(647f) Protein A-Based Immobilization of Antibody Onto Polymeric Microfludic Device for Enzyme-Linked Immunosorbent Assay

The immunoreaction sensitivity is a major challenge in the development of polymeric microfluidic device for enzyme-linked immunosorbent assay (ELISA). In this study, a biotin-derivatized poly(l-lysine)-g-poly (ethylene glycol) (biotin-PLL-g-PEG) and protein A-based technique was developed to immobilize antibody on the surface of poly(methyl methacrylate) (PMMA) microchannels. First, PMMA surface was activated by oxygen plasma, followed by poly (acrylic acid) (PAA) grafting to add functional carboxyl group for subsequent binding. After the Biotin-PLL-g-PEG molecules reacted with carboxyl groups through the electrostatic interactions, biotinylated protein A was immobilized on the surface through a linking molecule, netruavidin. To evaluate the applicability of this novel immobilization strategy, human interferon-gamma (IFN-ã) is used as a model protein. Since protein A can better control the immobilization orientation, and the combination of biotin-PLL-g-PEG and PLL-g-PEG can adjust the conformation of antibodies, antigen capture efficiency and detection signals are significantly improved on the microchips by using this strategy. The optimal grafting conditions are also experimentally determined. This new method has a great potential to improve the performance of current polymeric ELISA microchip. Meanwhile, this method can also be applied to highly efficient antibody immobilization for other immunoassay biosensor applications.