Nanostructured Modified Si-Electrodes for GOD and HRP Third-Generation Biosensors

The development of biosensors on miniaturized Si-platforms appears fundamental to overcome the extensive packaging, the complex electronic interfacing and the regular maintenance of conventional biosensors. In latest years, the functionalisation of solid electrodes with thin films of biocompatible materials revealed very attractive, since these configurations could provide a rapid translation of the biological processes occurring on the surface to electronic outputs. In this study we report the realization of nanostructured modified Si- electrodes for the immobilization of several enzymes and biological molecules. The electrode surface modification was based on the deposition of TiO2 layers on different Si substrates by MOCVD and Sol-gel technique. Chemical and structural properties of electrode surfaces before and after enzyme immobilization was investigated by X-ray Photelectron Spectroscopy (XPS) and Atomic Force Microscopy (AFM). Horseradish peroxidase (HRP) and glucose oxidase (GOD) were immobilized on the modified surfaces without the aid of a mediator. The Direct Electron Transfer of immobilised GOD and HRP was investigated by electrochemical methods. Process parameter revealed fundamental for the determination of electrode structural and chemical features, which influenced the enzyme immobilization. The immobilization of HRP and GOD led to the formation of homogeneous monolayers on electrode surface, with a surface coverage of about 8.0 x 10-10 mol/cm2 and 7.5 x 10-10 mol/cm2. GOD and HRP immobilized onto modified Si-electrodes exhibited a pair of well-defined and quasi-reversible voltammetric peaks. The electron exchange between the enzyme and electrodes was greatly enhanced by the presence of TiO2 nanostructured layers. The responses showed a surface-controlled electrode process with an electron transfer rate constant of (39.5±4.3)/s for HRP and (45.0±5.0)/s for GOD, determined in the scan rate range from 10 to 100 mV/s. The immobilised enzymes could electrocatalyse the reduction of H2O2 without the aid of an electron mediator, and this resulted in a great increase of the reduction peak current. Upon the addition of glucose substrate, GOD biosensor showed a high sensitivity (100 mA mol-1 L cm-2), a linear range from 2 to 500 ?M and a detection limit of 1 ?M at a signal-to-noise ratio of 3s. For HRP biosensors, the calibration range of H2O2 was with good linear relation from 2 to 200 ?M, and a detection limit of 2 ?M. The performances of these biosensors were tested also in terms of storage stability, which resulted over 3 months, excluding the interference of commonly coexisted uric acid, acetaminophen and ascorbic acid. In conclusions, the formation of nanostructured TiO2 layers promotes the direct immobilization of GOD and HRP on Si substrates. GOD and HRP biosensors realized using these Si-modified electrodes show very interesting properties, such as high sensitivity and long-term storage stability. The direct electrocatalytic activity of HRP and GOD towards H2O2 and glucose may have a potential perspective for the fabrication of low-cost miniaturizable third-generation biosensors.