Phenolic Compounds Determination Using Enzyme Modified Clark Type Electrode

Abstract

ABSTRACTPublic health and environmental protection concerns provoked by phenolic compounds pollution impose the development of sensitive, rapid and cost effective methods for in situ phenols monitoring. Given that biosensors based techniques could face these challenges, a variety of such devices was suggested and applied for phenolic compounds quantification. Their majority are based on the polyphenol oxidase (PPO) catalyzed phenols oxidation to catechol and then, to quinones, coupled with the registration of the quinones reduction current. Nevertheless, quinoid products polymerization involving electrode passivation corrupts the biosensors operational stability. Thus, to avoid this drawback, in this work is proposed another approach for phenolic compounds quantification based on the electrochemical detection of the oxygen depletion during PPO catalyzed catechol oxidation using a Clark type electrode with a disposable active enzyme membrane. The oxygen probe was modified in comparison to the commercial ones: its flat front allowed ensuring a good contact with the active enzyme membrane and the gold multicathode uniformly dislocated on the surface of the flat front permitted eliminating O2 diffusional constraints. The active enzyme membrane was prepared by drop-coating of a mixture of PPO and gelatin onto a gelatin-saturated cellulose filter. A linear calibration graph for catechol determination was obtained in the range up to 0.7 mM with a slope of 0.902 μA/mM, at pH 6.5 and ambient temperature. The steady-state response to catechol of the biosensor was reached in 120 s. The biosensor had an excellent reproducibility (RSD<3%) due to the reliable enzyme immobilization technique, allowing the preparation of active enzyme membranes with identical characteristics. The proposed biosensor provided stable response and free of interferences measurements since the unique possible electrochemical reaction is O2 reduction. Another biosensor advantage is associated with the use of disposable prefabricated active enzyme membranes.