Application Of Cationic Celluloses To The Separation Of Basic Proteins By Capillary Electrophoresis

Capillary electrophoresis (CE) has rapidly developed into an important technology in analytical biochemistry due to the advantages of high speed, great efficiency, low sample and reagent consumption and ease of automation. CE has made graven contributions to the accomplishment of Human Genome Project, and it becomes increasingly implemented in genomics, proteomics, glycomics, metabolomics, etc. However, the adsorption of protein onto the capillary wall seriously limits the application of CE to protein analysis, and it is one urgent problem to be solved in CE methodology.Capillary wall coating has been proved to be the most efficient approach for minimizing protein adsorption. The character of coatings plays a critical role for its antifouling ability. In order to develop new polymer coatings which are effective, environmentally friendly and biocompatible, in this dissertation cationic celluloses and its supported nanomaterial were explored as capillary coatings to separate basic proteins. The correlation between the molecular structure and the coating ability had been investigated. Main points of this dissertation are listed as following1. Quaternized celluloses (QCs) were used as new dynamic coatings in capillary electrophoresis for basic protein separation. QC can not only cover the silanol groups on the capillary inner surface, but also repel the basic proteins electrostatically to prevent protein adsorption. It was found that capillaries coated with QCs at the very low concentration were able to cause effective charge inversion and generate stable, reversed electroosmotic flows. The degree of substitution of quaternary ammonium group (DSQ) was the key factor for the coating ability of QC. With the growth of DSQ, the coating ability of QCs was enhanced, the analysis time was shortened and the protein adsorption was inhibited more effectively. Meanwhile, the EOF can be manipulated for various applications by using QCs with different DSQ, MW, concentration and at different pH conditions. Five basic proteins, that is, lysozyme, ribonuclease A, cytochrome C, bovine pancreatic trypsin inhibitorand chymotrypsinogen were baselinely separated in a wide pH range by using QC coated capillaries, and QCs were proved to be efficient coatings for the separation of basic proteins.2. A new method was developed for the determination of bovine lactoferrin (bLF) in milk. Bovine lactoferrin is a basic protein with diverse physiological functions. Due to its low content in milk, the conventional method for quantitative analysis of bLF was complicated. In this method, bLF was isolated from milk with cation exchange resin and then detected by dynamic coating capillary electrophoresis. The QC dynamic coating could inhibit the adsorption of bLF effectively and reduce the interference of other proteins in the milk. This method was fast, efficient and it was expected to be applied to the analyses of bLFs in other biological samples.3. QC supported gold nanoparticles (QC-Au NPs) were used as new dynamic coatings in capillary electrophoresis for basic protein separation. QC could disperse and stabilize AuNPs effectively to form the QC-Au nanocomposites. AuNPs could adsorb on the capillary inner surface and enhance the coating ability of QC-Au NPs. Meanwhile, the aggregation extent of QC chains might be reduced as the introduction of AuNPs, and then more quaternary ammonium groups could be exposed to interact with the capillary surface and repel the basic proteins. Thus QC-Au NPs combined the advantages of both AuNPs and QC, and it could generate stronger reversed EOF. Compared with QCs, QC-Au NPs could further improve the separation performance of basic proteins, especially at high pH conditions. QC-Au NPs were demonstrated to have great potential in protein analysis.4. Hydrophobically modified quaternized celluloses (HMQCs) were used as new dynamic coatings in capillary electrophoresis for basic protein separation. With the introduction of hexadecyl group to QC, besides the electrostatic interactions and hydrogen bonding interactions, HMQC could be attached to the capillary surface via enhanced hydrophobic interactions thus achieving better coating ability. The effects of the polymer concentration and the degree of substitution of hexadecyl groups (DSH) had obvious influence on the antifouling ability of HMQC. The results showed that, compared with QCcoating, HMQC coatings were able to generate stronger reversed electroosmotic flows and were more efficient in suppressing protein adsorption within a broad pH range. HMQC coating could improve the separation efficiency and reproducibity in the separation of basic proteins. The successful performance of HMQC coating was further demonstrated by the analysis of lysozyme isolated from egg white.5. A pressure-based method for the assessment of protein adsorption onto the capillary wall was developed. Generally, the antiadhesive ability of coatings is monitored by assessing the separation efficiency of proteins by electrophoresis. However, since the EOF can be altered with the use of different coatings, electromigration approach is not perfect for coating screening. In the pressure-based method, protein was driven by pressure and was propagated from different ends in the coated capillaries.The magnitude change of the protein detected along with the overall peak pattern was used to gauge the extent of protein adsorption and the antiadhesive ability of the coating. By using this simple method, we compared different coatings, and the results were consistent with the electrophoresis experiments. This pressure-based method used UV detector, and was expected to become a convenient approach for fast assessment of different coatings.