Investigating organic multilayers by spectroscopic ellipsometry: specific and non-specific interactions of polyhistidine with NTA self-assembled monolayers

Background: A versatile strategy for protein–surface coupling in biochips exploits the affinity for polyhistidine of the nitrilotriacetic acid (NTA) group loaded with Ni(II). Methods based on optical reflectivity measurements such as spectroscopic ellipsometry (SE) allow for label-free, non-invasive monitoring of molecule adsorption/desorption at surfaces. Results: This paper describes a SE study about the interaction of hexahistidine (His(6)) on gold substrates functionalized with a thiolate self-assembled monolayer bearing the NTA end group. By systematically applying the difference spectra method, which emphasizes the small changes of the ellipsometry spectral response upon the nanoscale thickening/thinning of the molecular film, we characterized different steps of the process such as the NTA-functionalization of Au, the adsorption of the His(6) layer and its eventual displacement after reaction with competitive ligands. The films were investigated in liquid, and ex situ in ambient air. The SE investigation has been complemented by AFM measurements based on nanolithography methods (nanografting mode). Conclusion: Our approach to the SE data, exploiting the full spectroscopic potential of the method and basic optical models, was able to provide a picture of the variation of the film thickness along the process. The combination of δΔ(i)(+1)(,i)(λ), δΨ(i)(+1)(,i)(λ) (layer-addition mode) and δΔ(†)(i)(',)(i)(+1)(λ), δΨ(†)(i)(',)(i)(+1)(λ) (layer-removal mode) difference spectra allowed us to clearly disentangle the adsorption of His(6) on the Ni-free NTA layer, due to non specific interactions, from the formation of a neatly thicker His(6) film induced by the Ni(II)-loading of the NTA SAM.