Liquid crystals (LCs) were used to amplify and transduce receptor-mediated binding of proteins at surfaces into optical outputs. Spontaneously organized surfaces were designed so that protein molecules, upon binding to ligands hosted on these surfaces, triggered changes in the orientations of 1- to 20-micrometer-thick films of supported LCs, thus corresponding to a reorientation of approximately 10(5) to 10(6) mesogens per protein. Binding-induced changes in the intensity of light transmitted through the LC were easily seen with the naked eye and could be further amplified by using surfaces designed so that protein-ligand recognition causes twisted nematic LCs to untwist. This approach to the detection of ligand-receptor binding does not require labeling of the analyte, does not require the use of electroanalytical apparatus, provides a spatial resolution of micrometers, and is sufficiently simple that it may find use in biochemical assays and imaging of spatially resolved chemical libraries.
Molecular exchange kinetics between a monolayer of antibody molecules formed on the air−water interface and the protein solution was studied by means of fluorescent labeling. It was shown that there is no inclusion of dissolved molecules in the previously formed monolayer during even 6 h of exposure regardless of monolayer surface density. The surface activity of IgG and horseradish peroxidase molecules was studied by means of surface compression isotherms, and the specific biological activity of the monolayers formed from these proteins was measured by enzyme and immunoassay techniques. It was shown that the surface activity of the proteins increases while specific biological activity decreases with exposure of the molecules on the water surface. Since the same effects were caused by denaturing agents, we propose that the surface activity of the proteins and the absence of surface−volume exchange are due to partial unfolding of the molecules which takes place on the water surface. Two models of the partial unfolding are discussed: complete denaturation of some part of the molecules and partial unfolding of each molecule. The process of surface denaturation was shown to be slow and controllable. One can achieve a pronounced increase of protein surface activity with low degradation of the specific biological activity of the monolayer; thus, it can be used in the practice of protein Langmuir film deposition.
This paper reports the electroless deposition of gold onto the surface of micrometer-sized particles of silica (silica gel) and the self-assembly of monolayers formed from ω-substituted alkanethiols on the surface of the goldcoated silica gel. Whereas the proteolytic enzyme subtilisin BPN′ adsorbs irreversibly from aqueous solution (100 mM Tris buffer, 10 mM CaCl 2 , pH 8.6) onto the surface of untreated silica gel, reversible adsorption of subtilisin BPN′ is measured on silica gel coated with gold and derivatized with self-assembled monolayers (SAMs) formed from HO(CH 2 CH 2 O) 2 (CH 2 ) 11 SH. We demonstrate the usefulness of gold-coated silica gel for the preparation of stationary phases with controlled surface properties by forming a variety of mixed SAMs through coadsorption of pNA-FPAA-suc-(CH 2 ) 11 SH and X(CH 2 ) 11 SH (where X ) CH 3 , OH, or COOH). These stationary phases were used to demonstrate that the extent of enzymatic hydrolysis of pNA from the surface-immobilized tetrapeptide depends on the functional group X as well as on the dilution of the substrate within the mixed SAM. We also demonstrate that SAMs presenting biotin (X ) biotin) can be used to form multilayer structures of biomolecules on the surface of the gold-coated silica gel. These results, when combined, demonstrate the usefulness of gold-coated silica gel for the preparation of well-defined, surface-functionalized supports for biological assays.
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