| Solid-phase immunoassays (SPIs) have become an indispensible tool for research, diagnostics, and therapeutics. Understanding the antibody-antigen interaction occurring at a solid-liquid interface is needed in order to develop more sensitive and selective sensors with lower limits of detection. In turn, this will provide a better understanding of the immune system, and lead to the discovery of more effective immunotherapy treatments. In this work, total internal reflection fluorescence (TIRF) microscopy is employed to investigate individual ligand-receptor interactions at a glass-water interface. The extreme sensitivity of TIRF imaging requires the measurement surfaces to resist the nonspecific adsorption of proteins in order to minimize backgrounds, while containing low density of optically resolvable capture sites. The nonspecific adsorption of antibodies to poly(ethylene glycol) (PEG) monolayers covalently bound to glass surfaces was characterized for two different PEG immobilization procedures. First, N-hydroxysuccinimide (NHS) active esters of 2000 g/mol PEG were reacted to the amine functionality of (3-aminopropyl)triethoxysilane (APTES) derivatized glass slides; secondly, in addition, 750 g/mol and 2000 g/mol PEG-amines were reacted to the epoxide functionality of (3-Glycidyloxypropyl)trimethoxysilane (GOPTS) modified glass slides. The protein resistant property of each PEG-modified surface was characterized by monitoring the adsorption of monoclonal mouse derived antibiotin using single-molecule fluorescence imaging. The protein resistant coating produced using epoxide-amine PEG chemistry was adapted to produce a glass modified surface containing a low density of biotin conjugation sites by reacting a mixture containing a lower concentration of biotin-labeled 2000 g/mol PEG-amine and a higher concentration of 750 g/mol PEG-amine to GOPTS modified glass slides. The density of biotin conjugation sites within the diluent PEG layer was characterized using fluorescently-labeled streptavidin, where the surface concentration of biotin increased linearly with biotin-PEG concentration in the reaction solution. Using the low density biotin capture surfaces, the activity of a surface immobilized rabbit-antigoat IgG receptor was investigated using TIRF imaging for four different antibody immobilization strategies: (i) passive adsorption to the mPEG surface, (ii) active bioaffinity immobilization directly to streptavidin capture sites, (iii) two-step sequential immobilization of streptavidin and protein A intermediate, and (iv) a one-step immobilization to a preassociated chimeric streptavidin-protein A complex. |
