This paper reports a strategy for the oriented immobilization of protein receptors on gold films possessing nanometer-scale topographies and the detection of protein binding events to these receptors by using liquid crystals. The approach revolves around the use of self-assembled monolayers (SAMs) formed from nitrilotriacetic acid (NTA)-terminated alkanethiols, 1, and tri(ethylene glycol)-terminated alkanethiols, 2. The SAMs are formed on ultrathin gold films that are deposited from a vapor onto silica substrates oriented at an oblique angle of incidence. Single-component SAMs formed from 2 on these gold films resist nonspecific protein adsorption (using cell lysates) and promote uniform planar anchoring of the nematic liquid crystal, 4-cyano-4′-pentylbiphenyl (5CB). Surprisingly, the azimuthal orientation of nematic 5CB is parallel to the direction of maximum roughness within the gold film when using SAMs formed from 2 but perpendicular to the direction of maximum roughness when tetra(ethylene glycol)-terminated SAMs are formed on the gold films. Mixed SAMs formed from 1 and 2 bind the hexahistidine-tagged protein MEK via specific complexation of the hexahistidine tags of MEK to the Ni II -NTA complexes on the surface. When gold films are prepared by oblique deposition at an angle of 30°from the normal, we measure bound MEK to disrupt the uniform orientation of 5CB, thus leading to an easily visualized change in the optical appearance of the liquid crystal. However, by using gold films deposited at an angle of 40°from the normal, we report that bound MEK does not disrupt the alignment of the liquid crystal whereas anti-MEK IgG bound to the MEK does lead to a nonuniform alignment. These results, when combined with appropriate control experiments, suggest that nanostructured surfaces presenting NTA and ethylene glycol terminated SAMs form a useful interface for imaging proteins bound to histidine-tagged, surface-immobilized receptors.

We report an investigation of the binding ability of a protein immobilized on surfaces with different orientations but in identical interfacial microenvironments. The surfaces present mixed self-assembled monolayers (SAMs) of 11-[19-carboxymethylhexa(ethylene glycol)]undecyl-1-thiol, 1, and 11-tetra(ethylene glycol) undecyl-1-thiol, 2. Whereas 2 is used to define an interfacial microenvironment that prevents nonspecific adsorption of proteins, 1 was activated by two different schemes to immobilize ribonuclease A (RNase A) in either a preferred orientation or random orientations. The binding of the ribonuclease inhibitor protein (RI) to RNase A on these surfaces was characterized by using ellipsometry and the orientational behavior of liquid crystals. Ellipsometric measurements indicate identical extents of immobilization of RNase A via the two schemes. Following incubation of both surfaces with RI, however, ellipsometric measurements indicate a 4-fold higher binding ability of the RNase A immobilized with a preferred orientation over RNase A immobilized with a random orientation. The higher binding ability of the oriented RNase A over the randomly oriented RNase A was also apparent in the orientational behavior of nematic liquid crystals of 4-cyano-4'-pentylcyanobiphenyl (5CB) overlayed on these surfaces. These results demonstrate that the orientations of proteins covalently immobilized in controlled interfacial microenvironments can influence the binding activities of the immobilized proteins. Results reported in this article also demonstrate that the orientational states of proteins immobilized at surfaces can be distinguished by examining the optical appearances of liquid crystals.

This paper reports the design of surfaces on which thermotropic liquid crystals can be used to image affinity microcontact printed proteins. The surfaces comprise gold films deposited onto silica substrates at an oblique angle of incidence and then functionalized with a monolayer formed from 2-mercaptoethylamine. Ellipsometric measurements confirm the transfer of anti-biotin IgG to these surfaces from affinity stamps functionalized with biotinylated bovine serum albumin (BSA), while control experiments performed using anti-goat IgG confirmed the specificity of the IgG capture on the stamp. On these surfaces, anti-biotin IgG caused nematic phases of 4-cyano-4'-pentylbiphenyl (5CB, Delta epsilon = epsilon(parallel) - epsilon(perpendicular) > 0) to assume orientations that were parallel to the surfaces (planar anchoring) but with azimuthal orientations that were distinct from those assumed by the liquid crystals on the amine-terminated surfaces not supporting IgGs. Following incubation of these samples for >8 h at 36 degrees C, we observed that the amine-terminated regions of the surface not supporting IgG cause 5CB to undergo a transition from planar to perpendicular (homeotropic). Because N-(4-methoxybenzylidene)-4-butylaniline (MBBA) (Delta epsilon < 0) does not undergo a similar transition in orientation, this transition is consistent with the effects of an electrical double layer formed at the amine-terminated surface on the liquid crystal. Following the transition to homeotropic anchoring, the liquid crystals provide high optical contrast between regions of the surface supporting and not supporting IgG. We conclude that amine-terminated surfaces (I) uniformly align liquid crystals when not supporting proteins and (II) have sufficiently high surface free energy to capture proteins delivered to the surface from an affinity stamp, and thus they form the basis of a useful class of surfaces on which affinity microcontact printed proteins can be imaged using liquid crystals.

The chemical heterogeneity of proteins makes development of general and facile surface-based methods for protein analysis a substantial challenge, particularly when analyzing transmembrane proteins. Here, we report a simple surface-based procedure that permits detection of transmembrane proteins from crude cell lysates and cell membrane extracts. The method relies on the use of thermotropic liquid crystals to amplify and report the presence of the transmembrane proteins captured by an affinity ligand on the surface of an elastomeric stamp. A merit of this approach is that the proteins can be imaged on surfaces without requiring the use of matched pairs of antibodies, labels, or complex instrumentation. Detection of epidermal growth factor receptor, a transmembrane glycoprotein, is demonstrated.

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