Protein internal reflection evanescent wave

To discriminate between surface bound protein molecules and those in bulk solution, total internal reflection fluorescence microscopy (TIRFM)41 55 was employed. TIRFM creates an evanescence wave that decays as a function of distance from the surface as ... 

The utihzation of fluorescence dyes for analytical measurements enhances the sensitivity for the detection of the molecules of interest. First, Cronick and Little made use of evanescent wave excitation for a fluorescence immunoassay, in 1975. By using totally internally reflected light, they excited the fluorescence of a fluorescein-labeled antibody which has become bound to a hapten-protein conjugate adsorbed on a quartz-plate in an antibody solution [41]. Contrary to the label-free high-refractive-index sensors where the mass of the molecule of interest is... 

Figure 2 Studying membrane fusion with supported bilayers. A supported bilayer is suspended from a quartz substrate (top, gray background) and illuminated by the evanescent wave of a totally internally reflected laser beam (angled cylinders red). A membrane vesicle is observed to approach, hemifuse, and then fully fuse with the supported membrane. Vesicle contents, lipids, or proteins may be labeled fluorescently to monitor this process.

Unfortunately, water has very strong absorption in this spectral range, exhibiting extremely wide bands. Most proteins require water as a solvent. To do precision IR spectra of proteins, the optical path of the water must be made very small, on the order of 10 fim or so. If the path length is that small, a very high protein concentration is required, on the order of 10 mM typically. There are two ways to achieve spectra at these protein concentrations, either by forming thin films or by using a cell where the IR beam probes via the evanescent wave of total internal reflection. 

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