微流控分析芯片

第八章微流控分析芯片Doyouknow?Biochip 生物芯片Lab-on-a-Chip 芯片實(shí)驗(yàn)室 LabchipMicrofluidicChip 微流控芯片MicroTotalAnalysisSystem (MicroTAS,

TAS) 微全分析系統(tǒng)Researchintominiaturizationisprimarilydrivenbytheneedtoreducecostsbyreducingtheconsumptionofexpensivereagentsandbyincreasingthroughputandautomation.Forexample,mostareawareoftheincreasingcostofhealthcare,driveninpartbythecostofimplementingthelatestdiagnosticassays.Theseassays,whichareusuallyperformedinmicrotiterplatesthatconsumehundredsofmicrolitersofreagents,wouldbenefitfromtheuseofmicrofabricatedarraysofnanolitervolumevials.Byreducingreagentconsumptionbyafactorof103–104,thesedevicescouldprovidedramaticsavingsfortherepetitiveassaysoftenperformedindiagnosticlaboratories.Whyminiaturization？尺寸效應(yīng)〔cm→100m〕尺寸 1/100分子擴(kuò)散時(shí)間 1/10,000(1h→0.36s)體積 1/1,000,000 試劑用量 1t→1g,ml→nl傳熱速度 1500oC/s由此將帶來：方法上的變革理論上的突破還有巨大的經(jīng)濟(jì)和社會(huì)效益陣列芯片微流控芯片微流控分析芯片微流控分析芯片目的是通過化學(xué)分析設(shè)備的微型化與集成化，最大限度地把分析實(shí)驗(yàn)室的功能轉(zhuǎn)移到便攜的芯片中。微流控分析芯片通過微機(jī)電加工技術(shù)把整個(gè)實(shí)驗(yàn)室的功能，包括采樣、稀釋、加試劑、反響、別離、檢測等集成在幾平方厘米的微流控芯片上，且可屢次使用，因而極大地減少了樣品和分析試劑的用量，降低了分析的本錢，加快了分析的速度，具有廣泛的適用性。參考文獻(xiàn)

D.Figeys,D.Pinto,Lab-on-a-Chip:ARevolutioninBiologicalandMedicalSciences,AnalyticalChemistry,2000,72,330AD.R.Reyes,D.Iossifidis,P.A.Auroux,A.Manz,MicroTotalAnalysisSystems.1.Introduction,Theory,andTechnology,AnalyticalChemistry,2002,74,2623P.A.Auroux,D.Iossifidis,D.R.Reyes,A.Manz,MicroTotalAnalysisSystems.2.AnalyticalStandardOperationsandApplications,AnalyticalChemistry,2002,74,2637T.Vilkner,D.Janasek,A.Manz,MicroTotalAnalysisSystems.RecentDevelopments,AnalyticalChemistry,2004,76,3373馬立人，蔣中華，生物芯片，化學(xué)工業(yè)出版社，北京，2002方肇倫，微流控分析芯片，科學(xué)出版社，北京，2003目錄8-1開展歷史8-2相關(guān)理論8-2-1根本概念8-2-2理論描述和模擬8-3根底技術(shù)8-3-1微制造8-3-2界面和連接8-3-3流體控制8-4單元操作8-4-1樣品制備8-4-2注射8-4-3流體和顆粒操作8-4-4反響器和混合器8-4-5別離8-4-6檢測8-5應(yīng)用8-5-1細(xì)胞培養(yǎng)和操作8-5-2免疫分析和臨床檢驗(yàn)8-5-3蛋白質(zhì)分析8-5-4核酸分析TheEarlyDays:1975-1989ThefirstanalyticalminiaturizeddeviceAgaschromatographicairanalyzerfabricatedonasiliconwaferTerry,S.C.Ph.D.Thesis,Stanford,Stanford,CA,1975Terry,StephenC.;etal.IEEETransactionsonElectronDevices,1979,ED-26(12),1880Aminiaturegasanal.systembasedontheprinciplesofgaschromatog.ThemajorcomponentsarefabricatedinSiusingphotolithog.andchem.etchingtechniques,whichallowssizeredns.ofnearly3ordersofmagnitudecomparedtoconventionallaboratoryinstruments.consistsofasampleinjectionvalve,a1.5-m-longcapillarycolumn.Athermalconductivitydetectorfabricatedonaseparatesiliconwafer.sepns.ofgaseoushydrocarbonmixts.areperformedin<10s.8-1開展歷史PhotographofagaschromatographintegratedonaplanarsiliconwaferfabricatedbyTerryandco-workersatStanfordUniversity.However,theresponseofthescientificcommunitytothisfirstsiliconchipdevicewasvirtuallynone,presumablybecauseofthelackoftechnologicalexperience(oftheseparationscientists)todealwiththiskindofdevice.theresearchworkrelatedtominiaturizationonsiliconfocusedonthefabricationofcomponentssuchasmicropumps,microvalves,andchemicalsensors.TheRenaissance:1990-1993thereemergenceofsilicon-basedanalyzersDesignofanopen-tubularcolumnliquidchromatographusingsiliconchiptechnologyManz,A.;etal.SensorsandActuators,B:Chemical(1990),B1(1-6),249Anovelconceptofhighpressureliquidchromatog.asiliconchipwithanopen-tubularcolumnandaconductometricdetector.A5×5mmchipcontaininganopen-tubularcolumnof6

m×2

m×15cmwasfabricated,whichhastheor.separationefficienciesof8000and25,000platesin1and5min,resp.Thetotalcolumnvolumeis1.5nLandthedetectioncellvolume1.2pL.MicrographofLiquidChromatographchipmanufacturedbyManzandco-workersatHitachiLtd.Theconceptof"miniaturizedtotalchemicalanalysissystem"or

TASwasproposedbyManzetal.themainreasonforminiaturizationwasthereforetoenhancetheanalyticalperformanceofthedeviceratherthantoreduceitssize.itwasalsorecognizedthatasmallsizepresentedtheadvantageofasmallerconsumptionofcarrier,reagent,andmobilephase.GrowingtoCriticalMass:1994-1997In1994,thenumberofpublishedpapersrelatedto

TASincreasedabruptlysincemoreresearchgroupsjoinedtheeffortstodevelopthearea.MicrofabricationDesignSeparationsBiochemicalReactorsDetection分類與特點(diǎn)分類：材料：硅、玻璃、石英、聚合物、復(fù)合材料功能：別離、采樣與前處理、檢測、化學(xué)合成等特點(diǎn)：高效、低耗、集成、一致性好、昂貴8-2相關(guān)理論

8-2-1根本概念遵循低雷諾數(shù)流動(dòng)的規(guī)律。除了組分間的擴(kuò)散，兩層或者多層流體可以相鄰流動(dòng)而不互相混合，使得樣品的混合變得困難。Anal.Chem.2002,74,45-51液體流動(dòng)的特點(diǎn)液體流動(dòng)的特點(diǎn)由于比外表積增大，外表張力、摩擦力的影響非常顯著。微通道中的液液界面與通道壁平行，因?yàn)橥獗韽埩湍Σ亮Υ笥谥亓?。Anal.Sci.2001,17,89-93液體流動(dòng)的特點(diǎn)液體的物理性質(zhì)發(fā)生變化，如表觀粘度變大純水通過微通道的時(shí)間：理論值 2.3ms實(shí)驗(yàn)值 10msAnal.Chem.2002,74,6170-6176Aq.Org.Aq.Org.Aq.Org.MicrochipMicrochannel多相流的模擬8-2-2理論描述和模擬擴(kuò)散的數(shù)字模擬BiophysicalJournal,2001,80,155–160不同尺寸通道的數(shù)字模擬MicrosystemEngineeringofLab-on-a-ChipDevices,5SimulationsinMicrofluidicsISBN:3-527-30733-8微結(jié)構(gòu)的形成

1．經(jīng)典的光刻技術(shù)Photolithographproceduresformakingglasstemplate.(a)Spinecoatingofphotoresist,

(b)coveredwithphotomask,

(c)exposure,(d)developing,(e)etching,and(f)removalofphotoresist.適合硅、玻璃、石英等材料，與傳統(tǒng)的半導(dǎo)體工業(yè)的方法一致。分為濕法和干法兩種，干法的分辨率較濕法高，相應(yīng)的制造本錢也高。Analyst,2004,129,305–3088-3根底技術(shù)8-3-1微制造微結(jié)構(gòu)的形成

2．模版澆注法

〔模塑法〕Processoverviewformassmanufacturingofplasticmicrofluidicsystems適合聚合物材料。大批量生產(chǎn)時(shí)本錢低。Anal.Chem.,2002,74,78A-86A微結(jié)構(gòu)的形成

3．模版熱壓法Schematicrepresentationofthefabricationmethodinvolvinghotembossingofthermoplasticpolymerpelletsandthermalbonding.適合熱塑性聚合物。AppliedPhysicsLetters,2002,80,3614-3616微結(jié)構(gòu)的形成

4．激光刻蝕法 用激光直接在聚合物或玻璃上加熱形成微結(jié)構(gòu).Anal.Chem.,1997,69,2035-2042芯片的封裝

1．熱鍵合 對玻璃和石英材質(zhì)刻蝕的微結(jié)構(gòu)一般使用熱鍵合方法，將加工好的基片和相同材質(zhì)的蓋片洗凈烘干對齊緊貼后平放在高溫爐中，在基片和蓋片上下方各放一塊拋光過的石墨板，在上面的石墨板上再壓一塊重0.5kg的不銹鋼塊，在高溫爐中加熱鍵合。玻璃芯片鍵合時(shí)，高溫爐升溫速度為10oC/分，在620oC時(shí)保溫3.5小時(shí)，再以10oC/分的速率降溫。石英芯片鍵合溫度高達(dá)1000oC以上。此方法對操作技術(shù)要求較高?，F(xiàn)代科學(xué)儀器，2001，4，8-12芯片的封裝

2．陽極鍵合在玻璃、石英與硅片的封接中已廣泛采用陽極鍵合的方法。即在鍵合過程中，施加電場，使鍵合溫度低于軟化點(diǎn)溫度。在500-760伏電場下，升溫到500oC時(shí)，可使兩塊玻璃片鍵合。在兩塊玻璃板尚未鍵合時(shí)，板間空氣間隙承擔(dān)了大局部電壓降，玻璃板可視為平行板電容器，板間吸引力與電場強(qiáng)度的平方成正比，因此，鍵合從兩塊玻璃中那些最接近的點(diǎn)開始，下板中可移動(dòng)的正電荷〔主要是Na+〕與上板中的負(fù)電荷中和，生成一層氧化物〔正是這層過渡層，使兩塊玻璃板封接〕，該點(diǎn)完成鍵合后，周圍的空氣間隙相應(yīng)變薄，電場力增大，從而鍵合擴(kuò)散開來，直至整塊密合?，F(xiàn)代科學(xué)儀器，2001，4，8-12芯片的封裝

3．室溫鍵合Anal.Chem.2004,76,5597-5602芯片的封裝

4．貼合

將聚合物薄片直接覆

蓋在玻璃或石英板上。

5．壓合Schematicillustrationofsealingandconnectionmethod.Thetopandbottomplatesarepressedbyascrewandholders.Anal.Chem.2002,74,1724-1728外表修飾

1．作為色譜固定相coatingofchannelwallswithsomething,e.g.octadecylsilanes,tocreatestationaryphasesAnal.Chem.2002,74,784-789Anal.Chem.2003,75,64A-69A外表修飾

2．固定化酶Schematicdiagramofstreptavidin-conjugatedalkalinephosphataseboundtoasupportedlipidbilayercontainingbiotinylatedlipids.Anal.Chem.2002,74,379-385外表修飾

3．固定化生物膜Langmuir2003,19,1624-1631外表修飾

4．親水或疏水修飾Analyst2004,129,284-287與外部溶液連接Anal.Chem.2000,72,1711-17148-3-2界面和連接與質(zhì)譜儀連接Basicfeaturesofanelectrosprayinterface.(A)Diagramofthelayoutofanelectrospray-chipincorporatingneedlesources(electrosprayemitters).(B)Scanningelectronmicrographofanactualchip.LabonaChip,2001,1,7N–12N外加壓力驅(qū)動(dòng)Anal.Chem.2000,72,1711-17148-3-3流體控制重力驅(qū)動(dòng)Anal.Chem.2005,77,1330-1337離心力驅(qū)動(dòng)CentrifugalMicrofluidicsPlatformAnal.Chem.2002,74,5569-5575毛細(xì)作用驅(qū)動(dòng)Microfilterdevicedesignanddetail:(a)topviewofgenericdevicedesignwithnarrowandexpandedchannels,(b)filterdetailareashowingfilterporesandexpandedchannellayout,(c)microfiltercrosssection.LabChip,2005,5,922-929利用電滲流驅(qū)動(dòng)與控制Generationandcontrolofelectroosmoticflowonmicrofabricateddevices.Anal.Chem.2000,72,330A-335A利用外表性質(zhì)控制Anal.Chem.2003,75,5097-5102單向閥Cylinderdiametersareapproximately100and25

m.Anal.Chem.2002,74,4913-49188-4單元操作

8-4-1樣品制備Anal.Chem.2002,74,1565-1571Sonication

AMinisonicatorToRapidlyDisruptBacterialSporesforDNAAnalysisAnal.Chem.,1999,71,4232-4236Theminisonicatorandplasticsporelysiscartridge.Samplesofsporeswereloadedintothechamberthroughachannelinthecartridgewallandsubjectedtosonicationfor30s.

AmicrofluidiccartridgetopreparesporesforPCRanalysisThesporedisruptioncartridgesystem.Componentsincludedthefluidiccontrolunit,thecartridge,andtheminisonicator.Biosensors&Bioelectronics,2000,14,849–852ExtractionSideviewschematicofminiaturizedaffinitydialysisandconcentrationsystemAnal.Chem.,2001,73,2048-2053Conceptofsequentialion-sensingsystemusingsinglemicrochip

Anal.Chem.

2001,73,5551SchematicillustrationofexperimentalsetupandionpairextractionmodelAnal.Chem.

2001,73,1382-1386determinationofCo(II)as2-nitroso-1-naphtholchelatesbysolventextractionandthermallensmicroscopy

LabChip

2001,1,72-75PreconcentrationMicrofabricatedPorousMembraneStructureforSampleConcentrationandElectrophoreticAnalysisAnal.Chem.1999,71,1815-1819

Electrokinetic

Preconcentration

ofProteinsonthinPDMSMembranesmicrTAS,2003,Vol.2,1171-1174SchematicdiagramsofCEprocedureswithpinchedinjection(toppanels),floatinginjection(middle),and(C)simplestinjectionmode(bottom).Anal.Chem.,2001.73,2656-26628-4-2注射

Schematicofmicrochipusedforgatedvalving.Thedoublelinesrepresentmicrofabricatedchannels,andthesinglelinesrepresentelectricalconnections.Thehigh-voltagerelayisinthedispense(open)position.Anal.Chem.,1999,71(15),3273-3276,Whitelightimageof(a)microchipvalveandfluorescenceimagesof(b)sampleloading,(c)dispensing,and(d)analysismodes.Theinjectiontimewas0.4s.Inthesampleloadingmode,550,550,and0Vwereappliedtothebuffer,sample,andwastereservoirs,respectively.Inthedispensingmode,550and0Vwereappliedtothesampleandwastereservoirs,respectively.(a)Schematicofthecapillaryelectrophoresismicrodeviceequippedwithexternalvoltageelectrodes:1,injectorwasteport;2,separationportinlet;3,sampleinletport/injectorport;4,externalvoltageelectrode;5,separationwasteport;6,externalvoltageelectrode.Totalsizeofthestructure:2.54×7.62cm.

(b)Micrographoftheinjection(150pL)zonewithintheseparationchannel.Thesampleisinjectedfromreservoir3toreservoir1andseparatedfromreservoir2toreservoir5usingappliedvoltagefields.

(c)Micrographofexternalvoltageelectrodesonthecapillaryelectrophoresismicrodevice.Electrodesareplaced50mawayfromthesurfaceofthechannel(30

mwide×10

mdeep).Anal.Chem.,2000,72(5),1088-1092(a)(b)(c)8-4-3流體和顆粒操作

MagneticField-ControlledMicrofluidicTransport

(A)Gravity-drivenflowintheabsenceofamagneticfield(B=0).(B)MHDflowatB=1.0T.J.Am.Chem.Soc.

2002,124,462-467

MicrofabricationInside

CapillariesUsingMultiphase

LaminarFlowPatterningScience1999,285,83MechanisticInvestigationofNanoparticleMotion

inPulsedVoltageMiniaturizedElectricalField

FlowFractionationDevicebyinSituFluorescence

Imaging

Anal.Chem.2004,76,2719-2724MicrochipFlowCytometryUsingElectrokineticFocusingAnal.Chem.1999,71,4173APicoliter-VolumeMixerforMicrofluidicAnalyticalSystemsAnal.Chem.

2001,73,1942-19478-4-4反響器和混合器RapidMicrofluidicMixingAnal.Chem.

2002,74,45-51UltrasonicMixinginMicrofluidicChannelsUsingIntegratedTransducersAnal.Chem.,2004;76;3694-3698ChemicalReactorsANewSyntheticMethodforControlledPolymerizationUsingaMicrofluidicSystemJ.AM.CHEM.SOC.2004,126,9880-9881Generalideaofpolymermembraneformationunderorganic/aqueoustwo-phaseflowinanX-shapedmicrochannel

Anal.Chem.2003,75,350-354PhotothermalTemperatureControlofaChemicalReactiononaMicrochipUsinganInfraredDiodeLaserAnal.Chem.2001;73(16);4037-4044MicrofluidicDevicesforEnergyConversion:PlanarIntegrationandPerformanceofaPassive,FullyImmersedH2-O2FuelCellLangmuir2004,20,6974-6976EnzymaticReactorsMicrofabricatedElectrophoresisChipsforSimultaneousBioassaysofGlucose,UricAcid,AscorbicAcid,andAcetaminophenAnal.Chem.

2000,72,2514-2518MeasurementofEnzymeKineticsUsinga

Continuous-FlowMicrofluidicSystemAnal.Chem.2003,75,3161-3167SubstratepermeationandsubsequentenzymereactionexperimentAnal.Chem.2003,75,350-354ImmunoassayReactors.Schematicillustrationsofmicrochip-basedimmunosorbentassay.Anal.Chem.2001,73,1213-1218Glassmicrochipforimmunosorbentassay:PostcolumnLabelingSchematicofmicrochipusedforproteinseparationwithpostcolumnlabelingAnal.Chem.;2000;72,4608-46138-4-5別離TopviewoftheHDCseparationoffluorescentnanoparticlesandamarkerAnal.Chem.;

2003;75(24);6761-6768ChromatographyElectrophoresis

SurfaceModificationoftheChannelsofPoly(dimethylsiloxane)MicrofluidicChipswithPolyacrylamideforFastElectrophoreticSeparationsofProteinsAnal.Chem.2004,76,2055-2061DistillationContinuouslaminarevaporation:micron-scaledistillationChem.Commun.,2004,266–267MicrofilterSensorsandActuatorsB672000203–208

Characterizationofmicromachinedsiliconmembranesfor

immunoisolationandbioseparationapplicationsSchematicofassembledbiocapsuleconsistingoftwomicromachinedmembranesbondedtogethertoformacell-containingcavityboundedbymembranes.JournalofMembraneScience159(1999)221-2318-4-6檢測Anal.Chem.;1999;71;5309-5314FluorescenceChemiluminescenceATwo-ChannelMicrofluidicSensorThatUsesAnodicElectrogeneratedChemiluminescenceasaPhotonicReporterofCathodicRedoxReactionsAnal.Chem.2003,75,313-318Thethermallensmicroscope.ElectrochemistryCharacteristicElectrochemicalResponsesofPolymerMicrochannel-MicroelectrodeChipsAnal.Chem.2003,75,2086-2091MSChip-BasedP450DrugMetabolismCoupledtoElectrosprayIonization-MassSpectrometryDetectionAnal.Chem.2003,75,6430-6436AmplificationDetectionofViableCryptosporidiumparvumUsingDNA-ModifiedLiposomesinaMicrofluidicChipDevelopmentofaMicrochip

-BasedBioassaySystemUsingCulturedCellsConceptofthemicrochip-basedbioassaysystemAnal.Chem.,2005.77(7),2125-21318-5應(yīng)用8-5-1細(xì)胞培養(yǎng)和操作(A)Illustrationand(B)photoshowingthearrangementofPeltierelements.(C)Photoofthetemperaturecontroldevice(A)Illustrationand(B)photographofthebioassaymicrochip.(C)AnenlargedillustrationofthemicrochamberforcellcultureCompletesystemforthemicrochip-basedbioassay(A)Photoofthetemperaturecontroldevicewiththemicrochip.(B)ThermographofthemicrochipsurfaceMicrographsof(A)mouseperitonealmacrophagesand(B,C)J774.1cellsinthemicrochip.Thedamsuccessfullykeptcellsinthemicrochamber.MicrographsofJ774.1cellsculturedinthemicrochipfor2days(A)undercontinuousmediumflowconditionsand(B)staticconditions.Thecellsstainedinblueweredead.Chemicalprocessescarriedoutinthemicrochipforbioassayofmacrophage-stimulatingagentCalibrationcurvesofNOdissolvedinthemedium(A)determinedonthebulkscaleand(B)inamicrochip.TypicalresultsofNOreleasedfromJ774.1cells.

DesignofaCompactDisk-likeMicrofluidicPlatformforEnzyme-LinkedImmunosorbentAssaySchematicsof(a)aCD-ELISAdesignwith24sets