Abstract
A quantitative total internal reflection intrinsic fluorescence (TIRIF) method for determining the adsorption of proteins at optically suitable solid/liquid interface is presented. Intrinsic protein fluorescence is excited by evanescent part of the standing wave produced by total internal reflection. The TIRIF data are quantified using as an internal standard the fluorescence from nonadsorbed proteins which are present in the evanescent region. In order to account for the fraction of fluorescence excited by scattered light which propagates through and beyond the volume sensed by the evanescent wave, a set of nonadsorbing external standards has to be used. The combination of TIRIF and 125I-protein γ-photon detection system is described and applied to bovine serum albumin (BSA) and human immunoglobulin (IgG) adsorption at silica/electrolyte interfaces. The difference between the results obtained with different adsorption detection systems is discussed. An average fluorescence emission efficiency of adsorbed proteins can be evaluated by combining TIRIF adsorption data with the independent in situ quantitation of protein adsorption. It was found that in some cases adsorbed proteins emit fluorescence with significantly lower quantum yield.
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