Effects Of Polymer Additives On The Performance Of Cyclic Olefin Copolymer Microchip Electrophoresis And Their Analytical Applications

Cyclic olefin copolymer?COC?,which exhibits the advantages of excellent chemical stability,high optical transparency,high resistance to chemicals including polar organic solvents,strong acids and bases,and good processibility,is an ideal material for microfluidic chips.However,like other polymer materials used for microfluidic chip fabrication,its surface is highly hydrophobic.When COC microchip electrophoresis?MCE?is used in the analysis of sample containing proteins and other biological macromolecules,the non-specific adsorption of these components will cause uneven surface charge,broadening of the sample band,unstable electroosmotic flow,which will degrade the analysis efficiency and reproducibility.The channels used for chip electrophoresis are generally short,the unbalanced liquid levels and the different meniscus shapes between each resevoirs will cause unexpected pressurized laminar flow of solution,leading to the dispersion of the sample zone,negatively affect the reproducibility of quantitation and separation efficiency,skillful operators are required to obtain reliable results.And it is impossible to achieve the specific separation of the proteins in complex biological samples by MCE,the specific extraction of the proteins before MCE analysis is necessary.In addition,when MCE-laser-induced fluorescence used for the detection of the sample with no fluorescence or weak fluorescence,fluorescence derivation is normally needed and the time of most derivative reactions is far longer than the time of MCE separation.It is necessary to develop fast and efficient fluorescence labeling methods.Therefore,the goal of this dissertation is to explore simple and low-cost methods to improve the electrophoretic analysis performance of COC microchips.The main outputs of this dissertation include:?1?Neutral hydroxypropyl cellulose?HPC?,polyanionic sodium carboxymethyl cellulose?CMC?and polystyrene sulfonate?PSS?were used as multifunctional buffer additives,the effects of these additives on the electrophoretic behavior of ephedrine and pseudoephedrine were systematically studied.?2?The interactions of hydroxyethyl cellulose?HEC?,polyoxyethylene?PEO?,polyvinylpyrrolidone?PVP?,polyacrylamide?PA?,HPC and PSS with the surface of COC microchips and their effects on electrophoretic separation of proteins when using as multifunctional buffer additives were systematically studied.?3?Instant formation of solid chitosan/?-glycerol phosphate?CS/?-GP?hydrogel within separation microchannels of COC microchips by electric field was achieved.The used hydrogel could be swiftly removed with acetic acid also by an electric field,and fresh hydrogel was formed after a simple rinse with water.The method was used for the detection of transferrin and lactoferrin in human serum samples.?4?FITC was used to rapidly label proteins at elevated temperature.It only takes5 minutes including the derivatization and the MCE-LIF.This method was used to monitor the purification process of enhanced green fluorescent protein?EGFP?in E.coli lysates.?5?The magnetic composite nanomaterials of Fe₃O₄@PDA@Ni-MOF was successfully prepared by hydrothermal reaction.It was used for the specific extraction of His-GFP in HeLa cell lysates.The sensitive detection of His-GFP was realized by MCE-LIF detection.This dissertation contains six chapters:Chapter 1:MCE analysis system was briefly introduced,which include:sample injection,separation mode and detection methods,common problems in MCE,methods for improve the analytical performance of MCE,application of polymers in MCE,and the application of MCE in biochemical analysis.Chapter 2:Using ephedrine?E?and pseudoephedrine?PE?as model analytes,the effects of neutral HPC,polyanionic CMC and PSS as multifunctional buffer additives on electrophoretic behaviors were studied.HPC,CMC and PSS all could increase the surface hydrophilicity of the COC chips.When HPC was used as buffer additive,the analyte migrated to the anode,and when CMC or PSS were used as buffer additive,the analytes migrate to the cathode.When 0.3%HPC?pH 9.5?and 0.3%CMC?pH9.0?were used as buffer additives,E and PE could be baseline separated?Rs were 1.2and 1.1,respectively?.When PSS was used as the background electrolyte,E and PE could not be resolved,and the separation could only be realized with the addition of?-cyclodextrin.The concentration of PSS and the pH of the running buffer had a great influence on the migration times and the plate numbers.The results showed that HPC and CMC played excellent roles in surface modification,buffer viscosity regulation,electroosmotic flow control and analyte separation.Chapter 3:By measuring the contact angle of the COC microchip surface after soaked in the water soluble polymer?HPC,HEC,PSS,PEO,PA and PVP?solutions at25 and 80?,it was found that,except HPC,heating?up to 80??could enhance the interaction between the polymer and COC.All the six polymers could increase the hydrophilicity of the surface of COC chips.The electroosmotic flow?EOF?difference between a COC chip treated with PSS and the untreated one is not significant,and the other five polymers could effectively inhibit the EOF.Using HSA and lysozyme as model proteins,the inhibition of non-specific adsorption of proteins on microchannel surface of COC chips with these polymers was studied by measuring streaming potentials.The results proved that PEO,PVP,HPC and HEC could effectively inhibit non-specific adsorption of proteins on COC surface,but PA and PSS had poor inhibition efficiency.When polymers were used as buffer additives to separate proteins and peptides,the repeatability was poor with PSS and PA,and the interaction of hydrophobic groups of PVP and HPC with proteins reduced the separation efficiency.HEC and PEO could effectively inhibit nonspecific adsorption of proteins because of their stronger hydrophilicity.Three variants of EGFP could be baseline separated in 20 s with 1 cm of effective separation length,plate numbers of proteins could be>1.0×10⁶/m.Chapter 4:A rapid one-step method for in-situ formation and replacement of chitosan/?-glycerol phosphate?CS/?-GP?hydrogel within microchannels of COC microchips with an electric field same as the electric field used for electrophoretic separation was described.The advantages of fast formation,precise control of location and a well-shaped hydrogel interface were confirmed.The CS/?-GP hydrogel was formed in the separation microchannel within 15 s of the application of a voltage of 2000 V.The presence of hydrogels in the separation channel could effectively eliminate the pressure flow caused by the difference of liquid levels,and guaranteed a narrow sample zone?105?m?.The hydrogel was compatible with organic solvents,and when the content of organic solvents?methanol,acetonitrile,DMSO,ethanol and isopropanol?in running buffer reached to 90%,the hydrogel remained stable.The separation efficiency of CS/?-GP hydrogel maintained almost constant for over 200runs of FITC-labeled HeLa cell lysates and RSDs of migration times and peak areas were no more than 4.7%and 3.9%,respectively.The used hydrogel could easily be removed with acetic acid that was electrically driven into the microchannel,and a fresh hydrogel could be re-formed after a simple rinse with water.This method was used for the analysis of transferrin and lactoferrin in human serum samples and the recovery results were in the range of 90.0-104.9%.Chapter 5:A method for fast labeling of proteins with FITC and their analysis with COC MCE-LIF was described.The labeling efficiency of five fluorescent dyes(Alexa Fluor 488,NBD-F,NBD-Cl,FITC,Chromeo^TM P503)for recombinant protein A were compared.FITC was highly efficient,but the reaction time at 25??12 h?was far longer than the time of electrophoretic separation?2 min?.The reaction time could be reduced to 3 min by elevating reaction temperature to 95?.The labeling efficiency of trypsin and trypsin inhibitor at 95?for 3 min was higher than at 25?for 12 h.A sample could be analyzed within 5 min,which include the labeling process and MCE-LIF.This method was successfully used to monitor the purification process of EGFP in E.coli lysate.Chapter 6:In this work,a core-shell structured nanocomposite Fe₃O₄@PDA@Ni-MOFs containing a magnetite core of Fe₃O₄ coated with polydopamine?PDA?and a Ni-based MOFs shell have been synthesized by solventthermal method at 120?for 10 h.The Ni-based MOFs shell was synthesized with terephthalic acid as ligand,Ni?NO₃?₂·6·H₂O as the source of metal and DMF as the solvent.The Fe₃O₄@PDA@Ni-MOFs was characterized by TEM and FT-IR,the hysteresis loop was measured.Compared with Fe₃O₄,this material had better dispersibility and stability in aqueous solution,and it showed superparamagnetism,could be separated rapidly with an external magnet.The adsorption kinetics of His-GFP and the recycling efficiency of the material were systematically studied.The adsorption efficiency for proteins did not change significantly after recycled five times.This material was used for the extraction of His-GFP in HeLa cell lysate,and its sensitive detection with MCE-LIF.