Surface attachment of protein fibrils via covalent modification strategies.

Chemical control of surface functionality and topography is an essential requirement for many technological purposes. In particular, the covalent attachment of monomeric proteins to surfaces has been the object of intense studies in recent years, for applications as varied as electrochemistry, immuno-sensing, and the production of biocompatible coatings. Little is known, however, about the characteristics and requirements underlying surface attachment of supramolecular protein nanostructures. Amyloid fibrils formed by the self-assembly of peptide and protein molecules represent one important class of such structures. These highly organized beta-sheet-rich assemblies are a hallmark of a range of neurodegenerative disorders, including Alzheimer's disease and type II diabetes, but recent findings suggest that they have much broader significance, potentially representing the global free energy minima of the energy landscapes of proteins and having potential applications in material science. In this paper, we describe strategies for attaching amyloid fibrils formed from different proteins to gold surfaces under different solution conditions. Our methods involve the reaction of sulfur containing small molecules (cystamine and 2-iminothiolane) with the amyloid fibrils, enabling their covalent linkage to gold surfaces. We demonstrate that irreversible attachment using these approaches makes possible quantitative analysis of experiments using biosensor techniques, such as quartz crystal microbalance (QCM) assays that are revolutionizing our understanding of the mechanisms of amyloid growth and the factors that determine its kinetic behavior. Moreover, our results shed light on the nature and relative importance of covalent versus noncovalent forces acting on protein superstructures at metal surfaces.

Fabrication and bio-functionalization of tetrahedral amorphous carbon thin films for bio sensor applications

Tetrahedral amorphous carbon (ta-C) thin films are a promising material for use as biocompatible interfaces in applications such as in-vivo biosensors. However, the functionalization of ta-C film surface, which is a pre-requisite for biosensors, remains a big challenge due to its chemical inertness. We have investigated the bio-functionalization of ta-C films fabricated under specific physical conditions through the covalent attachment of functional biomolecular probes of peptide nucleic acid (PNA) to ta-C films, and the effect of fabrication conditions on the bio-functionalization. The study showed that the functional bimolecular probes such as protected long-chain ω-unsaturated amine (TFAAD) can be covalently attached to the ta-C surface through a well-defined structure. With the given fabrication process, electrochemical methods can be applied to the detection of biomolecular interaction, which establishes the basis for the development of stable, label-free biosensors. © 2011 Elsevier B.V. All rights reserved.

Preparation and characterization of a novel pyrrole-benzophenone copolymerized silica nanocomposite as a reagent in a visual immunologic-agglutination test.

Biological sensing is explored through novel stable colloidal dispersions of pyrrole-benzophenone and pyrrole copolymerized silica (PPy-SiO(2)-PPyBPh) nanocomposites, which allow covalent linking of biological molecules through light mediation. The mechanism of nanocomposite attachment to a model protein is studied by gold labeled cholera toxin B (CTB) to enhance the contrast in electron microscopy imaging. The biological test itself is carried out without gold labeling, i.e., using CTB only. The protein is shown to be covalently bound through the benzophenone groups. When the reactive PPy-SiO(2)-PPyBPh-CTB nanocomposite is exposed to specific recognition anti-CTB immunoglobulins, a qualitative visual agglutination assay occurs spontaneously, producing as a positive test, PPy-SiO(2)-PPyBPh-CTB-anti-CTB, in less than 1 h, while the control solution of the PPy-SiO(2)-PPyBPh-CTB alone remained well-dispersed during the same period. These dispersions were characterized by cryogenic transmission microscopy (cryo-TEM), scanning electron microscopy (SEM), FTIR and X-ray photoelectron spectroscopy (XPS).

Differences between FRP bond behavior in cracked and uncracked regions

Based on an analysis of the experimental results of a proposed bond test method, significant differences are shown to exist between the local FRP bond stress-slip relationships in the uncracked anchorage regions and in the regions between cracks. The proposed method simulates the bond behavior between the flexural cracks and anchorage regions of a flexurally FRP-strengthened RC beam. The boundary conditions, including the presence of cracks and steel, are shown to have significant effects on the local bond stress-slip models. The results showed that, at the same force, the bond stresses in the regions between cracks were lower than in regions outside the cracks, so the debonding formed in the anchorage regions. The local bond stress-slip models in the anchorage regions can be obtained from the conventional bond test methods but these do not mimic the conditions between the cracks.

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry.

The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.

Spectroscopic characteristics and cellular compatibility of protein wrapped single wall carbon nanotubes

Non-covalent functionalization of CoMoCAT single-wall carbon nanotubes (SWNTs) by bovine serum albumin (BSA) was achieved. Photoluminescence spectra for the functionalized nanotubes showed good dispersion by BSA functionalization. Raman spectra were taken for the sonicated SWNT-BSA solution to establish the signal versus concentration correlation. Cellular uptake of functionalized carbon nanotubes by mouse macrophage (RAW264.7) was then investigated using Raman spectroscopy. For a seeding density of 50% confluence in a culture solution containing 10 μg/ml of BSA-SWNTs, uptake of 200 μg/ml by the macrophages was recorded after 23hr incubation, indicating an active uptake of SWNTs. © 2012 IEEE.