Fluorometric enzymatic autoindicating biosensor for H2O2 determination based on modified catalase

Our general aim is to develop reversible optical biosensors which can be used for continuous monitoring. In this paper we propose a biosensor for H2O2 determination. The bioreceptor is catalase (Cat) previously linked to a Ruthenium O2-sensitive fluorophore (Cat–Ru). It is based on the reversible H2O2 disproportionation into O2 and H2O. First, the fluorescent-enzymatic system was optimized for batch measurements (linear response ranges from 1×10−4 to, at least, 1×10−3M H2O2). Because of its reversibility, the same enzyme aliquot can be used for performing the whole calibration step (and the subsequent determination). Secondly, the optical sensor was prepared by Cat–Ru immobilization in a polyacrylamide film. The sensor permits H2O2 determination in a similar concentration range as in batch mode and can be used during at least 1 month. A mathematical model has also been developed which permits the effect of the experimental parameters to predict. The model also explains the sensor behavior if different fluorophores are used, and shows that the analytical signal only slightly depends on the initial concentration of the O2 in the sample. Finally an alternative sensor is presented based on a commercially available O2 fluorescence sensor linked to catalase. This system gives an analytical behavior similar to that shown for the Cat–Ru sensor. ► An optical biosensor for H2O2 continuous monitoring has been developped. ► The sensor is based on catalase, which decomposes H2O2 giving O2. ► O2 is detected by an O2-sensitive fluorophore linked to catalase and entrapped into polyacrylamide (PAA). ► Alternatively, catalase entrapped into PAA can be coupled to a commercial O2 fluorescence sensor. ► This work opens the door to a new family of optical monitoring systems based on catalase.