Elaboration and characterization of spatially controlled assemblies of complementary polyphenol oxidase-alkaline phosphatase activities on electrodes

The electrooxidation of a biotin pyrrole has allowed the formation of bioti nylated polypyrrole films. Gravimetric measurements based on a quartz cryst al microbalance demonstrate the efficient coupling of avidin, biotinylated polyphenol oxidase (PPO-B) and avidin-labeled alkaline phosphatase (AP-A) w ith the underlying biotinylated polymer film. The estimated mass increase c orresponds to the anchoring of 1.6-1.8 equivalent layer of proteins. A step -by-step construction of bienzyme multilayers composed of PPO-B and AP-A wa s carried out on the electrode surface modified by the biotinylated polypyr role film through avidin-biotin bridges. A spatially controlled distributio n of the two enzymes was performed by the formation of one AP-A layer on 1, 5, and 10 PPO-B layers. The resulting bienzyme electrodes were applied to the determination of phenyl phosphate on the basis of amperometric detectio n of enzymically generated o-quinone at -0.2 V. Their analytical performanc es were analyzed in relation to the design of the enzyme architectures and in comparison with the amperometric behavior of the monoenzymatic electrode s (PPO-B electrode and AP-A electrode). It appears that at the 10-layer-PPO -B polypyrrole electrode only 4% of phenol is transformed, whereas 42-69% o f phenyl phosphate is enzymatically consumed and detected at the AP-A polyp yrrole electrode, depending on the enzyme activity, For the bienzymatic AP- A/PPO-B polypyrrole electrodes, the activity of each immobilized enzyme cle arly affects the biosensor performance, the main limiting factor being the very low efficiency of PPO-B at pH 8.8.