Evaluation of intact- and fragmented-antibody based immunosensors by total internal reflection ellipsometry

Total internal reflection ellipsometry (TIRE) technique was used to investigate biological recognition layers of immunosensors in order to estimate orientation of immobilized intact- and fragmented-antibodies. Two differently prepared biological recognition layers formed on gold-substrate were investigated: one layer was based on intact-antibodies (intact-Ab), second layer was based on chemically fragmented specific-antibodies (frag-Ab), which were obtained by reduction of intact-Ab. It was shown that TIRE enables to resolve differences in nanostructures of intact-Ab and frag-Ab layers. A multilayer model applied in our calculations shows that the distance between fragmented-antibody active sites and gold-surface after the immobilization process is lower than theoretical dimension of fragmented-antibodies. Moreover, it was calculated that analytical sensitivity of the Δ(λ) parameter of TIRE was 5.89 times better if compared to the sensitivity of the Ψ(λ) parameter, which is in fact similar to the sensitivity of surface plasmon resonance (SPR) based immunosensors.