Surface Patterning Of Antibody And The Orientation Analysis

In the past decades, protein immobilization technology is emerged in the field ofprotein microarray, biochips, affinity chromatography and so on. Proteinimmobilization technology makes the fast and sensitive testing possible in diagnosticassays, environmental testing and drug screening. In fact, oriented immobilization is thekey of protein immobilization technology to the success in binding the target. It isimportant to control and probe the orientation of the protein immobilized on the surface.However, the structure and composition of protein are so complex that some surfacecharacterization methods, such as Atomic Force Microscope (AFM), SurfacePlasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM) can only beused to investigate the orientation by measuring the amount of binding target.Therefore, new strategy for orientating the protein on surface and probing theorientations has attracted more and more attention.In this dissertation, we develop a novel method to control the antibodyorientation on co-patterned surfaces and Time-of-Flight Secondary Ion MassSpectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy werecombined to probe the orientation of antibody on the surface. Our detailedworks are mainly concentrated on the following aspects:1, Protein A and fluorescein were co-patterned on NHS activity glass slideby lithography. Specific affinity between Fab-fluorescein and Fc-protein A wereconfirmed by investigating the binding behavior of Fab fragments and Fcfragments on co-patterned surfaces. It is proved that two orientations of Y shapeantibody bound on the different region of co-pattern surface was achieved.2, ToF-SIMS analysis, sensitive to surface chemistry in the top1-3mm ofthe surface, differentiates "heads-up" and "tails-up" orientations of theantibodies patterned on the surface. Principal components analysis (PCA)processing of ToF-SIMS ion fragment data for such patterned antibodiesdistinguishes the natural amino acid composition differences in the antibody Faband Fc domains present and exposed differently in these two patterned regions.A multivariate ration of amino acids characteristic of the Fab and Fc fragment was used to provide an image "map" of antibody compositional variations andorientations across the patterned surface.