Designing surface chemistries for in situ AFM investigations of biomolecular reactions with proteins at the nanoscale
In situ atomic force microscopy (AFM) characterizations and lithography can be applied to investigate the orientation, reactivity and stability of protein molecules adsorbed on nanostructures of self-assembled monolayers at near-physiological conditions. Automated nanografting was used to fabricate regular arrays of nanopatterns of ù-functionalized n-alkanethiols with designated terminal chemistries. After writing nanopatterns, protein binding occurs selectively on carboxylate-terminated nanopatterns via covalent bonds that are formed using N-ethyl-N'(dimethylaminoporpyl)-carbodiimide and N-hydroxysuccinimide activation. The amine groups of lysine residues of proteins bond covalently to nanopatterns of carboxylate-terminated alkanethiol self-assembled monolayers, to form a robust surface attachment for sustained contact-mode AFM imaging during biochemical reactions. Staphylococcal protein A (SpA) furnishes a generic foundation for binding immunoglobulins for nanometer scale sandwich assays. The self-assembly of á,ù-alkanedithiols onto Au(111) was investigated using AFM. When SAMs of 1,8-octanedithiol or 1,9-nonanedithiol are grown naturally from solution, different surface orientations are observed in comparison to methyl-terminated n-alkanethiols. Local views from AFM images reveal a layer of mixed orientations in which the majority of á,ù-alkanedithiol molecules adopt an orientation parallel to the surface with both thiol endgroups bound to Au(111). Results from AFM studies reveal that the chemisorption of thiol endgroups of dithiols inhibits the phase transition from a lying-down to a standing orientation during natural self-assembly. Another method for producing protein nanostructures is particle lithography. Monodisperse mesospheres can be applied to rapidly prepare millions of exquisitely uniform nanometer-sized structures of proteins on flat surfaces using conventional benchtop chemistry steps of mixing, centrifuging, evaporation and drying. The natural self-assembly of monodisperse spheres provides a high throughput and efficient route to prepare circular geometries over millimeter scale areas. The spontaneous assembly of silica or latex mesospheres into organized crystalline layers on flat substrates supplies a structural frame to direct the placement of proteins. Nanopatterns of ferritin, apoferritin, immunoglobulin G and bovine serum albumin were produced with particle lithography. The applicability of particle lithography to generate arrays of protein nanostructures on surfaces such as mica(0001), glass and Au(111) was demonstrated. The morphology and diameter of the protein nanostructures can be tailored by selecting the ratios of protein-to-particles and the diameters of spheres.
Atomic Force Microscope, Self-assembled Monolayer, Nanopatterns, Assay
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