Abstract
An efficient approach to enhance the performance of colorimetric biosensors has been developed. The biosensor is based on the co-immobilization of the reagent 3,3′,5,5′-teramethylbencidine (TMB) and the enzyme horseradish peroxidase (HRP) in a PDMS-TEOS-SiO2NPs support. The HRP, in presence of H2O2, catalyzes the oxidation of TMB, producing a blue color. The generated biosensor, doped with the substrate (TMB) and the enzyme (HRP) (entrapped or adsorbed), has been used to determine H2O2 in real samples. Firstly, the immobilization of TMB and HRP in the composite has been studied in order to find the best suitable configuration. The kinetic parameters Vmax (maximum reaction rate) and Km (substrate affinity) of the different assayed systems have been determined and compared. Secondly, the analytical properties of the H2O2 method have been obtained. From the analytical point of view, no significant differences were observed between the adsorption and entrapment immobilization procedures used for the HRP. This method is simple, inexpensive, highly sensitive and selective for the determination of H2O2, with detection limits of 1.3μM and a good linearity over the range 4.2–72μM. The LOD can be improved to 0.4μM by acidifying the solution with sulphuric acid. Precision was also satisfactory (relative standard deviation, RSD<10%). Results were compared to those obtained by the conventional derivatization method in solution. The developed biosensor is a reagent-release support which significantly simplifies the analytical measurements, because it avoids the need to prepare derivatization reagents and sample-handling, and it allows in situ measurements.
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