Nanomaterials Immobilized Urease Biosensor Research

The biosensors are cross-region and multi-subject advanced diagnosis instruments. It can realize the biosensors in vivo and on-line determination of minim biological samples has importance role to the development of life science. However, nanostructure materials and nanotechnology-based biosensor not only have good effect on it,but also impel biosensor to be a high sensitivity, handy and inexpensive instrument for real-time and on-site analysis.The urease was adsorbed on different size particles of TiO₂(5nm, 25nm, 2.4μm ).Under conditions of 35℃and pH 7 , the activity of urease absorbed on TiO₂ particles was measured by potentiometry. A layer nanoporous TiO₂ film was deposited on the titanium surface , and urease was directly adsorbed on the TiO₂ film . Based on the pH response of TiO₂ film developed a cheap and microminiature pH sensitive urease sensor . The adsorption of urease immobilized on the self-cleaning TiO₂ ,films was investigated by Quartz crystal microbalance (QCM),FT-IR spectra and UV spectra , and the potentiometry was used to measured the property and influence factors of urease biosensors. The results show that the activity of urease can be enhanced via nanostructure materials, urease adsorbed on the self-cleaning TiO₂ films could have good reproducibility and long-term stability , under conditions of 1.0 mM PBS (pH 7-8),and 35℃, the urea biosensor was sensitive to urea in the linear range 8.5×10^-5-1.5×10^-1 M ,the correlation coefficient and the detection limit were 0.99373 and 6×10^-5 M respectively. The QCM results show that the adsorption amount was 0.03 g/g ,and the adsorption equilibrium constant was about 3.15×10⁵ L/mol.In this study, a urea biosensor was developed using molecularly imprinted TiO₂ film for the immobilization of urease. At the same time, we have tested four different methods for enzyme immobilization to determine the most reliable one. The quartz crystal microbalance (QCM) and the potentiometry results demonstrated that the biosensor based on molecular imprinting technique (MIT) are more efficient and stabler than that based on other immobilization methods. The adsorption amount of urease prepared by MIT was 128.3mg/g, it was more than 2.8 times of immobilized urease via non-imprinting technique. The potentiometry investigated show that, under conditions of 1.0 mM PBS (pH 7.0 ) , and 25℃, the life-time of the urea biosensor can be remained 94% after 35 days, the sensitive was 72.11mV, and the urea biosensor was sensitive to urea in the linear range 1×10^-2.5-1.5×10^-1.0M ,the correlation coefficient was 0.97913.