Scanning Tunneling Microscopy Studies of Glucose Oxidase on Gold Surfaces

Three immobilization methods have been used for scanning tunneling microscopy (STM) studies of glucose oxidase (GOD) on gold. They are based on (a) physical adsorption from solution, (b) microcontact printing, and (c) covalent bonding onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA). The STM images are used to provide information about the organization of individual GOD molecules and more densely packed monolayers of GOD on electrode surfaces, thus providing information on the role of interfacial structure on biosensor performance. The use of atomically flat gold substrates enables easy distinction of deposited enzyme features from the flat gold substrate. Microcontact printing is found to be a more reliable method than adsorption from solution for preparing individual GOD molecules on the gold surface. STM images of printed samples reveal two different shapes of native GOD molecules. One is a butterfly shape with dimensions of 10 ± 1 nm × 6 ± 1 nm, assigned to the lying position of the molecule, while the second is an approximately spherical shape with dimensions of 6.5 ± 1 nm × 5 ± 1 nm assigned to a standing position. Isolated clusters of five to six GOD molecules are also observed. With monolayer coverages, GOD molecules exhibit a tendency to organize themselves into a two-dimensional array with adequate sample stability to obtain high-resolution STM images. Within these two-dimensional arrays repeating clusters of five to six enzyme molecules in a unit are clearly seen. Cluster structures are observed at both high and low coverages despite the fact that native GOD is a negatively charged molecule under physiological conditions. GOD monolayers covalently immobilized onto SAMs (MPA) are considerably more difficult to image than when the enzyme is adsorbed directly onto the metal.