Surface plasmon resonance spectroscopic characterization of antibody orientation and activity on the calixarene monolayer

Abstract

Attachment of proteins to a solid support is fundamental to development of advanced biosensors, biochips, implantable electronic devices, and many diagnostic techniques. Profound knowledge of the orientation and specificity of an intricate protein in a particular antibody and adsorption to solid surfaces are of great importance in fabrication of nanoscale interface with maximized biological performance. Here we investigate antibody surface coverage and antibody layer thickness utilizing surface plasmon resonance (SPR) on the ProLinker™, a well-known protein artificial receptor molecule. The immunoactivity of immobilized antibodies was compared using two different methods. SPR revealed that antibodies were vertically immobilized in a dense monolayer when directly immobilized on the ProLinker™ SAM in an orientation comparable to that of protein G immediate antibody immobilization. Considering that surface coverage and native activity of the immobilized antibody are affected by antibody orientation, it is clear that the antibody is mainly adsorbed on the ProLinker™ SAM in an end-on orientation, which was verified by atomic force microscope (AFM). Aside from its original function, i.e., analyte monitoring, we used simple SPR spectroscopic analysis to monitor differences in orientation and concentration of immobilized bio-moieties at the molecular level.