Application Of Mixed Polymer Brushes Based On Poly(2-methyl-2-oxazoline) And Poly(acrylic Acid) In On-line Preconcentration Of Lysozyme By Capillary Electrophoresis

Lysozyme,also known as muramidase,is a natural alkaline protein.Lysozyme can not only hydrolyze peptidoglycan of bacterial cell wall,leading to cell wall breakage and bacteria dissolution,but also bind to viral protein to make virus inactivated.Thus it has antibacterial,antiviral and anti-inflammatory properties.Moreover,it is considered as the model protein and used to study protein conformation,enzyme kinetics and its relation with molecular immunity.Nowadays,common techniques for lysozyme determination include enzyme linked immunosorbent assay(ELISA),high performance liquid chromatography(HPLC),capillary electrophoresis(CE),etc.CE has been widely used in protein separation and analysis due to its advantages of rapid speed,high efficiency,low solvent consumption,etc.Ultraviolet detector(UV)is the most common detection mode,however,the detection sensitivity of CE-UV is limited owing to the small injection volume and short optical path.Thus,on-line preconcentration techniques have been developed to enhance the detection sensitivity.On-line preconcentration is the method to make analytes enrichment through adjusting the states of target analytes and instrument parameters,and it has advantages of simple operation,low cost and so forth.In this work,a mixed polymer brushes coated capillary with switchable property toward lysozyme adsorption/desorption was developed and it was applied for on-line preconcentration of lysozyme by CE-UV in order to enhance detection sensitivity.The main contents are as follows:Firstly,poly(2-methyl-2-oxazoline)(PMOXA)and poly(acrylic acid)(PAA)with different chain lengths were synthesized through cationic ring-opening polymerization and reversible addition-fragmentation chain transfer(RAFT)polymerization,respectively.Afterwards,glycidyl methacrylate(GMA)and PMOXA were used to prepare poly(2-methyl-2-oxazoline)-random-glycidyl methacrylate(PMOXA-r-GMA)via radical copolymerization,and poly(acrylic acid)-block-poly(glycidyl methacrylate)(PAA-b-PGMA)was obtained by RAFT polymerization of GMA and PAA with different chain lengths.Mixed solution of PMOXA-r-GMA and PAA-b-PGMA was then injected into capillary with certain mass ratio,and the mixed polymer brushes coated capillary based on PMOXA and PAA was fabricated after one-step annealing procedure.X-ray photoelectron spectroscopy(XPS)was used to characterize surface composition of capillary raw materials.The results showed that PMOXA-r-GMA/PAA-b-PGMA coating could be successfully obtained by one-step annealing protocol,and carboxyl contents in coatings increased with increasing chain length of PAA.Fluorescein isothiocyanate-labelled lysozyme(FITC-lysozyme)adsorption assay demonstrated that the coated capillary possessed switchable property toward lysozyme adsorption/desorption upon pH and ionic strength(I)trigger.At pH 7 and I=10-5 mol/L,it could adsorb large amount of lysozyme,and adsorbed lysozyme could be released when the condition changed to pH 3 and I=10-1 mol/L.Subsequently,the mixed polymer brushes coated capillary with switchable property toward lysozyme adsorption/desorption was applied to on-line preconcentrate lysozyme by CE.Under optimized conditions,on-line preconcentration performances of lysozyme on different coated capillaries were studied.It was observed that PMOXA-r-GMA/PAA-b-PGMA coated capillary possessed better enrichment effect compared to pure PAA-b-PGMA coated capillary owing to the addition of PMOXA-r-GMA.Moreover,lysozyme enrichment effect on PMOXA-r-GMA/PAA-b-PGMA coated capillary was improved with increasing chain length of PAA.The best on-line preconcentration performance was obtained when the chain length of PAA was 2.2 times that of PMOXA with sensitivity enhancement factor in terms of peak area and peak height of 17.69 and 6.74,respectively.The limit of detection and quantitation could reach 8.7 × 10-5 g/L and 3.0 × 10-4 g/L,individually.The relative standard deviation(RSD)values of intra-day and inter-day for peak areas were 2.9%and 4.1%,respectively.The RSD values of intra-day and inter-day for migration time were 0.9%and 2.1%,respectively.The developed method for the preparation of the coated capillary with good stability only needs one step in this work,and this research will supply a simple and effective way to analyze trace lysozyme by CE.