SPONTANEOUS RECONFIGURATION OF ADSORBED LYSOZYME LAYERS OBSERVED BY TOTAL INTERNAL-REFLECTION FLUORESCENCE WITH A PH-SENSITIVE FLUOROPHORE

By conjugating proteins with a common pH-sensitive fluorescent label, fluorescein isothiocyanate (FITC), and controlling the ionic strength, we provide a means to decrease the characteristic length scale of the total internal reflection fluorescence (TIRF) technique by two orders of magnitude. The usual characteristic length scale for TIRF is an op tical length, specifically the evanescent wave penetration depth (on t he order of 100 nm). In our experiments the penetration depth is repla ced by the Debye screening length as the characteristic length scale. This is readily controlled to match the dimensions of an adsorbed prot ein layer (on the order of 1 nm). We achieve this length scale reducti on by coupling the well-known pH-sensitivity of fluorescence emission by FITC-labeled proteins with the variation of electrostatic potential near a negatively charged surface. Using this fine-resolution TIRF ca pability in combination with scanning angle reflectometry, we find tha t lateral repulsions induce a dramatic reconfiguration of adsorbed lys ozyme layers on negatively charged silica surfaces. This occurs as the surface concentration approaches the jamming limit for random sequent ial adsorption. The reconfiguration evidently optimizes electrostatic interactions in the adsorbed layer and decreases the effective exclude d area per lysozyme. The decrease in effective excluded area allows ad sorption to continue beyond the jamming limit to ultimately attain a h exagonal close packed monolayer of horizontally oriented lysozyme mole cules. The adsorption kinetics switch abruptly from being transport-li mited to surface-limited after the reconfiguration.