Engineered nanostructures for investigation and regulation of mast cell activation

This thesis research focuses on the application of atomic force microscopy (AFM) and AFM-base nanolithography in cell biology. The motive is to mimic, study and regulate the polyvalent interactions involved in the antibody-mediated activation of mast cells, a key cell type of the immune system. Knowing and being able to regulate these reactions help to design and develop better polyvalent drugs to treat allergic diseases or other diseases in the immune system.;In nature, the cross-linking of IgE bound onto the FcepsilonRI receptors by polyvalent antigens induces the clustering of these receptors, which further triggers the degranulaton of mast cells. These processes happened spontaneously on the cell membrane and it is difficult to investigate and regulate them in solution base. Furthermore, the antigen and antibody recognition, the receptor clustering all occurred at nanometer scale. Thus, it is desire to using nanotechnology to investigate and regulate these micro and nano biological processes.;The solution to the above scientific questions proposed by this thesis research is to pattern antigen molecules on surface with nanometer precision using AFM-based nanolithography, and thus to regulate the receptor clustering at molecular level. The activation of mast cells on this engineered antigen-presenting platform is then characterized using AFM combined with laser scanning confocal microscopy (LSCM), through which the information acquired helps to better understanding the requirement to trigger or inhibit the activation of mast cells.;Three milestones were placed during this thesis investigation. First, high-resolution cell imaging protocols using AFM and combined AFM and LSCM were successfully developed and demonstrated in the characterization of mast cell activation. A new imaging protocol using two AFM probes under optical guidance for cell imaging was established and enabled to capture both whole cell morphology and local membrane structures with great details in aqueous environment. AFM imaging in buffer revealed rich membrane structures associated with the activation of mast cells with remarkable details. These high-resolution surface characteristics were further correlated with cytoskeletal arrangement and intracellular organelles using a combined atomic force and confocal microscope, which enabled the disclosure of detailed degranulation mechanisms for the exocytosis of mast cells.;Second, a new fabrication methodology combining micromachining with AFM-based nanografting was successfully developed to produce hierarchical micro/nano structures on coverslips for cell study. Pre-defined micro-patterns on transparent coverslips that are visible from optical microscopes are used to guide the AFM probes during nanografting, and help register the optically invisible nanostructures in later-on applications. These fabricated hierarchical micro/nano structures have sufficient size and throughput to serve as ligand-presenting platforms for cellular studies in research labs. Furthermore, ligand nanostructures fabricated by nanografting allows multiple functionalities, complex geometry and in situ protein binding.;Third, engineered antigen nanostructures were successfully applied as cell-stimulating platform to investigate the activation of mast cells, and exhibited intrinsic superiority, in terms of cell activation, than antigen-presenting monolayers formed by natural growth. By varying DNP nano-line density on surface, engineered nanostructure patterns demonstrated regulatory effects on both the adhesion and activation of mast cells, as characterized using combined atomic force and confocal microscopy. High-resolution AFM characterization of antigen-presenting surfaces reveals the superiority of engineered nanostructures to naturally formed monolayers in activating mast cells lies in the high aspect ratio and geometrically guided presentation of ligand molecules on surface, which facilitates the antigen-antibody recognition, as well as receptor clustering.;The investigations in this thesis work demonstrate the revealing and regulatory power of AFM on the hypersensitive reactions of mast cells, and pave the way for the development of new therapies for treating allergic diseases. They also manifest as elegant examples on applying nanotechnology to solve biomedical problems.