Piezoelectric Electrochemical Studies On The Immobilization Of Enzyme Using The Polymer From The Oxidation Of Dopamine

Biosensors are promising biochemical analysis tools for clinical diagnosis as well as food and environmental monitoring. The immobilization of the involved biomolecules is the key step in constructing a biosensor. To date, many techniques have been utilized for enzyme immobilization, involving protocols of adsorption, covalent binding, cross-linking and entrapment. Among them, the entrapment with polymers has become a popular method to immobilize biomolecules.The electrochemical quartz crystal impedance analysis (EQCIA) performs a simultaneous and rapid measurement of the electroacoustic impedance for the piezoelectric quartz crystal (PQC) resonance, which can provide multiple chemical/physical parameters and material-relevant characteristics during an electrochemical perturbation, such as electrode-mass changes down to the nanogram level, the solution viscodensity, and the elasticity of modified films. In the present thesis, the EQCIA method has been used to track the processes of immobilization of involved biomolecules for constructing the biosensors.1. The concept and construction of the biosensors, mainly involving immobilization methods for biomolecules, as well as the principle and application of the electrochemical quartz crystal microbalance (EQCM), have been reviewed.2. Glucose oxidase (GOD) was codeposited into a polymer grown from oxidation of dopamine (DA) at an Au electrode in a neutral phosphate aqueous solution for the first time. The EQCIA method was used to monitor the GOD-immobilization process. Effects of concentrations ofphosphate buffer, DA and GOD were investigated, and the optimal concentrations were found to be 20.0 mM phosphate buffer (pH 7.0), 30.0 mM DA and 5.00 mg ml*1 GOD. A glucose biosensor was thus constructed, and effects of various experimental parameters on the sensor performance, including applied potential, solution pH and electroactive interferents, were examined. At an optimal potential of 0.6 V versus the KCl-saturated calomel electrode (SCE), the current response of the biosensor in the selected phosphate buffer (pH 7.0) was linear with the concentration of glucose from 0.05 to 9 mM, with a lower detection limit of 3 jiM (S/N=3), short response time (within 15 s) and good anti-interferent ability. The Michaelis constant (Kmapp) was estimated to be 9.6 mM. The biosensor exhibited good storage stability, i.e. 96% of its initial response was retained after 7-day storage in the selected phosphate buffer at 4 °C, and even after another three weeks the biosensor retained 86% of its initial response. In addition, the enzymatic specific activity and enzymatic relative activity of the GOD immobilized in the polymer from dopamine oxidation (PFDO) were estimated from the EQCIA method to be 1.43 kU g"1 and 3.7%, respectively, which were larger than the relevant values obtained experimentally using poly(o-aminophenol) and poly(N-methylpyrrole) matrices, suggesting that the PFDO is a better matrix to immobilize GOD.3. Glucose oxidase (GOD) was codeposited into a polymer grown from dopamine oxidation (DA) on a Prussian blue (PB) modified Au electrode in neutral phosphate aqueous solution. The EQCIA method was used to monitor various modification steps in constructing theenzyme electrode. A glucose biosensor with good performance was thus fabricated, via the quantity optimization for PB, and effects of experimental parameters on the sensor performance, including applied potential, solution pH and electroactive interferents, were examined. At an optimal potential of 0.6 V vs SCE, the current response of the biosensor in the selected phosphate buffer (pH 7.0) was linear with glucose concentration from 0.05 to 6 mM, with lower limit of detection of ~2 joM (S/N=3), short response time (within ~8 s) and good anti-interferent ability. The Michaelis constant (￡mapp) was estimated as 9.5 mM. The biosensor exhibited good storage stability, i.e. 76% of its initial response was retained after 4-week storage in pH 7.0 phosphate buffer at 4 °C. Enzymatic relative activity of the GOD immobilized in the polymer from dopamine oxidation (PFDO) were estimated from the EQCIA method to be 20.1%.4. A novel method to immobilize hemoglobin in a polymer grown from dopamine oxidation has been proposed. The growth of the polymeric films at the Prussian blue (PB) modified Au electrode during DA oxidation in weaker alkaline aqueous solutions (PBS, pH 9.18) and the immobilization of hemoglobin into the polymeric films during their growth have been traced by the EQCIA method. A hydrogen peroxide (H2O2) biosensor was thus constructed, and effects of experimental parameters on the sensor performance, including applied potential, solution pH and electroactive interferents, were examined. At an optimal potential of -0.25 V vs SCE, the current response of the biosensor in the selected phosphate buffer (pH 5.29) was linear with the concentration of H2O2 from 0.01 to 4.5 mM, with a lower limit ofdetection of 0.5 ^M (S/N=3), short response time (within 10 s) and good anti-interferent ability. The Michaelis constant (K^) was estimated as 3.80 mM. The biosensor exhibited good storage stability.