The Preparation Of Polydopamine-graft-PEG Coating And Its Application For Separation Of Biomacromolecules By CE

Capillary electrophoresis (CE), which shows high separation efficiency, short analysis time, low sample consumption, economic and environment process, has a wide range of application for the analysis of a wide variety of biomolecules such as polypeptide, protein and DNA. Unfortunately, in the bare fused-silica capillary environment, because of the coulombic and hydrophobic interactions, the analysis of biomolecules such as proteins have a propensity to strong adsorb onto the surface of the capillary, altering performance with potentially hazardous outcomes such as lower separation efficiency, poor repeatability and additional peak broadening. These phenomena restrict the application of CE.Recently, coating the capillary with polymer has been proved to be a very efficient and simple approach to control the wall-protein interactions. One of the most extensively studied antifouling polymers are PEG and its grafted copolymers, water soluble polymers with low toxicity and outstanding biocompatibility, which have widely been used to separate the protein, polypeptide and DNA by CE. However, PEG is ineffective to prevent the protein adsorption after several consecutive runs because of the PEG coating is easy to be washed away from the capillary wall due to its high hydrophilicity.The small molecule dopamine can be spontaneous oxidation to form a sticking polydopamine coating with oxygen over the pH range 7-9. The polydopamine coating coatains catechol and quinine functional groups, which can react with amine or thiol via Michael addition or Schiff base reaction. Therefore, through proper choice of secondary reactants, polydopamine coating can be transformed into surfaces that have specific chemical properties.In this paper, the antifouling PEG coating, attributed to the firmly anchored onto the capillary surface by sticking polydopamine coating, and was used for protein analysis and dsDNA separation. Working was on the following aspects:1. Procedure and characterization of polydopamine-graft-PEG coatingFirstly, the terminal hydroxyl groups of the PEG were converted into more reactive primary amino groups. The structure and composition of the amine functionalized PEG was characterized by 1H NMR and FTIR. Then, using a simple yet effective two-step approach, anchored the PEG onto the surface of capillary through polydopamine coating. During the process, polydopamine-graft-PEG copolymer was formed via Michael addition or Schiff base reaction. The copolymer coating was observed using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). And both of them indicated the formation of the copolymer coating.2. Polydopamine-graft-PEG coating for protein separation by CEThe PEG coating was anchored firmly onto the capillary surface by polydopamine coating which enhanced the stability of PEG on the capillary wall. The copolymer coating was shown to be capable of stabilizing and suppressing the EOF. Meanwhile, highly efficient and rapid separation of three basic proteins (i.e. Cytochrome C, Lysozyme, Ribonuclease A) has been obtained within a broad pH range. Furthermore, the separation performance of three basic proteins and six proteins mixture using a polydopamine-graft-PEG coated capillary and a commercial capillary was compared. To study the stable performance of polydopamine-graft-PEG coating, the 200 consecutive separations of basic proteins was performed by montoring the separation efficiency and migration time repeatability. The results proved that the polydopamine-graft-PEG coating, providing effective suppression of EOF and minimized adsorption of proteins, was a coating of excellent stability and good properties.3. Polydopamine-graft-PEG coating for qualitative and quantitative analysis of food proteins by CEPolydopamine-graft-PEG coating can provide minimized adsorption of proteins and excellent stability. So it has been used to qualitative and quantitative analysis of the three whey proteins:α-lactalbumin,β-lactoglobulin A andβ-lactoglobulin B in milk powder. Furthermore, the coated capillary is also well suitable for the analysis of egg white proteins and peanut proteins. The effectiveness of a polydopamine-graft-PEG coated capillary was evaluated in comparison with that of a bare capillary. The results demonstrated the coating has many advantages such as excellent reproducibility of migration times, lower relative standard deviation values and long service life.4. Polydopamine-graft-PEG coating for dsDNA separation by CEPolydopamine-graft-PEG and LPA can be potentially applied to separate dsDNA because LPA can provide superior sieving ability. Additionally, the copolymer polydopamine-graft-PEG coating has excellent antifouling ability which is important to stabilize and suppress the EOF during the separation. The effects of LPA solution concentration, LPA molecular masses and the applied electric field strength on dsDNA sample separation performances were studied in detail. The best separation result was achieved when the 4% (m/v) LPA (4.70 MDa) was applied to separate dsDNA.