NAv/biotin interaction was determined using gold SPR sensor, starting with immobilization of biotin-PEG-SH (BPT), followed with BSA to fill any non-specific binding pockets and exposure to neutravidin (NAv). Control experiments were performed to rule out the nonspecific binding of NAv on PEG-thiol layers in the absence of biotin functionality. BPTs with three different tether lengths was used, viz. 0.4nm, 1.4nm and 6nm which corresponds to BPT-200, BPT-400 and BPT-1k respectively (200 g/mol, 400 g/mol and 1000 g/mol). The length of the tether was calculated with the help of Cambridge Scientific Chem3D 19.0 software considering the distance between the sulfhydryl group and the last oxygen forming the EG monomer before the biotin complex. The 0.4 nm long tether is considered to have a comparatively rigid molecular structure given that 200 g/mol is around the molecular weight of a biotin molecule (see Table ). In this case, we considered the Flory radius to be the radius of biotin (around 0.37 nm). The variation of BPT tether densities as function of tether length was determined for two different BPT density regimes on surface, viz. denser (Rf/D ≅ 1.4) and sparser (Rf/D ≅ 0.53)