Interfacial intrinsic fluorescence induced by evanescent wave total internal reflection was developed to study protein ad sorption at solid-aqueous buffer solution interfaces. The tech nique has a number of advantages over conventional meth odologies for the study of adsorption including (1) continuous, real-time sampling with 0.1-s resolution; (2) in situ sensing; (3) application to biomedically relevant, flat, low surface area samples; (4) quantitation of the amount adsorbed calculated on the basis of an internal standard; and (5) ability to obtain fluorescence emission spectra of intrinsic tryptophan moieties that are sensitive to local microenvironmental changes pro duced during the protein adsorption process. These advan tages are illustrated for bovine serum albumin and γ-globulins adsorbed on hydrophilic quartz. A n understanding of protein adsorption behavior is applicable in numer ous fields including blood-synthetic materials interfaces, macromolecular-membrane interactions, receptor interactions, enzyme engineering, ad hesion, and protein separation on chromatographic supports. Many methods have evolved to study interfacial adsorption, but no single independent method seems adequate. The ideal technique should produce quantitative, real-time, in situ data concerning the amount, activity, and conformation of proteins adsorbed on well-characterized surfaces. All adsorption techniques are approximations to this optimum. Protein solution depletion via adsorption on finely divided substrates is quantitative, but applicability to low surface area materials of biomedical relevance is often minimal. Adsorption of radiolabeled macromolecules is In Biomaterials: Interfacial Phenomena and Applications; Cooper, S., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.352 BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS quantitative on low surface area substrates; however, the presence of an extrinsic label may alter protein physical properties and subsequent adsorp tion behavior. Automated ellipsometry can, in principle, provide in situ, real-time information on film thickness and refractive index, but the minute differences in substrate, film, and buffer refractive indices often preclude this approach. Multiple internal reflection infrared spectroscopy is complicated by strong water signals that obscure protein amide bands and, while Fourier transform analysis seems promising, the interpretation remains difficult and the equipment is expensive. Interfacial protein fluorescence induced by internal reflection evanes cent wave excitation offers a number of advantages over conventional adsorp tion techniques. The total internal reflection fluorescence (TIRF) concept was originally patented by Hirschfeld (1 ) and applied to protein adsorption by Harrick and Loeb (2). Since then TIRF has been utilized in a limited number of investigations to study the adsorption of extrinsic fluor-labeled plasma proteins on quartz (3, 4), hapten-protein conjugates (5), and polydimethylsiloxane films (6). O...
