pH-dependent orientation of physisorbed and chemisorbed antibodies on silicon determined with TOF-SIMS and its effect on in-flow capture assay monitored with WLRS sensor

The orientation of the IgG antibody adsorbed on the biosensor surface determines the performance of the bioassay and depends on the pH of the adsorption solution, the amount Γ of antibody adsorbed, and other factors. So far, the arrangement of IgG at various pH levels has been indirectly inferred from the antigen binding efficiency, evaluated from an assay after adsorption, often with limited control of Γ. Instead, in this work, the dominant orientation of the antibody layer adsorbed at pH 6–10, in the Γ range of vertical arrangements, is determined with Time-of-Flight Secondary Ion Mass Spectrometry combined with Principal Component Analysis. The proportions of molecules with tail-on and head-on alignment decrease with pH from 4:1 to 1:2 for physisorption to a 3-aminopropyltriethoxysilane (APTES) monolayer, and from 1:1 to 1:2 for chemisorption to glutaraldehyde-modified APTES. The impact of pH-controlled antibody orientation is then examined in two assays performed using high molarity PBS-based solutions (pH 7.4). The results of a static assay with an Fc-specific secondary antibody mimic those of TOF-SIMS for chemisorbed IgG molecules and show IgG reorientation for physisorption. They also anticorrelate with the antigen binding rate constant of the in-flow capture assay kinetics, monitored with a White Light Reflectance Spectroscopy sensor.