 Polyvalent Biochemical Interactions
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The overall objective is to develop new methodologies to produce arrays of nanostructures of antibodies to mimic immunological processes. Previous studies have shown that the two antigen-binding sites of an antibody are separated by 14.5 nm. The distance among nearest neighbor antibodies are on the order of several to tens of nanometers to trigger certain immune processes such as hypersensitive reactions (allergy) and antibody-mediated phagocytosis. Therefore, an effective and reliable way to mimic or regulate these immune processes is to control the position and orientation of antibodies with nanometer precision. The proposed approach utilizes scanning probe lithography (SPL) to produce nanostructures of ligands (antigens) followed by the divalent binding of antibodies to those nanostructures. Thousands of antibodies will be immobilized individually, while maintaining the bioreactivity. The position, perpendicular and lateral orientations of each antibody will also be precisely controlled.
Specific projects include: (1) The design and production of a series of nanostructures (or test platforms) in which the binding sites, i.e. antigens, are arranged precisely. Fabrication of nanostructures of ligands will be accomplished by SPL techniques developed in the PI’s laboratory. (2) Systematic investigation of the reactivity of these nanostructures as a function of ligand separation, flexibility and local environment. Real time imaging is planed to monitor the binding of antibodies onto these nanostructures in situ and in real time. (3) Determination of the optimal design of the test platforms for divalent immobilization. Experiments include the studies of the antibody-antigen reaction kinetics, the structure of the antibody-antigen complexes, and the binding strength and polyvalency. Systematic comparisons are planned to investigate the nanoengineering approach versus current methods which utilize mixed components or crystalline phased receptors. (4) Preliminary testing of the capability of artificially engineered assemblies of antibodies to trigger cell responses which mimic the corresponding immune processes.
This approach has fundamental advantages because the arrangement of the binding sites and the local environment of each site are engineered with nanometer precision, thus investigations may be carried out systematically, under well-defined conditions. SPM-based nanofabrication techniques are unique in that they provide the highest possible spatial precision. These high-resolution engineering and imaging studies should reveal nanoscopic and molecular level information of polyvalent interactions that are otherwise very difficult to obtain, e.g. the influence of size, separation, orientation and local environment.
Beyond enhancing our fundamental understanding of antibody-antigen and antibody-cell interactions, the proposed research will establish a new, nanoengineering-based paradigm with broader applications to the investigation of polyvalent biointeractions such as adhesion of proteins and viruses on cells, signal transduction, and viral infection. Engineering the binding sites with nanometer precision provides an opportunity to discover novel phenomena because the artificially designed nanostructures may differ from (and perhaps be better and more robust than) their natural counterparts. Nanobiotechnology should also benefit from this study as (1) nanofabrication techniques provide a means to further miniaturize biochip components; and (2) precise control of polyvalent interactions provides new strategies for immobilizing biomolecules with desired positions and orientations, a critical step in engineering biodevices and sensors.
The research and education plan focuses around nanobiotechnology, high-resolution imaging and lithography, size-dependent phenomena, and polyvalent biointeractions. Through the proposed research projects, students are expected to gain important technical skills in nanofabrication, and to acquire knowledge about the potential and limitations of nanotechnology. Technical training includes: (1) scanning probe microscopy; (2) nanolithography and high resolution imaging; (3) protein immobilization and surface chemistry; and (4) cell adhesion on artificially engineered surfaces. Communication and collaboration among research groups at the UCD-nanoscience center--NEAT (Nanophases in the Environment, Agriculture and Technology) should broaden the overall background of student researchers.
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