A reagentless optical biosensor based on the intrinsic absorption properties of peroxidase

During the reversible reaction between peroxidase (HRP) and H2O2, several peroxidase intermediate species, showing different molecular absorption spectra, are formed which can be used for H2O2 determination; when H2O2 is generated in a previous enzymatic reaction, the substrate involved in this reaction can also be determined. On this basis, a new family of fully reversible reagentless optical biosensors containing HRP is presented; glucose determination is used as a model. The biosensor (which can be used for at least 6 months and/or more than 750 measurements) is prepared by HRP and glucose oxidase entrapment in a polyacrylamide gel matrix. A mathematical model (in which optical, kinetic and transport aspects are considered) relating the measured absorbance with the substrate concentration is also presented together with a simple methodology for characterization of this kind of biosensor. Regarding the optical model, the Kubelka–Mulk theory of reflectance does not give good results and the biosensors are better described by the Rayleigh theory of polymer solutions. Under working conditions, linear response ranges from 1.5 × 10−6 to 3.0 × 10−4 M glucose and CV was about 4%. This biosensor has been applied for glucose determination in fruit juices and synthetic serum samples without sample pretreatment.