Visualizing two-component protein diffusion in porous adsorbents by confocal scanning laser microscopy

The use of confocal scanning laser microscopy (CSLM) has recently been described for the visualization of intraparticle protein profiles during single-protein finite bath uptake experiments. By coupling of fluorescent molecules to proteins the penetration of porous media by labeled macromolecules could be detected by scanning single adsorbent particles for fluorescence emission after laser excitation. Thus the internal protein distribution profile, which is a central element in modeling of protein transport in porous adsorbents, became experimentally accessible. Results from the simultaneous visualization of two proteins by this technology are shown here. The use of two different fluorescent dyes for protein labeling and two independent detectors in the CSLM allowed for the first time ever the direct observation of a two-component diffusion process within a porous stationary phase. The finite bath uptake of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) to two different ion exchange adsorbents (SP Sepharose Fast Flow and Source 30S) and to an affinity adsorbent (Protein A Sepharose) was measured using Cy5 and Oregon Green as labels. Single adsorbent particles were scanned for intensity distribution of fluorescence emission from the two fluorophors. The intraparticle profiles obtained from the confocal images were translated into a relative protein concentration thus allowing the calculation of protein uptake kinetics from direct measurement in the stationary phase. The confocal technique may prove to be a very powerful means of data generation for modeling of multi-component mass transfer phenomena in protein adsorption.