Immobilization Of Glucose Oxidase And Its Electrochemical Performance

With the improvement of people’s living standards, diabetes as a kind of "rich people’s diseases" has been widely existed in the world. Glucose biosensor provides a convenient and fast method for concentration determination of glucose in blood and has attracted more and more attention. The enzymatic immobilization applied to the enzyme biosensor has been one of the hot spots in recent years. This paper prepared a new working electrode using glucose oxidase supported by columnar coal-based activated carbon, which has advantages such as large fixing amount, wide linear range, strong anti-interference capacity and long service life.The coal-based activated carbon and the coconut shell activated carbon with different pore size were used as carriers. Firstly, controllable oxidizing for activated carbons was conducted using nitric acid. Then surface treatment was conducted using a silane coupling agent,3-aminopropyl-trimethoxysilane(APTMS), and last cross-linked with glutaraldehyde. FT-IR spectrum suggested that the glucose oxidase (GOx or GOD) was immobilized successfully. GOx fixing amount were74.75mg GOx/g on the coal-based activated carbon and36.85mg GOx/g on the coconut shell activated carbon, respectively. The optimal active temperature of immobilized enzyme was38℃and the optimal active pH was6.0. Compared with free enzyme, the suitable range of reaction temperature and stable pH range of immobilized enzyme were widened significantly. The Michaelis constant (Km) of free enzyme was0.219mol/L, while the Michaelis constant (Km) of the coal-based activated carbon and the coconut shell activated carbon were1.321mol/L and0.393mol/L, respectively, which suggested that affinity of GOx with substrates did not significantly decrease after immobilization of GOx on the activated carbon. The coal-based activated carbon used as the support of GOx having advantages over the coconut shell activated carbon, such as the larger fixing amount, the wider range of optimal temperature and stable pH. We chose the coal-based activated carbon as the carrier to immobilize GOx in this experiment.The coal-based activated carbon was made into a cylinder, which had a diameter of3mm, a length of22mm. The iodine adsorption was800mg/g, the specific surface area was1000m2/g and the ash content was≤4%. The cylindrical coal-based activated carbon was used as a new type of working electrode, after a controllable oxidizing, a reaction with the silane coupling agent (APTMS) a crosslinking with glutaraldehyde and the immobilization of Gox. using Pt as a counter electrode and saturated calomel electrode as the reference electrode.0.1mmol/L hydroxy ferrocene PBS (pH=7.0) solution was used as the electrolyte. CV (cyclic voltammetry) curves showed pairs of redox peaks in this experiment increasing with the sweep speed increase, which suggested that the GOx-ACAGM electrode had a good response to the enzymatic reaction.The nano-cobalt coating was prepared by selecting appropriate electroplating conditions on the GOx-ACAGM electrode. Then replace the nano-cobalt with gold nanoparticles. GOx was loaded onto the electrode using ice bath with a simple stir. SEM and EDS results suggested that nano-cobalt and gold nanoparticles were successfully formed on the working electrode surface. The fixing amount of GOx on electrode was82mg GOx/g measured by glucose oxidase standard curve. CV curves showed that nano-gold modification working electrode had a significant current response to glucose. Chronoamperometry results indicated that the electrode had a linear relationship between2.5and20.31mmol/L with the glucose solution. It had strong anti-interference ability to uric acid and ascorbic acid and strong specificity to glucose. The electrode had high capability of current response and stability and remained more than90%capability a month later.