Covalent immobilization of immunoglobulins G and Fab' fragments on gold substrates for scanning force microscopy imaging in liquids

Scanning force microscopy offers the possibility of observing protein molecules under liquid environment. The main difficulty in obtaining reproducible images is given by the low adhesion of the molecules to the substrate. Physisorbed molecules are displaced by the scanning tip or are resuspended in the medium. We have therefore performed a covalent immobilization of immunoglobulin G (IgG) or its monovalent Fab' fragment on gold surfaces thanks to thiol groups, For this purpose, multiple thiol groups were chemically introduced into the IgG molecule by treatment with Traut's reagent. As an alternative, for a Fab' fragment, we prepared molecules with a single thiol group located close to the C terminus of the truncated heavy chain. Both immobilization techniques enable us to observe clearly discernable individual molecules in liquid media. The grafting of Fab' fragments on gold surface open new opportunities to study protein interactions. (C) 1996 American Vacuum Society.