Thiol mixed SAMs

We investigated the efficiency of NSC expansion on surfaces with EGF-His immobilized in the correct orientation. NSCs were obtained from neurosphere cultures prepared from fetal rat striatum harvested on embryonic day 16. NSCs were cultured for 5 days on EGF-His-immobilized substrates prepared with mixed SAMs of different COOH-thiol contents. Cells adhered and formed network structures at a density that increased with the COOH-thiol content of the surface. As a control, cells were seeded onto surfaces without immobilized EGF-His. This resulted in poor cell adhesion during the entire culture period. In addition, when EGF-His adsorbed to SAMs with 100% COOH-thiol or SAMs with NTA-derivatized COOH that lacked Ni2+ chelation, we observed poor initial cell adhesion, and the cells formed aggregates within 5 days. Interestingly, the substrate used to covalently immobilize EGF-His with the standard carbodiimide chemistry was not a suitable surface for cell adhesion and proliferation. The control experimental results contrasted markedly with results from EGF-His-chelated surfaces. 

We also conducted experiments to compare our culture method with the standard neurosphere culture. In the standard neurosphere culture, cell number increased approximately nine times over 5 days. Immunostaining showed that the neurosphere cultures contained 54 5.3% nestin+ cells and 41 7.4% nestin+ pIIF cells. This demonstrated that the standard neurosphere culmring method was less efficient than EGF-immobilized substrates for selectively expanding NSCs. Thus, the EGF-immobilized substrates prepared from mixed SAMs with 10% COOH-thiol provided the most efficient method for selective NSC expansion. 

Based on those results, we concluded that, when cultured on the EGF-His-immobilized surface prepared from a mixed SAM of 10% COOH-thiol, highly enriched NSC populations could be produced in large quantities. Over a 5-day culture on the substrate, cells were expanded 32 times. These expanded cells consisted of 98% nestin+ cells that retained multipotency for differentiation into neuronal and glial lineages. This suggested that selective expansion could be repeated for large-scale production of highly enriched NSC cells. 

In a scheme complementary to the one just presented where thiols are removed by reductive desorption of thiols, molecules can also be removed during stripping of a UPD layer. This was demonstrated by Shimazu et al. [221] where an alkane thiol SAM was deposited onto a Au(l 11) that had been modified with Pb. Oxidative stripping of the lead also caused thiols to be removed. The empty sites were then subsequently filled with mercaptopropionic acid (MPA). A remarkable result is that the binary SAMs exhibit only one desorption peak. From this it was concluded that a well-mixed layer forms that is very different from the mixed SAM obtained by adsorption from solution containing both types of thiols. In this case the layer exhibits singlecomponent domains that are refiected by two desorption peaks. 

In addition to uniform monolayers formed by using a single thiol compound, mixed SAMs are increasingly used for the immobilization of biomolecifles. The aim of combining different thiols is to avoid known disadvantages of uniform monolayers. 

In principle, the molar ratio of different thiols in a mixed SAM is the same as their original molar ratio in the solution which was used for the formation. In other words, for a mixture of two thiol compounds which does not show demixing tendencies (phase segregation), a random attachment of both compounds onto the surface can be assumed [16]. This observation offers the potential to mix a cw-substituted alkane thiol with short-chain nonsubstituted thiols. As the result, anchor molecules are available for which steric hindrance is minimized (cf. Fig. 3). 

Fig. 4 A schematic illustration of pure and mixed SAMs a thiol-derivatized single-stranded DNA only and b mixed SAM layer of thiol-derivatized single-stranded DNA and 6-hydroxy- 1-hexanethiol...

In the next example, a mixed SAM is discussed which aims to utilize photoinduced energy and electron transfer processes to create a photocurrent in an approach which is reminiscent of the natural photosynthetic process. Figure 5.33 illustrates the molecular structures of the components of interest, i.e. the molecular triad ferrocene-porphyrin-fullerene (Fc-P-C6o) and a boron dipyrrin thiol (BoDy) [67]. Mixed monolayers were generated by coadsorption onto vacuum-deposited gold... 

Here, the specific and strong chelating interaction between 6 x Histidine and the tetradentate nitrilotriacetic acid (NTA) mediated by Ni2+ [43] was used for the immobilization of a reconstituted LHCII mutant, whose c-terminus was extended by 6 x Histidine residues via genetic engineering (cf. Fig. 13(A)). NTA terminated thiols and OEG (spacer) thiols were used to built up a mixed SAM (X = 0.4) on a substrate coated by 23 nm silver and 5 nm Au (the gold layer was used to protect the silver film from being oxidized). 

Ligands A, B, and C have similar chemical structures. They all carry an end thiol functional group (for attaching onto a gold surface of a QCM device), an alkane chain (to form the well-packed SAM ), and a triethylene glycol linker (to prevent nonspecific adsorption ). The resulting 1 1 mixed SAMs... 

---