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ORIGINALAdsorptive BSA Coating Method for CE to SeparateBasic ProteinsYan ManXueFei LvJaved IqbalFuchao JiaPeng XiaoMurtaza HasanQin LiRongji DaiLina GengHong QingYulin DengReceived:2 June 2012/Revised:28 September 2012/Accepted:1 October 2012/Published online:5 December 2012?Springer-Verlag Berlin Heidelberg 2012AbstractA novel and simple coating method wasdeveloped by coating bovine serum albumin(BSA)ontothe inner surface of a fused-silica capillary,to avoid theadsorption of analytes during CE.The advantage presentedhere was that the coating process is more simple,fast,stable,and reproducible.The coated capillary avoided theadsorption of analytes onto the inner surface of a fused-silica capillary and might be a promising candidate forseparation of complex biological samples with furtherdevelopment.Meanwhile,the efficiencies of the coatedcapillary were evaluated by EOF,chromatographic peakshape,and theoretical plate number(N m-1)of RNase A.The optimal coating conditions were obtained from theresults.The pH value of coating buffer PB was 4.2,thestanding time was 12 h at 4?C,and the coating concen-tration of BSA was 1.5 mg mL-1.The stability of thecoating on the inner wall of the capillary and the repro-ducibility of the coated capillaries were good.The theo-retical plate number values of RNase A were over1.3 9 105(N m-1)in the coated capillary.After succes-sive electrophoresis for 48 h using the coated capillary,theRSD values of EOF and the theoretical plate number were4.14%and 9.14%,respectively.In addition,the RSDvalues of EOF and the theoretical plate number(N m-1)inthe coated capillaries were 13.19%and 8.96%,respec-tively.Finally,the coated capillary was successfullyapplied to separate the mixture of four basic proteins(RNase A,lysozyme,trypsin and myoglobin).KeywordsCapillary electrophoresis?BSA coating?Adsorption?Basic proteins separationIntroductionCapillary electrophoresis(CE)has been developed into apowerful analytical technique for separations of peptides,proteins,and nucleic acids.It can provide a variety ofadvantages,such as high efficiency,short analysis time,ease of automation,and low sample consumption.ThoughCE possesses is of so many advantages,there still exists theadsorption between sample and the wall,which wasinduced by the anionic nature of the silanol groups on theinner surface of the capillary.As we all know,proteins canbe easily adsorbed to many different kinds of surfaces,containing the inner wall of the fused-silica capillary.Tobe exact,the adsorption mainly originates from the strongelectrostatic interaction between proteins,especially basicproteins and the inner wall.For CE analysis,the adsorptionwill lead to poor peak shape,irreproducible migrationtimes,and irregular electro-osmotic flow(EOF).So one ofthe main objective for CE analysis is to minimize proteinwall interactions by masking or modifying the silanolgroups on the inner wall of the capillary 1,2.Over the past years,several methods have been appliedto reduce the adsorption of proteins on the inner wall of thecapillary.Like extreme pH values 3,4,high salt con-centrations 5,or coating the inner wall of the capillary.However,under the conditions of extreme pH and high saltY.Man,X.Lv contributed equally to this work.Y.Man?X.Lv?J.Iqbal?P.Xiao?M.Hasan?Q.Li?R.Dai?L.Geng?H.Qing?Y.Deng(&)School of Life Science,Beijing Institute of Technology,Beijing 100081,Chinae-mail:F.JiaUniversite de Strasbourg,IPCMS(Strasbourg),UMR7504,Strasbourg,France123Chromatographia(2013)76:5965DOI 10.1007/s10337-012-2337-yconcentrations,proteins might undergo conformationalchanges,even denaturation 6,7.Coating is the most common method to avoid proteinadsorption onto the inner wall of the capillary.Such coat-ings can be classified as covalently bonded polymers andphysically adsorbed polymers coatings.Covalent coatingshave often been used in separation of proteins due to its higheffectivity.Barron et al.8 covalently attached tri(ethyleneglycol)-terminated alkyltrichlorosilane to create a very thincoating on the inner surface of a fused-silica capillary,andthe coating displayed good resistance to the adsorption ofcationic proteins,providing clean separations of a mixtureof lysozyme,cytochrome c,RNase A,and myoglobin formore than 200 consecutive runs.Xiao et al.9 developed anovel covalent coupling method for coating of capillarieswith liposomes.The intra-and inter-capillary variations inEOF were 4.02%RSD(n=30)and 6.72%RSD(n=4),respectively,and the coated capillaries can be used to per-form analysis at least for 1 month without any performancedeterioration when stored at 4?C.It should be noted that theproduction of covalent coatings is tedious,which requiresmultiple time-consuming steps;moreover,this covalentcoating is not reproducible and uses the expensive or toxicorganic solvents.Compared with the covalent coatings,the physicallyadsorbed polymer coating shows several advantages 1,10,11:easily prepared,no use of organic solvents,andreproducible,for instance.Physical coating can be obtainedby either a dynamic or a static approach 12.Dynamiccoating is done by adding additives to the BGE,such asamines,surfactants,and some neutral polymers 13,andcan be quite effective to avoid protein adsorption.Forexample,five additives,potassium chloride,morpholine,cetyltrimethylammoniumbromide(CTAB),poly(vinylalcohol),and polyethyleneimine were used by Cifuenteset al.14 to reduce the adsorption of the proteins onto thecapillary inner wall.But when CE is coupled with massspectrometry(MS),the dynamic coating may deterioratethe ionization of the analyte.Haselberg et al.15 usedpolybrene and dextran sulfate to form a semi-permanentand non-covalent triple-layer coating,and was used toefficiently separate basic proteins by coupling the triple-layer coated capillaries with time-of-flight mass spec-trometry(TOF-MS).Peng et al.16 coated the inner wallofcapillarywiththehydroxyethylcellulose-graft-poly(N,N-dimethylacrylamide),which was used to separatebasic proteins successfully and were also used as a sievingmatrix to separate 11 fragments of the double-strand DNA(dsDNA)sample.Wang et al.17 developed another quitestable non-covalent coating,poly(1-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate),for the separation offour basic proteins:lysozyme,cytochrome C,ribonucleaseA,and a-chymotrypsinogen A.Apart from the above-mentioned coatings,there is stillanother kind of physical coatingprotein coating,but onlyfew reported.Coating one characteristic protein easilyadsorbed onto the inner surface of the capillary to inhibitthe adsorption of protein sample.VanTassel et al.18placed a thermally treated monolayer of adsorbed fibrino-gen on the internal surface of the fused silica capillary,andthe measured capillary surface potential and electro-osmotic mobility are reduced roughly 30%by the adsor-bed layer and are shown to be stable over the course ofseveral runs.Janssens et al.19 used the proteins,such asalbumin or hemoglobin.to form a polycation coating tosaturate the sites of adsorption on the inner surface of thecapillary to prevent adsorption of cationic species.Bonventet al.20 used one characteristic protein,ferritin(Ft),which was adsorbed to fused-silica capillaries,to prove thebinding of proteins to the capillary wall using atomic forcemicroscopy.The aforementioned studies demonstrate that it isimperative to find a simple coating method to avoid theadsorption of analytes during capillary electrophoresis.Here we introduce a simple coating of BSA to inhibit theadsorption of protein samples during CE.BSA is the mostabundant proteins in the blood,and the BSA coating candecrease the non-specific adsorption of proteins.A physicaladsorption process was used to coat the BSA onto the innerwall of the capillary.The pH of coating buffer,the standingtime of coating,and the coating concentration of BSA wereoptimized.Moreover,the stability of the coating on theinner wall of the capillary and the reproducibility of thecoated capillaries were evaluated.Finally,the performanceof the capillary coated with BSA in the inner wall wasvalidated by separation of four mixed proteins(RNase A,lysozyme,trypsin and myoglobin).Materials and MethodsMaterials and ChemicalsFused-silica capillary(375 lm o.d.9 75 lm i.d.)with apolyimide outer coating was purchased from Hebei prov-ince Yongnian County Ruipu Chromatogram EquipmentCO.,LTD(Hebei,China).Bovine serum albumin(BSA,purity C98%),ribonuclease A(bovine pancreas),myo-globin(equine skeletal),lysozyme(chicken egg white),trypsin,and dimethyl sulfoxide(DMSO)were orderedfrom Amresco Reagent Company(Cleveland,OH,USA).Methanol,hydrochloric acid(HCl),and disodium hydrogenphosphate dehydrate(NaH2PO4?2H2O)were produced byBeijingChemicalWorks(Beijing,China).Sodiumhydroxide(NaOH)and disodium hydrogen phosphate(Na2HPO4?12H2O)were bought from Beijing Chemical60Y.Man et al.123Reagent Company(Beijing,China).All of the chemicalswere of analytical grade.The pH value of phosphate buffer(PB)wasmeasuredwithmicroprocessorpHmeter(HNANA instruments,Woonsocket,RI,USA).The waterwas purified and deionized with a Milli-Q system(Milli-pore,France).All the solution was filtered with 0.45 lmsyringe filter(MEMBRANA,Wuppertal,Germany)beforeuse.The Procedure of Capillary CoatingPrior to the coating,about a 2 mm optical detection win-dow was prepared on the fused-silica capillaries by burn-ing.The detection window was protected by a plastic pipe(o.d.is about 375 lm).The effective length of the capillaryis 385 mm.PretreatmentNew fused-silica capillaries were initially pretreated by thefollowing steps:first of all,the capillary was rinsed insequence with flowing HCl(1 mol L-1)for 90 min anddeionized water for 30 min.Second,they were flushed withNaOH(1 mol L-1)for 120 min,deionized water for40 min,and nitrogen gas for 30 min,sequentially.CoatingA physical coating procedure was employed.BSA wasdissolved in 10 mM PB(pH 4.2).First,the capillary wasflushed with BSA solution for 15 min and then was storedat 4?C for 120 min with two ends capped.Second,thecapillary was flushed with the same BSA solution foranother 15 min and then was stored at 4?C 12 h with twoends capped.After this,the capillary was dried withflowing nitrogen for 30 min.Finally,the capillary wasequilibrated with 10 mM PB(pH 7.0)for 30 min andstored with two ends capped analysis at 4?C.For theregeneration,the capillary was easily redone following thepretreatment procedure and the coating procedure men-tioned above.Capillary Electrophoresis SystemExperiments were employed on the HP3Dcapillary electro-phoresis instrument(Agilent Technologies,Wilmington,DE,USA)equipped with a diode array UV detection.Thetotallengthofthefused-silicacapillarywas470 mm,andtheeffective separation length(from the inlet to the detectionwindow)was 385 mm.The capillary of CE was installed inthecartridge,andthetemperaturewassetat25?Cduringalltheexperiments.Theproteinsamplewasinjectedat50 mbarfor 3 s.The separation voltage was set at 15 kV,and theabsorbance wavelength of the UV detector was set at200 nm.The protein sample used in the experiment con-tained 0.6 mg mL-1RNaseA,0.6 mg mL-1lysozyme,1 mg mL-1trypsin,and 1 mg mL-1myoglobin,whichwere all dissolved in PB(pH 7.0).DMSO was chosen as theEOF mobility neutral marker with the concentration of1:7000(v/v)in PB.Between runs,the capillary was flushedfor 3 min(50 mbar)with the PB(pH 7.0),and the PB bufferof separation vials(the inlet and outlet vials)were changedevery four runs.Electropherograms were analyzed usingAgilent Chemstation B.02.01.The number of theoreticalplates(N m-1)was calculated based on peak width at halfheight.Results and DiscussionIn order to achieve the optimal effectiveness of coating,thecoating conditions,such as the pH value of the buffer,theconcentration of BSA,and the standing time were opti-mized in our study.The stability of the coatings on theinner wall of the capillary and the reproducibility of thecoated capillaries were evaluated.Finally,four basic pro-teins were successfully separated using the new capillarywith BSA coating under the optimal conditions.The per-formance of the BSA coated capillary was validated.Optimizing the pH Value of Coating Buffer(PB)and the Concentration of BSAIn this section,pH 4.2,and pH 7.0 of the coating bufferwere chosen according to properties of protein;meanwhile,a series of different coating concentrations of BSA(0.1,0.75,1.5,3,6,10,and 20 mg mL-1)were set.The samplecontaining RNase A and DMSO was used as analysis tar-get.The chromatographic peak shape and theoretical platenumber(N m-1)of RNase A and the migration times ofDMSO were used to evaluate the efficiency of the coatedcapillary.The computational formula of theoretical platenumber(N m-1)is N 5:54tRW12?2,where tRis themigration time of RNase A(min);W1/2is the peak width athalf height(cm).All the results were shown in Fig.1 including thecontrol result.The capillary of the control was the non-coated capillary.The Optimal Coating Buffer pHFrom the results of control group,shown in Fig.1 sample ait can be concluded that the separation efficiency of theRNase A and DMSO was not good according to the poorAdsorptive BSA Coating Method61123peak shape of RNaseA.The results of CE separation usingthe capillary coated with BSA under pH 7.0 of the coatingbuffer were shown in Fig.1a.Although RNase A andDMSO were not well separated in most of different con-centrations of BSA,there did exist two exceptions likesamples d and e.It should be noted that the baseline wasnot stable because of adsorption;besides that,the peakshape of RNase A of samples f,g,and h was poor,and themigration time of DMSO was irregular.All these provethat it was not reasonable for CE separation and to coatonto the inner surface under pH 7.0.But for CE separationusing the capillary coated with BSA under pH 4.2 of thecoating buffer was different,as unambiguously describedin Fig.1b,RNase A and DMSO represented by peak 1 andpeak 2 were already separated thoroughly.And all of thepeak shape of RNase A was much better than that inFig.1a.We had temporarily ignored the problems ofbaseline induced by adsorption,only from the shape ofpeak,it can be easily concluded that pH 4.2 was muchmore proper than that of pH 7.0 in the process ofseparation.In order to better understand the influence of pH value tothe separation,the theoretical plate numbers of RNase Aseparated in capillaries coated under different pH value ofcoating buffer were calculated,and the results are shown inFig.1c.When the pH value of coating buffer was 7.0,thetheoretical plate number of RNase A can be only calculatedat the concentrations of BSA of 1.5 and 3 mg mL-1,because the theoretical plate number of RNase A at theother concentrations of BSA cant be calculated due to thepoor shape of the peaks.It was not difficult to find that thetheoretical plate numbers of RNase A at the pH value of4.2 was at least 3 times higher than that of pH 7.0.The separation result of the coated capillary under thecoating buf