Production of Glucose Oxidase Nanocomposite Electrodes for Enzymatic Biofuel Cell Operation

The practicality and demand for small, continuously powered energy sources have increased in recent decades. These energy sources could be used to power a wide range of environmental sensors and implantable medical sensors. The ability of these power sources to be left for long periods of time without being replaced is one of the major design specifications that consumers of these sensors have put forward. These energy sources must also be capable of producing a sufficient amount of electrical energy to operate the sensors. One such energy source that has been developed is the enzymatic biofuel cell. These cells generally convert glucose and other naturally abundant biomaterials into electrical energy by utilizing the ability of some naturally occurring enzymes to perform oxidation-reduction reactions on the biological substrates. However, there are a number of technical obstacles that must be overcome to allow for electrical energy to be produced. One such obstacle is the short lifetime of enzymes in solution. While the lyophilized powder form of an enzyme might be kept for multiple years without a loss in activity, enzymes in solution have a life span of merely a few days or a few weeks because of their tendency to denature and lose their reactive conformation in solution. In addition, these enzymes generally transfer the produced electrons necessary for the generation of electrical energy from the oxidation reaction to a natural oxidizing agent such as oxygen to produce hydrogen peroxide. This is especially the case for the enzyme that was utilized in this research, Glucose Oxidase from Aspergillus niger (GOx). In order to overcome these issues, a number of techniques were used to augment the natural tendencies of the enzyme. The first technique was the incorporation of carbon nanoparticles, mainly acid treated graphitized mesoporous carbon, into the enzyme solution. This allowed a pathway for the electrons to transfer from the active site of the enzymes to the electrode on which they were immobilized, allowing for electrons from the reaction to generate an electrical current in the electrode. However, this treatment did nothing to decrease the tendency of the enzymes in solution to denature and also demonstrated the inability of large amounts of enzyme to be immobilized on the electrode surface. To increase the stability and amount of enzymes that could be loaded onto electrode, another technique had to be employed. This technique was the use of glutaraldehyde, an enzyme cross-linker, to cause the protein shells of the enzymes to bond to each other. This bonding of the protein shells encouraged the conformation of the enzymes to remain constant, thus keeping the enzyme from denaturing. This treatment would cause the GOx to form relatively large aggregates of enzymes which would also become attached to the carbon nanoparticles that were later introduced to the system during the preparation of this solution. These aggregates demonstrated extended lifetimes and allowed more enzyme to be loaded onto an electrode. However, a reduction in the overall activity of the enzyme solution was observed when the enzymes were aggregated. This arose from the fact that when the enzymes were aggregated, not all of the enzyme active sites were exposed directly to the solution that contained the substrate. Also, given the protein nature of the aggregate, the transfer of substrate into the aggregates and the transfer of electrons out of the aggregates could have been limited. In order to remedy this, a nanocomposite material consisting of gold nanoparticles with long chain carbon ligands attached to them was introduced into the enzyme solution before the aggregation process occurred. The procedure used to prepare the electrodes for the biofuel cell then made the ligands on these nanoparticles in the solution attach to the GOx before aggregation occurred. Then, during the aggregation process, the gold nanoparticles would be incorporated into the GOx aggregates. This then caused a marked increase in enzymatic activity, which could be due to the higher electrical conduction of the gold nanoparticles.