their oleuropein amounts such as polar fractions and relatively less polar fractions. Therefore, biosensor performance was investigated for fractions containing different type of phenolics. HPLC analyses of the fractions were also performed in this study. In addition total phenol content and antioxidant capacity of the fractions were determined by conventional methods.