Electrochemical Quartz Crystal Impedance Analysis Studies On Glucose Oxidase Electrodes

Biosensor has become one of the important research fields of analytical chemistry. Enzyme electrodes have been widely employed in clinical diagnose and preclinical medicine, especially in the diagnosis of diabetic patients. Quartz crystal microbalance (QCM), which is capable of ultrasensitive mass measurements down to the nanogram level, the solution viscodensity and the elasticity of modified films, has been extensively used in the fields of electrochemistry and labeling-free biosensors. Piezoelectric quartz crystal impendance analysis (PQCI) method, as a powerful multiparameter QCM, can perform a simultaneous and rapid measurement of the electroacoustic impedance, therefore multidimensional information on the resonant frequency and the parameters of a Butterworth-van Dyke (BVD) equivalent electrical circuit can be obtained. However, there are very limited reports on the QCM's being employed to study enzyme electrodes, involving the monitoring of various modification steps in constructing an enzyme electrode, and especially the quantification examination on the immobilized enzyme for assessment of its specific activity. In view of the aforementioned status, we have thus conducted some innovation investigations as follows:1. The recent researches using piezoelectric quartz crystal microbalance and scanning electrochemical microscopy are briefly reviewed.2. The immobilization of glucose oxidase (GOD) via its dip-dry coating on a Prussian blue (PB) modified Au electrode followed by deposition of an outer poly(o-phenylenediamine) (PoPD) film and then glutaraldehyde (GA) cross-linking was investigated for amperometric glucose sensing, based on the reduction of enzymatically generated H₂O₂ catalyzed by the electroactive PBlayer of the PoPD/GOD-GA/PB/Au electrode. The quartz crystal microbalance (QCM) was used to monitor various electrode-modification processes, and the effective enzymatic specific activity (ESA, defined as enzymatic activity per gram of enzyme) of immobilized GOD was estimated based on the mass of immobilized CjOD (from QCM) and the quantity of hydrogen peroxide as the product of the enzymatic reaction (from amperometric detection) for the first time. The ESA of native GOD under our experimental conditions was also estimated via amperometric detection of enzymatically generated H2O2 at a PB-modified Au electrode. Effects of various experimental parameters on glucose sensing, including the applied potential, solution pH and electroactive interferents, were investigated. At an optimal potential of -0.05 V versus saturated KC1 calomel electrode (SCE), the current response of the biosensor in the selected phosphate buffer (pH 7.0) was linear with glucose concentration from 0.05 to 10 mmol L"1, with detection limit of 8 jimol L1, short response time (within ⁵ s) and good anti-interferent ability. The Michaelis constant (Kmapp) of the immobilized GOD was estimated as 19.6 mmol L"1. The biosensor exhibited good storage stability, i.e., 88% of its initial response was retained after 30-day storage in pH 7.0 phosphate buffer at 4 °C. Since the present sensor-fabrication platform is rather general and advantageous in saving biochemical reagents, it is recommended for the development of other biosensors based on other substrate electrode materials, e.g. glassy carbon.3. A novel glucose biosensor based on the electrochemical detection of enzymatically generated H2O2 was constructed by the effective immobilization of glucose oxidase (GOD) via glutaraldehyde cross-linking with a poly(thionine) (PTH)-modified Au electrode, followed by coating witha Nafion outer layer to obtain high selectivity. An electrochemical quartz crystal microbalance (EQCM) was used to track various modification procedures. The mass of deposited PTH via cyclic voltammetric electropolymerization of thionine in aqueous H2SO4 was estimated from the "dry" frequency shift after fully removing the PTH layer via its acidic degradation, allowing us to evaluate the electroactivity per unit mass of PTH for the first time. A Nafion/glucose oxidase-glutaraldehyde/poiy(thionine)/Au electrode was thus constructed for glucose sensing. We found that the PTH film is a good matrix to immobilize more GOD, and the performance of the constructed sensor is significantly improved compared with its absence, though its mediator effect on the enzyme electrode is negligible. Influence of various experimental parameters on glucose sensing, including the applied potential, solution pH and electroactive interferents, was investigated. At an optimal potential of 0.7 V versus the KCl-saturated calomel electrode (SCE), the glucose biosensors were linear with glucose concentration from 0.005 to ⁵ mmol L"1, with high sensitivity (13.5 A mol"1 cm"1), short response time (within ca. 10 s) and good anti-interferent ability. The Michaelis constant (ATmapp) of the immobilized GOD was estimated to be 5.47 mmol L"1. The biosensor exhibited good storage stability, i.e., 83% of its initial response was retained after one-month storage in pH 7.0 phosphate buffer at 4 °C. The effective enzymatic specific activity (ESA, defined as enzymatic activity per gram of enzyme) of immobilized GOD was estimated to be 6.85 kU g1 on the basis of the mass of immobilized GOD (from EQCM) and the quantity of hydrogen peroxide as the product of the enzymatic reaction (from amperometric detection), and the leakage of H2O2 from the Nafion/GOD-GA/PTH/Au enzyme electrode at the detection potential, ascharacterized by an scanning electrochemical microscope (SECM), was found to be an important factor influencing the ESA estimation. 4. Glucose oxidase was immobilized via glutaradhyle cross-linking with self-assembled 4-aminothiophenol on the PB modified Au electrode. The influence of the thickness of PB film on the reduction current of hydrogen peroxide was examined. In addition, we also studied the effects of various experimental parameters, such as the applied potential, solution pH and electroactive interference, on glucose sensing. At optimized experiment conditions, the response characteristic and stability of the enzyme biosensor was investigated. The linear calibration range of the biosensor was from 0.01 to 2mmol L'1, with a linear correlation coefficient of 0.9945. The detection limit of the biosensor was 1 ^mol L"1 at a signal to noise ratio of 3. Besides, the storage stability of the enzyme electrode was tested over a period of three weeks. When the electrode was stored in the phosphate buffer of pH 7.0 at 4 °C in a refrigerator, the response of the enzyme electrode to 1-mmol L1 glucose decreased to 93% of its initial value in the first week, and the biosensor still retained 84% of its initial response after another three weeks.