Immobilizing Antibody Biorecognition Layers on Au-TiO2 Thin Films: Direct (physisorption) vs. DSP-crosslinking (chemisorption) Surface Functionalization

Abstract

The immobilization design strategy plays an important role in biosensor development and its sensing performance. Both adsorption (physisorption) and cross-linker functionalization (chemisorption) are common approaches for immobilizing a bioreceptor layer. In this work, these two approaches were studied and compared, envisaging a functional and strongly attached bioreceptor layer onto sputtered Au-TiO2 thin films. DSP cross-linker (Lomant's reagent) was used in the thin film’s functionalization, and the effect of different concentrations on the development of an adhesion layer was investigated. Surface morphology analysis of functionalized thin films suggested the development of uniformly coated self-assembled layers. However, DSP islands with a fractal structure were found for a concentration of 4 mg mL-1. Infrared spectroscopy confirmed the cross-linker functionalization at the thin film’s surface. Confocal microscopy of immobilized fluorescent antibodies revealed that DSP islands improve the chemical surface area available for bioreceptor immobilization. Moreover, an immunoassay using mouse IgG interaction with fluorescent anti-mouse IgG (Fab specific), working as capture and detection antibody, respectively, showed that DSP functionalization favors antibodies orientation and adhesion strength to the surface, when compared to physisorption.