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Separation of carboxylic acids in human serum by isotachophoresis using a commercial field-deployable analytical platform combined with in-house glass microfluidic chips

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Separation of carboxylic acids in human serum by isotachophoresis using a commercial field-deployable analytical platform combined with in-house glass microfluidic chips
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  AnalyticaChimicaActa 755 (2012) 115–120 ContentslistsavailableatSciVerseScienceDirect Analytica   Chimica   Acta  journal   homepage:www.elsevier.com/locate/aca Separation   of    carboxylic   acids   in   human   serum   by   isotachophoresisusing   a   commercial   field-deployable   analytical   platform   combinedwith   in-house   glass   microfluidic   chips Petr   Smejkal a ,Michael   C.Breadmore a ,   Rosanne   M.   Guijt b ,Jakub   Grym c ,Frantiˇ sek   Foret c , ∗ ,Fritz   Bek d ,   Mirek   Macka a , ∗∗ a UniversityofTasmania,ACROSSandSchoolofChemistry,Hobart,Australia b UniversityofTasmania,ACROSSandSchoolofPharmacy,Hobart,Australia c InstituteofAnalyticalChemistryof    TheASCR,v.v.i.,Brno,CzechRepublic  d  AgilentTechnologies,Waldbronn,Germany h   ig   h   l   igh   t   s ◮  A   glassITPmicrofluidic   chip   wasdesigned   foranalysis   of    lactateinhuman   serum. ◮  ITP   chip   wascompatiblewith   a   field-deployable   commercial   platform. ◮  LIF/LEDIF   indirectfluorescencedetection   was   used. ◮  Lack   of    research-flexible   commercialmicrofluidic   separation   platform   isaddressed. gra   phi   ca   l   a   b   s   t   ra   cta   r   t   i   c   le   i   nf   o  Articlehistory: Received30August2012Receivedinrevisedform9October2012Accepted12October2012 Available online 23 October 2012 Keywords: IsotachophoresisMicrofluidicchipFluorescenceIndirectdetectionCarboxylicacidsSerum ab   s   t   ra   ct Portable   andfielddeployable   analytical   instrumentsare   attractive   inmany   fieldsincludingmedicaldiag-nostics,   where   pointof    care   and   on-sitediagnostics   systemscapable   of    providing   rapid   quantitativeresultshavethe   potentialtovastly   improve   the   productivity   andthe   qualityof    medicalcare.Isota-chophoresis   (ITP)   isa   well   known   electrophoreticseparation   techniquepreviously   demonstrated   assuitablefor   miniaturization   inmicrofluidic   chip   format(chip-ITP).   In   thiswork,a   purpose-designed   ITPchip   compatible   with   a   commercial   end-usedtargetedmicrofluidic   systemwas   usedto   studydifferentinjection   protocols   and   toevaluate   the   effect   of    the   lengthof    the   separation   channelonthe   analyticalperformance.   Thein-houseITPchipswere   made   fromCorning   glass   andcompared   tothe   commercialDNA   chip   for   the   ITP   separationof    anions   from   the   hydrodynamic   injection   of    human   serum.Using   thein-house   ITPchip   theisotachophoretic   step   of    lactate   from   human   serum   wasapproximately   two   timeslonger.   Theresultsof    this   research   suggested   that   microfluidic   ITP   with   indirect   fluorescence   detection   isa   viable   technique   for   separation   of    organicacidsinhuman   serum   samples,   especially   when   achip   withsuitabledesign   is   used. © 2012 Elsevier B.V. All rights reserved.  Abbreviations: R6G,rhodamine6G;PVP,polyvinilpyrolidone;LE,leadingelectrolyte;TE,terminatingelectrolyte. ∗ Correspondingauthorat:Instituteof    AnalyticalChemistryASCR,v.v.i.,Veveri97,   60200Brno,CzechRepublic. ∗∗ Correspondingauthorat:PrivateBag   75,SchoolofChemistry,UniversityofTasmania,SandyBay,Hobart7005,Australia.Tel.:+61362266670;fax:+61362262858. E-mailaddresses: foret@iach.cz (F.Foret),Mirek.Macka@utas.edu.au(M.   Macka).0003-2670/$–see   frontmatter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2012.10.022  116  P.Smejkaletal./AnalyticaChimicaActa 755 (2012) 115–120 1.Introduction Oneofthekeyareasofinterestfortheapplicationof    portableandfielddeployableanalyticalinstrumentationisinmedicaldiag-nosticsaspointofcareandon-sitediagnosticssystemscanvastlyimprovetheproductivityandqualityof    medicalcare.Althoughmanyon-sitescreeningtestsprovideonlyqualitativeorsemi-quantitativeresults,miniaturizationof    analyticaltechnologyhasacceleratedthedevelopmentof    portableandfielddeployableinstrumentationtoenablemorecomplex,quantitativeassays.Inlastdecadefewcommercialmicrofluidicplatformsusinga   capil-laryelectrophoresisonchip(chip-CE)foranalysisof    DNA,RNAandproteins[1]wereintroduced.Oneofthosesmalldimensionsandhighefficiencyinstrumentsis   AgilentBioanalyzer2100.TheAgilentBioanalyzer2100isa   commerciallyavailable,portable,andfielddeployableanalyticalinstrumentbasedonachip-CEplatform.ThecommercialapplicationsincludeDNA,RNA,proteins,andcellanalysis,asready-to-useanalytics-orientedmethods.Ourresearchisfocusedonexaminingthisinstrumentasaflexibleanalyticalplatformforthedevelopmentof    newmethodsforotherareas,includinganalysisoflowmolecularweightana-lytes.PreviouslywedemonstratedtheuseoftheBioanalyzerforscreeningtestsof    diseasesassociatedwithchangedglycanpro-filesinpatients[2].Lloydetal.   usedtheBioanalyzer2100fortheseparationof    amphetamineslabeledwithfluoresceinisothio-cyanate[3].Nuchtavornetal.separatedNilebluestainedcellsof fivedifferentpathogenmicroorganisms[4].   Theseworksfocusedontheanalysisof    fluorescentlylabeledmoleculesormicroor-ganisms.However,derivatisationis   oftenundesirablebecauseitrequiresfunctionalgroupsforlabelingandthelabelingreactionincreasesthelengthof    theanalyticalprotocol.Recently,weusedisotachophoresiswithindirectfluorescencedetectionforsepara-tionofcarboxylicacidsontheBioanalyzer2100DNAchip[5].Theseparationofuptoeightcarboxylicacidswasdemonstratedandthemethodwasappliedforthequantitationofbenzoateinsoftdrinks.Whilesuccessful,softdrinksrepresenta   relativelysimplesam-ple,andthequestionwaswhetherthemethodcouldbesuitablefortheseparationof    ionsinsampleswitha   morecomplexmatrix.Inthiswork,wedevelopedamethodforon-chipITPfortheanal-ysisofsmallionsinserumtoassessitscapacityfortheanalysisofcomplexsampleslike   thosefrombiologicalorigin.Theratio-nalebehindusingITP   fortheseparationof    thistypeofcomplexsamplesisinthepotentialsimplicityoftheanalyticalmethod.Samplepre-treatmentis   typicallythemosttimeconsumingpartoftheanalyticalprocedure,andavoidingsamplepretreatmentwillsignificantlyspeedup   totalanalysistime.Thisisparticularlyimpor-tantforthedevelopmentof    pointofcareandon-sitediagnosticssystems.AlreadymorethanthreedecadesagoITPwasusedforsepara-tionofionsfromsamplessuchasserumorplasmawithoutanypre-treatment[6–11].WhiletheITPtheoryandinstrumentationiswelldevelopedanddocumented,mostof    thepracticalapplica-tionsrelyoncapillarysystemswithuniversalcontactconductivitydetection[12].Coupledcolumnisotachophoreticchipwithconduc-tivitydetectionhasbeenpreviouslydevelopedbyKanianskyetal.[13].Unfortunately,contactlessconductivitydetectionis   notavail-ablewiththeBioanalyzer.Moreover,sincetheITPzonesofminorsamplecomponentsmay   oftenbeonlylessthanamillimeterlong,thecommoncontactlessconductivitydetectors,developedforcap-illaryzoneelectrophoresis[14]areunsuitableduetotheirpoorspatialresolution.Ontheotherhandtypicalfluorescencedetec-torprovidesdetectionspotsizein   tensof    micrometersresultinginexcellentspatialresolutionsuitableforshortITPzones.SincethequantitationinITPisbasedonmeasuringthezonelengthratherthanmonitoringthesignallevel,indirectfluorescencedetectionis   asuitableuniversalwayformonitoringtheITPanalysis[15,16].   Here,wedescribetheanalysisof    organicacidsinserumbyITPwithindi-rectfluorescencedetectionusingtheAgilentBioanalyzer2100.Wedevelopeda   newglassmicrochipforITPcompatiblewiththeBio-analyzer2100to   improveanalyticalperformanceoverthestandardcommerciallyavailableDNAchips.Themicrochipdesignincludedfourdifferentchannellengths,25.7,34.5,62.8,and125.8mm,   forcomparisonof    pressure,vacuumandtwo   waysofelectrokineticinjection.Theoptimaldesignusedthe25.7mmITPchannelwithpressureinjection,wheretheITPstepsforlactatefromhumanserumweretwiceaslongasthoseobtainedinthestandardDNAmicrochip. 2.Experimental  2.1.ITP    in-housechipfabrication 2.1.1.Instrumentationandchemicals TheITPchipwasdesignedbyusingthefreesoftwareDraftSight(DassaultSystemes,VelizyVillacoublay,France).Themicroflu-idicchipsweremadefromsoda-limeglassslidesobtainedas76mm   × 26mm × 1mm   microscopeslides(MenzelGläser,Braun-schweig,Germany)or75mm   ×   50mm   × 1mm   microscopesslides(Corning,NY,USA).ChipsweremadebyemployingmasklessphotolithographyusingaLaserPatternGenerator(  PG101,Hei-delbergInstruments,Heidelberg,Germany)andwetetchingtoadepthof    13  m.   Detailedinformationregardingmaskfabricationandglasswet   etchingareincludedinsupportinginformation.Toaccesstheetchedchannels,16holesweredrilledunderalayerofcoolingwaterinthecoverglasssheetbyusingdiamonddrillbits.Thermalbondingat590 ◦ C   (LaboratoryFurnace5013L,Cla-sic, ˇ Revnice,CzechRepublic)wasusedtosealtheetchedchannelswithaglasslid,whichcontainedthe16holes(2mmindiameter).Afterbonding,eachchipwascutwitha   DicingSaw(EC-400,MTIcorp.,Richmond,USA)intoa   17.5mm   square.TheglasschipsweregluedintotheblackPMMA   plasticframealsousedforthecom-mercialchips(AgilentTechnologies)bytwo   minuteepoxyglue(obtainedfroma   localhardwarestore)depositedasalayertoensurethatallreservoirsentrancesinglasschipwerewellsep-aratedandthatthein-housechipwasfittedwellintheplasticcaddy.  2.2.Isotachophoresisofhumanserum 2.2.1.Instrumentationandchemicals TheAgilentBioanalyzer2100(AgilentTechnologies,PaloAlto,CA,USA),isa   smalldesktopinstrumentusingchipsinareasmallerthan3cm 2 .   Thisdimensiondoesnotallowto   incorporateanyvents.Therefore,to   attaina   zeroflowateachof    theunusedchannels,thechipreservoirswerefilledintothesamelevelandsystemwasoper-atedduringtherunundera   zerocurrentsettoallunusedelectrodes.ThecommercialDNAchipsandin-houseITPchipswereusedforallITPexperiments.ITPzoneswerevisualizedbyindirectfluorescencedetectionasdescribedpreviously[5].   Fluorescencemicroscopywasperformedwithaninvertedmicroscope(NikonEclipseTi-U,Tokyo, Japan)usingSemrock390/482/563/640nmBrightLinequad-bandbandpassexcitationfilter,SemrockDI01–R405/488/561/635nmdichroicfilterandSemrock446/523/600/677nmmultibandemis-sionfilter(Rochester,NY,USA).Hydrochloricacid(32%)andpropionicacid(99%)werepur-chasedfromMerck(SanDiego,CA,USA).Sodiumpyruvate(99%),sodiumlactate(syrup,60%(w/w)),sodium3-hydroxybutyrate(99%),  -alanine(99%),polyvinilpyrolidone(1.3MDa),rhodamine6Gchloridesalt(99%)wereallpurchasedfromSigmaAldrich(St.Louis,MO,   USA).Milli-Qwaterwasusedforallexperiments.  P.Smejkaletal./     AnalyticaChimicaActa 755 (2012) 115–120 117  2.2.2.Leadingelectrolyte,terminatingelectrolyteandsamples ConditionsforITPofcarboxylicacidsin   humanserumwereadoptedfromourpreviouswork[5].   Theleadingelectrolyte(LE)wasprepareddailybymixingtwostocksolutionsin   a   ratioof 1:1(v/v).Thefirststocksolutioncontained100  MRhodamine6G(R6G)dissolvedinwater,andthesecondstocksolutionwas   pre-paredbydissolving2%(w/v)polyvinilpyrolidone(PVP)in40mMhydrochloricacid,adjustedtopH3.3with  -alanine.Theterminatingelectrolyte(TE)waspreparedbydilutingpro-pionicacidtoa   concentrationof    50mM.Amodelsamplewaspreparedbydissolvingpyruvate,lactateand3-hydroxybutyrateinwater.Humanserumwas   collectedfromahealthyvolunteerbystaffatHobartPathologyandwas   pipettedintoplasticvialsandstoredinthefreezerat − 20 ◦ C. 3.Resultsanddiscussion  3.1.DNAchip:electrokineticandpressureinjectionforITPof serumsample Previouslywe   developedanITPsystemfortheanalysisofben-zoateinsoftdrinksontheBioanalyzer2100usingthecommercialAgilentDNAchip[5].   In   Fig.1Aisshownthat   thismethodfailedfor   theanalysisofcarboxylicacidsin   humanserum,withnostepsobservedwitha200sinjectionandonlyoneminorstepafterinjectiontimesof500and1000s,   respectively.Theelectrokineticinjectionusedto   injectsampleintothemicrochipswassuitableforthelowconductivitysamplesusedbefore,butnot   forhighconduc-tivitysampleslikeplasma,containinganabundanceofchloride,approximately100mM,   whichis   ∼ 100timeshigherthanthecon-centrationofthetargetanalytes.Further,aschloridehasamuchhighermobilitythantheorganicacidspresentinserum,chlorideionsarepreferentiallyinjected.Thusunderelectrokineticinjec-tionconditions,themajorityofionsinjectedintothemicrochannelarechlorideionsbecauseof    itshighermobilityandnaturallyhighabundance,makingelectrokineticinjectionunsuitableforthissam-pletype.HydrodynamicinjectionwasconductedonaDNAchipbyapply-ingavacuumasillustratedinFig.2.InthefirststeptheLE   wasloadedintothereservoir(+).Pressurewasappliedto   thisreservoirbya1mL    plasticsyringewithrubbersealing(0.5mL    for10s),andLEfilledallthechannelsin   theDNAchip.All   reservoirsexceptthosereservedforTEandsamplewerefilledwithLE.   ThreereservoirswerefilledwithTE(blue)andthreewithsample(red).Vacuum Fig.1. ITPofhumanserumona   commercialDNAchipbyBioanalyzer2100using(A)electrokineticand(B)vacuuminjection.Conditions:LE=   20mMHCl+  -alanine(pH   3.3)+1%PVP+   50  MR6G;TE   =50mMpropionicacid;sample=humanserum;injectionmethods(A)electrokinetic(serum/TE − 0.45/ − 0.15  A,waste+0.6  A,details[5])and(B)   hydrodinamicbyvacuum(30sby   1mL    plasticsyringe,formoredetails   Section3.1);currentusedforITPseparationwas − 0.3  A. Fig.2. (A)Amanualvacuuminjectionfor30swasusedto   filltheDNAchipwithsam-ple   andTE(dartsmarkingflowdirectionof    theelectrolytes).Afterapplyingvacuum,thevacuumreservoirwasfilledwithLE.ThefilledchipwasplacedintheBioana-lyzer2100andITPseparationwasstartedbyapplyingcurrent( − 0.3  A)   betweenreservoirs(+)and( − ).(B)Imagecapturedwitha   fluorescencemicroscopeshowsthevacuuminjection. wasappliedtothereservoirmarkedasvacuumfor30sbyusinga1mL    plasticsyringewithrubbersealingandafterthat,thereser-voirwasfilledwithLEtopreventhydrodynamicflow.ThefilledchipwasplacedinsidethebioanalyzerandITPseparationwasstartedbyapplyingseparationcurrentbetweenthereservoirs( − )( − 0.3  A)and(+)(+0.3  A).Allotherelectrodesweresetto   currentof0  A.Thishydrodynamic(nodiscriminative)injectionmethodintroduceda   serumsampleintoaDNAchipandresultedinanisota-chopherogramasshowninFig.1B.However,theslopingtransitionsbetweenthefirststepssuggestthat   thestepswerenot   fullydevel-opedandsteady-statehadnotbeenreachedwhenthezonespassedthedetector.Thisshowstheneedfora   purposedesignedITPchip.  3.2.In-houseITPchipdesign TheresultsusingthecommercialDNAchipsfortheexperimentsaboveindicateda   numberof    deficienciesinthechipdesignwhenusedforITPof    serum.Theseparationof    ionsbyITPon   a   chipcanbeimprovedbyusingspecificallydesignedchips[17–21],   anapproachundertakenheretoimprovetheperformancecapabilityof    ITPin   thebioanalyzer.ToensurecompatibilityoftheITPchipwiththeBioanalyzer2100instrumentanditsdetectionsystem,theglassmicrochipneededtobebondedtotheplasticchipholderusedfortheDNAchipstopositionelectrodesin   thefluidreservoirsin   theinstrument.Furthermore,theseparationchannelneededtobeinthesameposi-tionasthat   usedinthecommerciallyproducedchipssuchthatopticalalignmentcouldbeperformed.Thepositionsofthenon-flexiblecomponentsof    themicrochipareshowninFig.3Aandwereobtainedfromtheliterature[22,23]andmeasurementsusingamicroscope.Fig.3Bshowsthelayoutof    thein-houseITPchipswhichcanbe   dividedintofourquadrants,eachcontaininga   singleITPchannel.EachITPchannelwas   composedof    a   wideandanar-rowsection.Thewidesectionwasthesameforallfourchannels(5mmlongand154  mwide)andalloweddifferentmethodsforloadingsamplesintoa   chip.Thenarrowchannelwas   10  mwideanditslengthwas   differentforeachfourchannels.Theshortestchannelwas20.7mm   long,thesecondwas   29.5mm,   thethirdwas57.8mm,andthelongestchannelwas   120.8mm.   Thetotallengthof    separationchannelswas   25.7,34.5,62.8and125.8mm.AstheDNAchipsweremadefromsoda-limeglass,thesamematerialwas   selectedforuse.Soda-limeglassisa   non-standardizedmaterialanditspropertiescanbedramaticallydifferentnot    justbetweenproducers,butalsofrombatchto   batch.AsshowninFig.4,therewas   asignificantdifferenceinITPseparationsof    organicacids  118  P.Smejkaletal./AnalyticaChimicaActa 755 (2012) 115–120 Fig.3. Chipdesign;(A)dimensions(inmillimeters)whichareimportantforthecompatibilityofin-housechipswiththeAgilentBioanalyzer2100;(B)masterpat-tern   usedforchipfabrication.Reservoirsattheendof    eachchannelaremarked:S–sample,T–terminatingelectrolyteandW–waste(leadingelectrolyte).Theverticalnumbersrepresentthelengthof    eachITPchannel(inmillimeters)fromtheinjec-tion   point(junctionS–T).Dimensionsof    thewideandnarrowchannelsare154and10  m. Fig.4. ITPseparationof    modelacidmixture,performedinchipsmadefromsoda-limeglassobtainedfromtwo   differentsuppliers.Sample,comprising1mMpyruvate,1mM   lactateand1   mM3-hydroxybutyrate,wasinjectedintotheshort-estchannel(25.7mm   inlength)byapplyingpressure(1barfor1s).ITPseparationperformedwithaconstantcurrentof  − 0.2  A. obtainedforthetwodifferentglassmanufacturers.Isotachophero-gramsobtainedbyusingchipsmadefromMenzelGlasersoda-limeglassmicroscopeslideshadlonger,slopedITPsteps.Thiswas   mostlikelycausedbyresidualEOFevenattheelectrolytepHused(pH3.3)andwithacontentof    1%PVPin   LE.ShorterandsharperITPzoneswereformedusinga   chipmadefromCorningsoda-limeglassmicroscopeslides.Becauseof    theirsuperiorseparationper-formance,thechipsmadefromCorningglassweresubsequentlyemployedforallotherexperiments.  3.3.Sampleinjection Thein-housedesignedchipsweredesignedto   testdifferentinjectionmethods,asshowninFig.5.Fourdifferentapproachesto   injectionwerepossible,twobasedonelectrokineticinjection(AandB),andtwobasedonhydrodynamicinjection(CandD).Thesediffernotonlyin   thewayinwhichthesampleisinjectedintothechip,butinthevolumeinjectedandwhetherornotthesolu-tionswithinthechipneedtobechangedbetweeninjectionandseparation.Theonlymethodevaluatedthatcouldbeimplementedwithoutrefillingthechipwastheon-linemodeelectrokineticinjec-tion.Thisusedelectricfieldstodefinetheinjectionvolume,asdepictedinFig.5A.All   of    theother3methodsrequiredremovalof thesamplefromtheinjectionwellandreplacementwithTE.Injec-tionswerevisualizedusinga   fluorescencemicroscopebydirectlymonitoringthefluorescenceofR6GinLE.To   visualizethesample,adilutesolutionofCy5dissolvedin   Milli-Qwater,was   used.TheTE Fig.5.   Schemeof    electrokineticinjectionby   (A)on-lineand(B)off-linemode.Hydrodynamicinjection(C)by   vacuum(V)andhydrodynamicinjection(D)bypressure(P).ForB–D,solutioninthesample/terminatorreservoirwas   physicallychanged. didnot   containanyfluorescentdye,butitszonewas   slightlyvisi-bleduetomigrationof    R6GthroughtheITPsystemasacounterionfromtheLE.Thevideoobtainedfromthefluorescencemicroscopeandmoredetailsabouteachoftheinjectionmethodsareaccessiblein   thesupportinginformation.Whilethesimplestmethodforinjectionis   on-linemodeelec-trokineticinjection(A)asitis   performedwithoutrefillingthechip,itfailedtointroducea   sufficientamountofsampleoftheorganicacidsduetothehighabundanceandmobilityof    chloride,aspre-viouslydiscussedfortheDNAchip.Electrokinetic(B)andvacuum(C)injectionwerehighlyirreproduciblebecauseofthelossof    thesamplewhenflushingtheinjectingreservoirwithTE(ascanbeseeninvideomentionedabove).Themostpromisingresultswereobtainedwiththepressureinjection(using1mLplasticsyringewithrubberseal).Thisinjectionintroducedthelargestamountof sample,whichincreasedthesystemsensitivity.Thepressureandtimeneededtoinjectapproximatelythesameamountof    asampleintothedifferentchannelsinthein-housechipswereexaminedbyusingfluorescencemicroscopy.Thevolumeof    airin   thesyringeandtimeusedforpressureinjectionwas   strictlyspecificforeachchan-nel.Forexample,tofillthewidepart   oftheshortestchannelthepressure(20  L    in   thesyringe)wasappliedfor1s,thesecondshort-estchannelneededto   applythesamepressurebutfor3s.Thethirdchannelwas   filledbyapplyingpressure(100  L    in   thesyringe)for3s,andthelongestchannelusedthesamepressurebutappliedfor5s.Theinjectionpressureandtimeweredifferentnotjustbetweenchannelsdimensionsbutevenbetweenthesamechannelsontwodifferentin-housechips.InFig.6canbe   seenthatthepressureinjectionintroducedthelargestamountof    samplebetweentheLEandTE,   andtheresultingisotachophoregramsdemonstratedthelongeststepsfromalltestedmethods.ThisensuredthatminorlosswhenflushingtheinjectingreservoirwithTEwasinconsequential  P.Smejkaletal./     AnalyticaChimicaActa 755 (2012) 115–120 119 Fig.6. Differentlengthof    pyruvatestepobtainedbyusingdifferentinjectionmeth-odsonanin-houseITPchip.Thebarsindicateconfidenceinterval( ˛ =   0.95)basedon   threereplicates. whencomparedtothevolumethathadbeeninjected.Despitetherelativelylargestamountof    introducedsamplein   thechipwiththepressuredriveninjection,itexhibitedrelativelypoorrepro-ducibility.ThecalculatedRSDbasedontenreplicateswas15.54%forpressuredriveninjection.  3.4.Microchannellength As   mentionedabove,thenarrowpartof    eachITPchannelin   thechiphaddifferentlengthsinorderto   studythelengthrequiredfortheITPsystemtoreachsteadystate.InaccordancewiththeITPprinciples[12]theresultsfromtheisotachopherogramsinFig.7 showedthatthelengthoftheseparationchannelonlyinfluencedthetotalanalysistime   anddidnotaffectthelengthof    thesteps.Theshortestchannel(25.7mm)   andpressureinjectionwerechosenforITPanalysisofthehumanserumsample.  3.5.ITPofhumanserum Theprocessofanalysisof    thehumanserumstartedwithfillingthechipwithLE,TEanduntreatedserum,asdescribedabove.Thefilledchipwasplacedin   thebioanalyzer,andtheprotocolrunningtheITPseparationwasstarted.Aconstantcurrentof    − 0.3  AwasappliedbetweenreservoirwithTE-T1andreservoirwithLE-W5(seeFig.3B),thefirstITPstepappearedafter830swiththeentireseparationcompletedwithin900s,ascanbe   seenin   Fig.8.   Incon-trast,theseparationshowninFig.1B,usingthecommercialDNAchipwithvacuuminjection,thefirststepappearedafter350sandwascompletein   425s. Fig.8. ITPof    humanserumusingtheshortestchannel(25.7mm)   of    thein-housechip   andpressureinjection.Conditions:LE=   20mMHCl+    -alanine(pH3.3)+   1%PVP+50  M   R6G;TE   =50mMpropionicacid;sample=   humanserum;Aconstantcurrentof    − 0.3  Awasusedforseparation,unusedchannels0    A. IsotachopherogramfromDNAchipshownin   Fig.1B,andfromin-houseITPchipshowninFig.8showsimilarresults.OntheDNAchip,thefirstsignificantITPstepwaslonger.Thesecondsignificantstep,lateridentifiedaslactate(standardaddition),was   longerintheisotachopherogramobtainedfromthein-houseITPchip.Thestepof    lactatewas   34.2slonginthein-housechipand17.95swiththeDNAchip,indicatingthatpressureinjectiononthecustomdesignedITPchipintroduceddoubletheamountof    sampleintothechannelwhenusingtheDNAchip.Moreimportantly,aswasmen-tionedbefore,thesloppytransitionsbetweenstepsontheDNAchipsuggestedthatthestepswerenotfullydevelopedandsteady-statehadnotbeenfullyreached.ItislikelythatthiswasthereasonwhythefirststepwaslongeronDNAchip.Whilethisindicatestheimprovementsfromthein-housedesignedchip,thelargedif-ferencebetweentheinjectedamountsresultedin   adoublingof thetotalanalysistimeforthein-housechip.Thistimedifferencecouldtheoreticallybereducedbyapplyinga   higherconstantcur-rent,however,thiswas   notpossibleduetothedimensionsandhighresistivityof    thenarrowchannels,andthelimitedhighvoltagethatcanbesuppliedbythebioanalyzer(1500Vmax.).FromFig.8,theadditionalstepbehindthelactatestepwasregardedasamixedzone.Thiswouldsuggestusingthelongersep-arationchannelonthein-housechip.Theattempttorepeatthesameconditionanalysisonalongerchannelfailedduetoinabilitytokeeprequiredconstantseparationcurrent − 0.3  A.Whiletheusingofin-houseITPchipsuccessfullydemonstratestheincreaseddetectionlimitsbya   factorof    two,therepeatabilitywaspoorduetoaninabilitytoaccuratelycontrolpressureinjection. Fig.7. Effectofdifferentlengthsofseparationchannel.Conditions:LEcontained100mMHCl,1%PVP,50  MR6Gand  -alanine(pH3.3),TEcontained100mMpropionicacid.Thesamplecontained10mMpyruvate,10mMlactateand10mM3-hydroxybutyrate.The   timeandpressureusedforthesampleinjectionweretestedineverychannelseparatelybeforecommencingtheexperiments.Aconstantcurrentof  − 0.5  A   wasusedforallexperiments.(A)isotachopherogramsshowtimedelaybetweendifferentseparationchannelsand(B)alignedisotachopherogramsshowthatsimilardatawereobtainedfromallfourITPchannels.
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