Surface density dependent orientation and immunological recognition of antibody on silicon: TOF-SIMS and surface analysis of two covalent immobilization methods

Antibodies are commonly used as detection elements in biosensors. Antibody orientation on transducer surface determines immunological recognition and biosensor performance. Although a relation between antibody orientation and adsorbed amount Gamma was predicted, assuming close packing of proteins, and even applied to infer the preferred orientation from indirect surface analysis, the issue has not been so far examined experimentally with the surface techniques probing directly antibody orientation for a wide Gamma range covering all possible antibody orientations. In this work, time-of-flight secondary ion mass spectrometry combined with Principal Component Analysis probes the orientation of immunoglobulin G (aIgG) immobilized on silane-modified silicon surface with surface density 0 <= Gamma <= 3 mg/m(2), determined with ellipsometry. Two covalent immobilization methods: with NHS-silane and with amino-silane followed by glutaraldehyde (GA) activation, are compared. Atomic Force Microscopy reveals surface density dependent nanostructure of aIgG layers. AFM and PCA of TOF-SIMS clearly distinguish between the G ranges of flat-on, side-on and vertical aIgG orientation, that accord with random molecular packing. For vertical aIgG arrangement, a dominant head-on orientation and a mixed tail-on/head-on orientation is concluded from PCA for GA- and NHS-surface modification, respectively. In addition, molar binding ratio of antigen to antibody accords with the determined surface density dependent aIgG orientation.