The double selective synthetic approach is proposed for easy preparation of asymmetrically N-substituted derivatives of 1,4,7-triazacyclononane (tacn, [9]aneN(3)) with the same or different pendant arms. The synthetic routes simplify the synthesis of "tacn" ligands for functionalization of other carboxy terminated molecules/supports and also facilitate access to azamacrocycle functionalized self-assembled monolayer (SAMs) or nanopatterns for specific and stable Histidine-tagged protein immobilization.

The chemically programmed non-fouling surfaces were used to observe the surface assembly behaviour of hexahistidine tagged Green Fluorescent Protein (His-GFP) as a model protein. In this particular case, siteselective physisorption of His-GFP was achieved in the absence of metal ions. This preference does not arise from surface charge, wettability or topographic differences between regions. We found that His-GFP has a tendency to centre into an array of marked squares and acquires a template shape on the entire nonfouling surface when both the internal and surrounding areas present carboxylate groups. This surfacedirected organization of protein in assemblies is an unusual example of non-specific molecular interactions transfer to a higher scale objects organization. Furthermore, we performed a proof-of-concept study for the autonomous formation of protein microarrays with uniform orientation of the tagged protein molecules on the surface. Periodic protein microarrays were formed spontaneously within about one minute after the deposition of a few drops of protein solutions on the substrate. We propose a simple, gentle and cost-effective approach to fabrication of protein microarrays, which can be done by the enduser. This phenomenon of collective protein clustering into large scale patterns may help to assess experimentally how the peripheral proteins arrange into separate domains of the cell membrane.

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