Probing fibronectin adsorption on chemically defined surfaces by means of single molecule force microscopy

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII^8-14 and full-length fibronectin (FN) on CH₃-, OH-, COOH-, and NH₂-terminated alkane-thiol self-assembled monolayers (SAMs). Force-distance curves acquired between hexahistidine-tagged FNIII^8-14 immobilised on trisNTA-Ni²⁺ functionalized AFM cantilevers and the OH and COOH SAM surfaces were predominantly 'loop-like' (76% and 94% respectively), suggesting domain unfolding and preference for 'end-on' oriented binding, while those generated with NH₂ and CH₃ SAMs were largely 'mixed type' (81% and 86%, respectively) commensurate with unravelling and desorption, and 'side-on' binding. Time-dependent binding of FN to SAM-coated QCM crystals occurred in at least two phases: initial rapid coverage over the first 5 min; and variably diminishing adsorption thereafter (5-70 min). Loading profiles and the final hydrated surface concentrations reached (~ 950, ~ 1200, ~ 1400, ~ 1500 ng cm^-2 for CH₃, OH, COOH and NH₂ SAMs) were consistent with: space-filling 'side-on' orientation and unfolding on CH₃ SAM; greater numbers of FN molecules arranged 'end-on' on OH and especially COOH SAMs; and initial 'side-on' contact, followed by either (1) gradual tilting to a space-saving 'end-on' configuration, or (2) bi-/multi-layer adsorption on NH₂ SAM.