ω-functionalized self-assembled monolayers chemisorbed on ultraflat Au(111) surfaces for biological scanning probe microscopy in aqueous buffers

Two immobilization procedures for ultimately carrying out scanning probe microscopy of native biological macromolecules in buffer solution are presented. They are based on the preparation of ultraflat template-stripped gold surfaces and subsequent chemisorption of bioreactive ω-functionalized self-assembled monolayers. Immobilization was achieved either via amide bond formation or diazo linkage. The first was carried out using a long-chain N-hydroxysuccinimide-ester functionalized disulfide, which binds amino-group-containing biomacromolecules under mild conditions. The latter was performed via chemisorption of a nitrobenzylalkylthiol, followed by in situ transformation to the active diazo intermediate. Typical targets were activated aromatic hydrogens, such as phenolic or imidazole groups. The preparation of these monolayers, their characterization by scanning tunneling microscopy and radiolabeling, and as an example the in situ atomic force microscopy study of clathrin cages under native conditions in appropriate buffers are presented.