cytochrome-c-t and octadecyltrichlorosilane

Isoelectric focusing (IEF), a powerful technique for protein separation and enrichment, was successfully integrated into microfluidic paper-based analytical devices (μPADs) in this work. The μPADs for isoelectric focusing were fabricated by octadecyltrichlorosilane (OTS) silanization and subsequent region-selective plasma treatment. The system of IEF on μPADs could be easily assembled. And a series of conditions of the system were investigated, including the suitable concentration of ampholyte to create good pH gradient, the effect of polyvinylpyrrolidone (PVP) on electroosmotic flow (EOF) suppression, and focusing voltage applied on the paper channel. After optimization, simultaneous separation and enrichment of protein sample containing myoglobin and cytochrome C was successfully demonstrated. Besides, parallel IEF on multichannels were also achieved for the separation of multiple protein samples on one single chip, and their performance was compared with that of the conventional gel-IEF system. The developed IEF on μPADs exhibits appealing features such as low cost, simplicity, and disposability and are believed to have great application potentials.

Topics: Cytochromes c; Electroosmosis; Hydrogen-Ion Concentration; Isoelectric Focusing; Microfluidic Analytical Techniques; Myoglobin; Paper; Povidone; Silanes

We have employed in situ X-ray reflectivity (IXRR) to study the adsorption of a variety of proteins (lysozyme, cytochrome c, myoglobin, hemoglobin, serum albumin, and immunoglobulin G) on model hydrophilic (silicon oxide) and hydrophobic surfaces (octadecyltrichlorosilane self-assembled monolayers), evaluating this recently developed technique for its applicability in the area of biomolecular studies. We report herein the highest resolution depiction of adsorbed protein films, greatly improving on the precision of previous neutron reflectivity (NR) results and previous IXRR studies. We were able to perform complete scans in 5 min or less with the maximum momentum transfer of at least 0.52 Å(-1), allowing for some time-resolved information about the evolution of the protein film structure. The three smallest proteins (lysozyme, cytochrome c, and myoglobin) were seen to deposit as fully hydrated, nondenatured molecules onto hydrophilic surfaces, with indications of particular preferential orientations. Time evolution was observed for both lysozyme and myoglobin films. The larger proteins were not observed to deposit on the hydrophilic substrates, perhaps because of contrast limitations. On hydrophobic surfaces, all proteins were seen to denature extensively in a qualitatively similar way but with a rough trend that the larger proteins resulted in lower coverage. We have generated high-resolution electron density profiles of these denatured films, including capturing the growth of a lysozyme film. Because the solution interface of these denatured films is diffuse, IXRR cannot unambiguously determine the film extent and coverage, a drawback compared to NR. X-ray radiation damage was systematically evaluated, including the controlled exposure of protein films to high-intensity X-rays and exposure of the hydrophobic surface to X-rays before adsorption. Our analysis showed that standard measuring procedures used for XRR studies may lead to altered protein films; therefore, we used modified procedures to limit the influence of X-ray damage.

Topics: Adsorption; Cytochromes c; Hemoglobins; Hydrophobic and Hydrophilic Interactions; Immunoglobulin G; Muramidase; Myoglobin; Serum Albumin; Silanes; Silicon Dioxide; Surface Properties; X-Rays