RESUMO
We have used miniature planar IR waveguides, consisting of Ge strips 30-50 microm thick and 2 mm wide, as evanescent-wave sensors to detect the mid-(IR) evanescent-wave absorbance spectra of small areas of biomolecular monolayers and multilayers. Examples include picomolar quantities of an integral transmembrane protein (bacteriorhodopsin) and lipid (dimyristoyl phosphatidylcholine). IR bands due to the protein and lipid components of the plasma membrane of individual 1.5-mm-diameter devitellinized Xenopus laevis oocytes, submerged in buffer and sticking to the waveguide surface, were also detected. A significant improvement in sensitivity was observed, as compared to previous sizes and geometries of evanescent-wave sensors (e.g., commercially available internal reflection elements or tapered optical fibers). These measurements suggest the feasibility of using such miniature supported planar IR waveguides to observe structural changes in transmembrane proteins functioning in vivo in single cells.
Assuntos
Membrana Celular/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Bacteriorodopsinas/química , Fenômenos Biofísicos , Biofísica , Dimiristoilfosfatidilcolina/química , Feminino , Técnicas In Vitro , Lipídeos de Membrana/química , Proteínas de Membrana/química , Oócitos/química , Xenopus laevisRESUMO
We have fabricated miniature planar IR waveguides with thicknesses of 30-50 mum, consisting of 12-mm long, 2-mm wide strips of Ge supported on ZnS substrates. Evidence for efficient propagation of broadband IR light through these waveguides is provided by the presence of characteristic high- and low-frequency optical cutoffs of Ge; by the observation of an oscillatory interference pattern in the transmittance spectrum, which exhibits a dependence on waveguide thickness and propagation angle that closely matches waveguide theory; and by the detection of strong evanescent-wave absorption from small (2 mm(2)) droplets of liquid, e.g., water, on the waveguide surface. As also predicted by theory, the surface sensitivity (detected light absorbance per unit area of sample-waveguide contact) is shown to increase as a function of incidence or bevel angle.