Total internal reflectance fluorescence data

W. M. Reichert, J. T. Suci, J. T. Ives, and J. D. Andrade, Evanescent detection of adsorbed protein concentration-distance profiles Fit of simple models to variable-angle total internal reflection fluorescence data, Appl. Spectrosc. 41, 503-507 (1987). 

One of the simplest methods to study adsorption at the oil water interface is to measure the variation of interfacial tension as a function of concentration. If the polymer used for adsorption is monodisperse, then the Gibbs equation (51) may be used to estimate the surface excess. However, if the polymer is polydis-perse, this method will give erroneous values of the surface excess because the larger molecules will tend to adsorb preferentially, and the equation is imable to account for this adsorption behavior. As a result, most of the data available in the literature report the change in the interfacial tension as a function of concentration without attempting to convert it into an adsorbed amoimt. Apart from interfacial tension measurements, other techniques such as total internal reflection fluorescence microscopy (52) and scintillation measurements from radiolabeled polymers (53) have also been used to measure the adsorption at the liquid-liquid interface. 

When light traversing an optically dense medium approaches an interface with a more optically rare medium at an angle exceeding a critical value, Bent = sin (rerare/ dens), total internal reflection occurs and an evanescent wave of exponentially deca5ung intensity penetrates the rarer medium. This phenomenon is at the heart of certain spectroscopic methods used to probe biomolecules at interfaces (199). In total internal reflection fluorescence (TIRF) spectroscopy (200-202), the evanescent wave excites fluorescent probes attached to the biomolecules, and detection of the emission associated with their decay provides information on the density, composition, and conformation of adsorbed molecules. In fourier transform infrared attenuated total reflection (FTIR-ATIR) spectroscopy (203,204), the evanescent wave excites certain molecular vibrational degrees of freedom, and the detected loss in intensity due to these absorbances can provide quantitative data on density, composition, and conformation. 

Figure 7 shows a human (milk) Lyso adsorption isotherm recorded on hydrophobic silica slides treated with dimethyldichlorosilane (DDS) [29]. Total internal reflectance fluorescence (TIRF) spectroscopy was used to assess the adsorbed mass of Lyso. The isotherm experiment was carried out at pH 7.4, with protein dissolved in a phosphate-buffered saline (PBS) (0.013 M KH2PO4, 0.054 Af Na2HP04) buffer including 0.1 M NaCl. Tables 8 and 9 show the relevant thermodynamic and regression data determined for this isotherm. 

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