Using the DPI techniques described above, it is possible to obtain thickness and refractive index data for layers deposited upon the surface of the sensing waveguide and calibrate the refractive index values for bulk solutions such as the phosphate-buffered saline running buffers. Given that the refractive index increments of proteins are quite consistent (Arwin 2000; Davis and Wilson 2000; Huglin 1972; Wen and Arakawa 2000), with typical values of 1.465, at a density of 0.71 g cm-3 it is possible to determine the mass of material deposited on the sensor surface using Eq. 1
where pL is the adsorbed layer density, pp is the protein density, nL is the adsorbed layer refractive index, np is the protein refractive index, ns is the solution (bulk) refractive index, mL is the mass loading per unit area and tl
Fig. 16. DPI. A bench-top instrument for the study of protein structure in real time (Cross et al. 2004)
is the adsorbed layer thickness. From the mass loading it is straightforward to calculate the area per molecule according to Eq. 2:
where A is the area per molecule, Mw is the protein molecular weight and Nais Avogadro's number.
By using the measured values for the refractive index of the bulk solution the volume fraction of the layer occupied by protein (0p) can also be calculated using Eq. 3:
After calculation of the parameters described above, it is possible to draw inferences not only regarding the gross structures of the deposited protein layers, but also the likely orientation of the protein molecules within the layers.
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