Download - Hybrid Integrated Circuit/Microfluidic Chips for the Control of Living Cells and UltraSmall
HybridIntegratedCircuit/MicrofluidicChips
fortheControlofLivingCellsandUltraSmall
BiomimeticContainers
ADissertationPresented
by
DavidAaronIssadore
to
TheSchoolofEngineeringandAppliedSciences
Inpartialfulfillmentoftherequirements
forthedegreeof
DoctorofPhilosophy
inthesubjectof
AppliedPhysics
HarvardUniversity
Cambridge,Massachusetts
May2009
©2009byDavidIssadore
Allrightsreserved.
iii
DavidAaronIssadoreAdviser:RobertWestervelt
HybridIntegratedCircuit/MicrofluidicChipsfortheControlof
LivingCellsandUltraSmallBiomimeticContainers
Abstract
Thisthesisdescribesthedevelopmentofaversatileplatformfor
performingbiologyandchemistryexperimentsonachip,usingtheintegrated
circuit(IC)technologyofthecommercialelectronicsindustry.Thiswork
representsanimportantsteptowardsminiaturizingthecomplexchemicaland
biologicaltasksusedfordiagnostics,research,andmanufacturinginto
automatedandinexpensivechips.
HybridIC/microfluidicchipsaredevelopedinthisthesisto
simultaneouslycontrolmanyindividuallivingcellsandsmallvolumesoffluid.
Takinginspirationfromcellularbiology,phospholipidbilayervesiclesareused
topackagepLvolumesoffluidonthechips.Thechipscanbeprogrammedto
trapandposition,deform,setthetemperatureof,electroporate,andelectrofuse
livingcellsandvesicles.ThefastelectronicsandcomplexcircuitryofICsenable
thousandsoflivingcellsandvesiclestobesimultaneouslycontrolledonthechip,
allowingmanyparallel,well‐controlledbiologicalandchemicaloperationstobe
performedinparallel.
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Thehybridchipsconsistofamicrofluidicchamberbuiltdirectlyontopof
acustomIC,thatusesintegratedelectronicstocreatelocalelectricandmagnetic
fieldsabovethechip’ssurface.Thechipsoperateinthreedistinctmodes,
controlledbysettingthefrequencyoftheelectricfield.ElectricfieldsatkHz
frequenciesareusedtoinduceelectroporationandelectrofusion,electricfields
atMHzfrequenciesareusedfordielectrophoresis(DEP)totrapandmove
objects,andelectricfieldsatGHzfrequenciesareusedfordielectricheating.In
addition,magnetophoresis,usingmagneticfieldscreatedwithDCcurrentonthe
chip,isusedtodeformandtopositionobjectstaggedwithmagnetic
nanoparticles.
TodemonstratethesefunctionstwocustomhybridIC/microfluidicchips
andadropletbasedPDMSmicrofluidicdevicewithexternalelectronicsare
presented.Thelaboratoryfunctionsdemonstratedonthesechipsprovide
importantbuildingblocksforaversatilelab‐on‐a‐chipplatformthatcanbebuilt
onthewell‐developedICtechnologyofthecommercialelectronicsindustry.
v
TableofContents
Abstract……………………………………………………………………………………………iiiAcknowledgements………………………………………………………………….............viiAbbreviationsandSymbols……………………………………………………………….ixListofTableandFigures………………………………………………………...…………xiChapter1.Introduction……………………………………………………….……………1
1.1Lab‐on‐a‐Chip–Motivation…………………………………...…………11.2HybridIC/MicrofluidicChips–Concept…………………………..41.3OverviewofThesis…………………………………………………………..7
Chapter2.Theory:DielectricandMagneticControlofMicroscopicObjects………………………………………………………………………….16
2.1TheDielectricPropertiesofWaterandSolutions………………182.2Dielectrophoresis…………………………………………………………….222.3DielectricModelsforCellsandVesicles…………………………….232.4TransmembranePotentialDifferenceanditsApplications...282.5DielectricHeating…………………………………………………………….322.6Magnetophoresis……………………………………………………………..34
Chapter3.HybridIntegratedCircuit/MicrofluidicChips…………………...36
3.1Overview…………………………………………………………………………363.2DielectrophoresisChip(Fabutron1.0)……………………………...37
3.2.1OperatingPrincipals……………………………………………383.2.2CharacteristicTimes………………………………………...…393.2.3IntegratedCircuitDesign…………………………………….413.2.4Capabilities………………………………………………………...46
3.3ExperimentalApparatus…………………………………………………..483.3.1Fluidics……………………………………………………………….483.3.2Electronics…………………………………………………………493.3.4Optics………………………………………………………………….503.3.5ThermalManagement…………………………………………513.3.6ComputerControl……………………………………………….54
Chapter4.AHybridIntegratedCircuit/MicrofluidicPlatformtoControlLivingCellsandpLBiomimeticContainers…………………………….56
4.1Overview…………………………………………………………………………564.2Methods…………………………………………………………………………..60
4.2.1TheHybridIntegratedCircuit/MicrofluidicChipPlatform………………………………………………………………604.2.2UnilamellarVesicles……………………………………………60
vi
4.2.3DielectrophoresisofVesiclesSuspendedinWater………………………………………………….…614.3.4ElectroporationandElectrofusion……………………….63
4.3Demonstrations……………………………………………………………….674.3.1TrappingandMovingCellsandVesicles……………....674.3.2TriggeredReleaseoftheContentsofVesicles………704.3.3ElectroporationofCells……………………………………....704.3.4TriggeredFusionofVesicles………………………………..714.3.5DeformingVesicleswithDielectrophoresis………….74
4.4Discussion…………………………………………………………………….…76Chapter5.HybridMagneticandDielectrophoreticIC/MicrofluidicChip………………………………………………………………………..78
5.1Overview………………………………………………………………………....785.2DescriptionoftheChip……………………………………………………..80
5.2.1FieldSimulations………………………………………………..805.2.2ChipArchitecture………………………………………………..83
5.3Demonstrations……………………………………………………………….865.3.1Dielectrophoresis:TrappingandPositioningVesicles……………………………………………....865.3.2Magnetophoresis:TrappingandPositioningMagneticBeads………………………………...…895.3.3DielectrophoresisandMagnetophoresis:DeformingVesicles……………………………………………………...91
5.4Discussion…………………………………………………………………….…93
Chapter6.MicrowaveDielectricHeatingofDropsinMicrofluidicDevices……………………………………………………...…….95
6.1Overview…………………………………………………………………………956.2ModelofDielectricHeatingofDrops…………………………………986.3TheMicrofluidicDevice……………………………………………………1006.4Demonstration………………………………………………………………...1086.5Discussion…………………………………………………………………….…115
Chapter7.Conclusions…………………………………………………………………..…117
7.1Summary………………………………………………………………………...1177.2FutureDirections………………………………………………………….…119
WorksCited…………………………………………………………………………..…………121AppendixA.DataSheetandUsersGuidefortheDEPChip(Fabutron1.0)……………………………………………………………...126 AppendixB.DataSheetandUsersGuidefortheHV‐DEPMagneticChip(Fabutron2.0)………………...………………………………..………...130AppendixC.FabutronControlSoftware………………………………....………...134
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Acknowledgements
FirstIwouldliketoexpressmyappreciationandgratitudeformyadviser,Bob
Westervelt.Bob’sdeepunderstandingofphysicsandhisuniqueandoftentimes
unusualperspectiveonresearch,science,andtechnologyhavechallengedme
intellectuallyandhelpedmedevelopasascientist.Hehasbeenaconstant
sourceofinsightfulideasandthoughtfulfeedbackandhasgivenmethesupport
andfreedomthatIneededtodrivemyownresearch.IamverygratefulthatI
endedupinhislab.ItseemsthatIwasverylucky.
IwouldliketothanktheWesterveltLabfolksthatIhavehadthepleasureto
workwithandsharespacewithoverthelast5years.Firstandforemost,I’dlike
tothankTomHunt.Tomhelpedmefinddirectioninmyresearchandkeptme
ontrackwhenmyresearchwaslackingfocus.Healsotaughtmethepleasuresof
roadbiking.I’dliketothankKeithBrownwhojoinedthegrouptwoyearsafter
me.Keithhasbeengreattoworkwith,heprovidedmewiththekeyfeedback
thatIsooftenneededandhisearnestenthusiasmandscientificrigoroften
improvedmywork.Also,heisanadmirableWormsplayer.Jonathan,Ognjen,
Lori,andAlmalchiandtherestoftheundergradsthathaveworkedtheirway
throughtheWesterveltlabhavekeptlifeinterestingandwereapleasureto
workwith.Erin,Halvar,andJesseareawesomepeopletosharealabwithandI
amgratefultothemforkeepingmesomewhatknowledgeableaboutcold
temperaturephysics.Alex,Nan,andTinaarenewcomerstothegroupandseem
tobekeepingthegoodtraditionsalive.
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IwouldalsoliketothankthepeopleIhadthepleasuretocollaboratewith
outsideoftheWesterveltGroup.ThomasFrankeatUniversityofAugsburgin
Germany’sexpertiseinpreparingvesicleswaskeytothesuccessofthisthesis.
KatieHumphryintheWeitzGroup’sabilityinsoftlithographywasessentialfor
themicrowavedropheatingproject.RickRogersandRosalindaSepulvedaatthe
SchoolofPublicHealthprovidedcellsandgreatadviceonbiologyformanyof
theprojectsinthisthesis.
The5yearsthatI’vespentinCambridgehavebeenavariedlot,andIowealotto
myfriendsandfamilyforgettingmethroughitinonepiece.Firstandforemost,
myparentshavealwayssupportedmeandhavebeensupremelyunderstanding,
evenattimeswhenI’msureIwasintolerable.I’dliketothankmybrotherfor
alwaysbeingsupportive,myGrandmomChipforremindingmewhat’s
importantinlife,andTheoforgenerouslysharingwithmehisenlightened
philosophyonlife.
Alan,myroommateof5years,isresponsibleformyeducationonallofthetopics
thatIwasn’tlearninginschoolsuchastheworkingsofevilhedgefunds,
bosanovamusic,andDjango.MikewasafantasticadditiontoMagdaddyand
significantlyimprovedourqualityoflife.BenandClemensbothmightaswell
havelivedwithusandaregreatfriends.Andfinally,Imani.It’dbeinappropriate
towritehereallthatIamthankfulforaboutyou.But,Iamverygratefulandmy
lifeissomuchfullerbecauseofyou.
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AbbreviationsandSymbols
a Particleradius
€
B MagneticField
C Capacitance
Cmem Specificmembranecapacitance
CM ClausiusMosottiFactor
€
χ MagneticSusceptibility
CMOS ComplimentaryMetalOxideSilicon
D DiffusionConstant
DC Directcurrent
DEP Dielectrophoresis
ΔT ChangeinTemperature
€
E ElectricField
€
ε RelativePermittivity
€
εo VacuumPermittivity
€
ε' RealComponentofthePermittivity
€
ε' ' ImaginaryComponentofthePermittivity
f Frequency
€
F Force
gm SurfaceConductivity
GUI GraphicalUserInterface
HV HighVoltage
€
η Viscosity
I/O Input/Output
IC IntegratedCircuit
j
€
−1 kB Boltzmann’sConstant
L Thelengthofapixel
x
LD CharacteristicLengthintheHeatingModelforDrops
MOSIS MetalOxideSemiconductorImplementationService
MUX Multiplexer
n Concentration
NMR NuclearMagneticResonance
PBS PhosphateBufferSaline
R Resistance
Re ReynoldsNumber
RF RadioFrequency
ROI RegionofInterest
SPICE SimulationProgramwithIntegratedCircuitEmphasis
SRAM StaticRandomAccessMemory
σ Conductivity
T Temperature
ΔTSS SteadyStateChangeinTemperature
τdiff CharacteristicTimeforanObjecttoDiffuseaHalfPixellengthL/2
τmem CharacteristicChargingTimeofaVesicleorCell’sMembrane
τmove CharacteristicTimetoMoveanObjectaPixelLengthL
τSS CharacteristicTimetoReachSteadyStateTemperature
τMW DielectricRelaxationTimeofavesicleorcell
τW DielectricRelaxationTimeofWater
€
µo VacuumPermeability
V Volume
V Voltage
VTM TransmembraneVoltage
ω AngularFrequency
xi
ListofFigures
Figure1.1aJackKilby’sIC………………….…………….……………………………….6
Figure1.1bAnIntelQuad‐CoreIC…..…....…….……………………………………..6
Figure1.1cASimpleMicrofluidicChip……………………………………..……….6
Figure1.1dAComplexMicrofluidicChip…………………….…………………….6
Figure1.1eAHybridIC/MicrofluidicChip……………………………………….6
Figure1.2TheDEPChipSpelling“LabonaChip”withYeastCells…..….9
Figure1.3aTheDEPHybridIC/MicrofluidicChip…..…………….………….15
Figure1.3bTheHighVoltageDEP/MagneticChip…….………………..…….15
Figure1.3cTheMicrowaveDielectricDropHeatingChip……………....…..15
Figure2.1FrequencyDomainPlotoftheEfficacyofDEP,Electroporation,andDielectricHeating………………...…………….….17
Figure2.2aPermittivityofWatervs.Frequency………………………………...21
Figure2.2bPermittivityofAqueousSolutionswithVaryingConductivityvs.Frequency…………………….…………...……….…………………….21
Figure2.3aLumpedCircuitModelforthePermittivityofaVesicle….…27
Figure2.3bClausius‐Mosottivs.InteriorConductivityforaVesicle... 27
Figure2.3cClausius‐Mosottivs.frequencyforaVesicle…………….……. 27
Figure2.4aTransmembraneVoltagevs.FrequencyforaVesicle.…….…31
Figure2.4bSchematicofElectrofusion……………………………………………..31
Figure2.5DielectricHeatingPowerDensityvs.Frequencyfora SolutionwithVaryingConduct...………………………………………..………………34
Figure3.1aPlotofSimulatedElectricFieldStrengthAboveTheDEPChip………………………………………………………………………….40
Figure3.1bPlotoftheForceExperiencedbyaVesicleonTheDEPChip…………………………………………………………..............40
Figure3.2aSchematicoftheArchitectureoftheDEPChip….……………...45
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Figure3.2bSchematicofanindividualDEPPixel………………………………45
Figure3.2cMicrographoftheDEPArray,ShiftRegister,andRowDecoder……………………………………………………………………………...45
Figure3.3aDemonstrationofTrappingandPositioningYeastontheDEPChip……………………………………………………………………….47
Figure3.3bDemonstrationofTrappingandPositioning DropsofWaterinOilontheDEPChip……………….............................................47
Figure3.3cTheDEPChipSpelling“Harvard”withYeastCells…...............47
Figure3.4aFlowChartoftheExperimentalApparatusSurroundingtheHybridIC/MicrofluidicChips……………………………………………………..53
Figure3.4bMicrographoftheDEPChip’sIC………………………………….….53
Figure3.4cPhotographoftheHybridIC/MicrofluidicChip initsChipCarrier………………………………………………………………….………..…53
Figure3.4dPhotographoftheExperimentalApparatus SurroundingtheHybridIC/MicrofluidicChips…………………….……………53
Figure3.5ScreenShotsoftheGUIthatControlsthe HybridIC/MicrofluidicChip…………………………………………………………….55
Figure4.1ASchematicofUnilamellarVesiclesandEmulsions……………59
Figure4.2aPlotoftheConcentrationGradientofaSubstance ReleasedfromaVesicle………………………………………………………………….....66
Figure4.2bPlotoftheConcentrationGradientofaSubstance ConsumedontheSurfaceofaCellorVesicle…………………………………...…66
Figure4.3aDemonstrationofIndependentlyTrappingand MovingSeveralVesiclesontheDEPChip…………………….………..........……..69
Figure4.3bTheDEPChipDrawing‘H’withVesicles…..………………………69
Figure4.3cSimultaneouslyTrappingandMovingYeastCellsandVesiclesontheDEPChip…………………………………………....69
Figure4.4aTriggeringtheReleaseoftheContentsofaVesicleontheDEPChip…………...………………………………………………….73
Figure4.4bElectroporatingaYeastCellontheDEPChip….………………..73
Figure4.4cElectrofusingTwoVesiclesontheDEPChip…..…………………73
xiii
Figure4.5DeformingVesiclesontheDEPChip…………………………..…..….75
Figure5.1aMicrographoftheHV‐DEP/MagneticIC…………………………83
Figure5.1bPlotoftheSimulatedElectricFieldStrengthAboveTheHV‐DEP/MagneticChip……………………………………………………………..83
Figure5.1cPlotoftheSimulatedMagneticFieldStrengthAboveTheHV‐DEP/MagneticChip………………………………………………………..……82
Figure5.2aMicrographoftheHV‐DEP/MagneticIC,showing thecircuitArchitecture……………………………………………………………………..85
Figure5.2bSchematicofaDEPPixelontheHV‐DEP/MagneticChip...85
Figure5.2cSchematicofaMagneticWireontheHV‐DEP/MagneticIC.………………………………………………………………........... 85
Figure5.3TrappingandMovingVesiclesontheHV‐DEP/MagneticChip…………………………………………………..……..88
Figure5.4TrappingandMovingMagneticBeadsontheHV‐DEP/MagneticChip…………...……………………………………….……90
Figure5.5DeformingVesiclesontheHV‐DEP/MagneticChip…...….…...92
Figure6.1aSchematicofMicrofluidicDielectricHeater……………………..102
Figure6.1bPlotofElectricFieldSimulationsfortheMicrofluidicDielectricHeater……………………………………………………………102
Figure6.1cCalibrationCurvefortheCdSeTemperatureSensor……..….102
Figure6.2aFlowDiagramoftheMicrowaveDielectricHeater…………...107
Figure6.2bMicrographoftheDropMaker………………………………………..107
Figure6.2cMicrographoftheDropSplitter………………………………………107
Figure6.2dMicrographoftheMicrowaveHeatingDevice………………....107
Figure6.2ePhotographoftheExperimentalSetupofthe MicrowaveDielectricDropHeater……………………………...………………..……107
Figure6.3aFluorescenceImageofDropsHeating……………………………..110
Figure6.3bLine‐averageofFluorescenceIntensityvs.Distance….……..110
Figure6.3cPlotofChangeinTemperaturevs.DistanceandTime……...110
Figure6.4aPlotofChangeinTemperaturevs.MicrowavePower….……114
xiv
Figure6.4bPlotofLog‐LinearplotofChangeinTemperaturevs.Time……………………………………………………………………….114
ListofTables
Table1.1ListofFunctionsthatarePerformedinthisThesis….............…13
Table7.1Lab‐on‐a‐ChipFunctionsDemonstratedinthisThesis………...118
1
Chapter1.Introduction
Labonachip:ATechnologythatminiaturizesandintegratesthecomplexchemical
andbiologicaltasksusedfordiagnostics,research,andmanufacturingonto
automated,portable,andinexpensivechips.
1.1LabonaChip–Motivation
Amajorchallengeofthe21stcenturyistobetterdiagnoseandtreatinfectious
diseaseforthelargeportionoftheworld’spopulationthatiscurrentlyunderserved.
Diseases,suchasHIV/AIDS,Tuberculosis,andMalaria,forwhichthereexists
interventions,continuetokillmillionsandinfectmillionsmoreeachyeardue,in
part,toourinabilitytodiagnosediseaseseffectivelyinpoorcountries.(Urdeaetal.,
2006)TheBillandMelindaGatesFoundationandtheNationalInstituteofHealth
havedeclaredtheassessmentofdiseaseinpoorcountriesaGrandChallengefor
PublicHealth.Thesechallengesread:(Varmusetal.,2003)
1. Developtechnologiesthatpermitquantitativeassessmentofpopulationhealth
statistics.
2. Developtechnologiesthatpermitquantitativeassessmentofindividualsfor
multipleconditionsorpathogensatpointofcare.
Withthesegoalsinmind,researchersaroundtheworldaredevelopingtechnology
tomeasurebiologicalandchemicalmarkersforinfectiousdiseaseininfrastructure‐
poorpartsoftheworld.(Ahnetal.,2004,Chinetal.,2007,andMartinezetal.,2008)
Thelaboratoriesthatperformthechemicalandbiologicalteststodiagnosedisease
2
intheworld’swealthiernationsrequireresourcesthatarenotreadilyfound
elsewhere,suchascentrallylocatedairconditionedbuildings,cleanwater,
electricity,accesstoexpensivereagents,andwell‐trainedtechnicians.(Chinetal.,
2007)Assuch,technologytoassessdiseaseinpoorcountriesfaceschallengesnot
ordinarilyfoundinamodernlaboratoryspace.
Lab‐on‐a‐chiptechnologyoffersapowerfultooltobringexistingmedicaland
environmentaltestsfromthelaboratoryintothefieldandtheclinic.(Figeysetal.,
2000,Petraetal.,2006,Chinetal.,2007)Attheforefrontofthistechnologyarethe
micro‐fabricatedpipes,valves,pumps,andmixersofmicrofluidicsthatareleading
tointegratedlab‐on‐a‐chipdevices.(Whitesidesetal.,2001andStoneetal.,2004)
Inadditiontothepotentialforlow‐costmedicine,theminiaturizationofthe
handlingofliquidandbiologicalsampleshasenabledadvancesinfieldssuchas
drugdiscovery,geneticsequencingandsynthesis,cellsorting,andsinglecellgene
expressionstudies.(Tabelingetal.,2005,Yageretal.,2006,andMartinezetal.,
2008)Theseintegratedmicrofluidicdevicesareleadingaparadigmshiftinfluid
handlingthatisanalogoustowhatintegratedcircuitsdidforelectronicshalfofa
centuryago.(Leeetal.,2007)However,alab‐on‐a‐chipthatcanperformthe
complex,multi‐stepexperimentsthatarecurrentlyperformedinlaboratories,akin
toamicroprocessorforfluids,remainsachallenge.(Leeetal.,2007)
Thepotentialimpactofalab‐on‐a‐chipmicroprocessorcouldbeenormous.Imagine
adevicethesizeofaniPodthatcostlessthan$100andcanperformnumerous
complexbiologicalandchemicaltestsonsmallsamplesofbodilyfluids,drinking
3
water,orair.Suchadevicewouldmakemedicalandenvironmentaltesting
inexpensive,portableandautomated.Testingcouldbeperformedinthefield,at
home,orataclinicbyanon‐expert.Theresultsoftestswouldbequantitativeand
consistentsuchthatrelevantdatacouldbecollectedforbothpersonalandpublic
healthstatistics.(Chinetal.,2007)And,thedevicecouldeasilybeconnectedtothe
Internetsuchthatrelevantdatacouldbeshared.Suchadevicecouldfundamentally
changethewaythatpeopleinteractwiththechemicalandbiologicalinformationof
theirsurroundingsandoftheirownbodies.
Inadditiontoitsapplicationsinthedevelopingworld,lab‐on‐a‐chipdeviceshavea
bigroletoplayinthefutureofhealthcareinTheUnitedStates.Currently,15.2%of
theUnitedState’sGDPisspentonhealthcareandthatfractionisexpectedtogrow
to19.5%by2020.(U.S.DepartmentofHealthandHumanServices,2007)This
amountofspendingisbelievedtobeunsustainable,especiallyasthepopulation
growsandages.(Keehanetal.,2008)Lab‐on‐a‐chipdevicescanbeusedtolower
thepriceofmedicaldiagnosticsandmonitoring,enablingdiseasestobedetectedin
theirearlierstageswheretreatmentiseasierandfarlessexpensive.(Tudosetal.,
2001)
Inthisthesisalab‐on‐a‐chipplatformisdevelopedthatcancontrolsinglecellsand
verysmallvolumesoffluidtoperformsimultaneous,programmableexperimentson
achip.Incontrasttotechnologythatattemptstomakelab‐on‐a‐chipdevicesultra‐
lowcostbybeinglowtech,(Martinezetal.,2008)thechipsdescribedinthisthesis
4
usecutting‐edgetechnology.Thechipsremaininexpensive,however,byusingthe
integratedcircuit(IC)technologyofthecommercialelectronicsindustry.
1.2HybridIC/MicrofluidicChipsConcept
Integratedcircuitsarecentraltomanyofthetechnologicalwondersofthe21st
century.Builtonaslabofcrystallinesiliconnobiggerthanasquarecentimeterin
area(roughlythesizeofaquarter)anICcontains100sofmillionsoftransistorsand
amazeofwiresthatconnectthetransistorsintocomplexcircuitstoperformbillions
ofoperationspersecond.ThetransistorsandwiresofICsarefabricatedwithnano‐
lithography(asopposedtooneatatime),whichallowICstobecomplex,small,fast,
andalso,inexpensive.(Lee,1998)Integratedcircuitsareubiquitousintoday’s
technology.Theyarethemicroprocessorsincomputers,theradiofrequency(RF)
circuitsincellphones,thefilmindigitalcameras,andthemicrocontrollersinheart
patients’pacemakers.Integratedcircuits,havingbeeninventedonly50yearsago,
haveprofoundlychangedthewayhumansuse,store,andcommunicateinformation.
InFig.1.1atheoriginalICisshown,builtbyJackKilbyin1958,itcontainsonlya
singletransistor.(Kilby,1976)InFig.1.1bamodernPentiumquad‐coreIC,thatcan
befoundintoday’slaptopsisshown,itcontains820milliontransistors.(Intel)
Inaspiritanalogoustotheminiaturizationofelectronicsthatleadtotoday’sICs,
modernresearchersareminiaturizingthefluid‐handlingtoolsofbiologyand
chemistrylaboratoriesintosmall,inexpensivechipstoperformautomated
experiments.Agrowinglibraryofmicrofluidicelementsforlab‐on‐a‐chipsystems
havebeendevelopedinrecentyearsfortaskssuchasthemixingofreagents,
5
detectingandcountingcells,sortingcells,geneticanalysis,andproteindetection.
(Whitesidesetal.,2001,Stoneetal.,2004,Tabeling,2005,andYageretal.,2006)
InFig.1.1catypicaltwo‐channelglassmicrofluidicchipusedtocontrollablymix
twosubstancestogetherfromMicronitCorp.isshown.Figure1.1dshowsan
exampleofoneofthemorecomplexmicrofluidicchipsbuilttoday,fromtheQuake
GroupatStanford,thatconsistsofhundredsofpneumaticallycontrolledgatesand
valvesandisusedtoperformgeneticanalysisonmicrobesinthehumanmouth.
(Marcyetal.,2007)
Thecomplexity,smallfeaturesize,andlowcostofICscanbeappliedtobiological
andchemicalapplicationsbycombiningICswithmicrofluidicstoformhybridIC/
microfluidicchips.Complimentary‐metal‐oxide‐semiconductor(CMOS)optical
sensorshavebeencoupledwithmicrofluidicstomakea“microscopeonachip”that
achievesenhancedresolutionandsensitivitybybringingmicroscopicobjects
directlytotheopticalsensors.(Eltoukhy,etal.,2006andCuietal.,2008)Electrical
sensorarrayshavebeenbuiltonIC/microfluidicchipstostimulateandmeasure
largearraysofindividualneuralandcardiaccells.(DeBusscherreetal.,2001and
Eversmannetal.,2003)And,hybridIC/microfluidicchipshavebeenusedtotrap
andmovedielectric(Gascyoneetal.,2004andHuntetal.,2008)andmagnetic
objects(Leeetal.,2006)alongprogrammablepathsforchemistryandbiology
experimentsonachip.Figure1.1eshowsahybridIC/microfluidicchipthattraps
andmovesobjectsalongarbitrarypathswithDEPusingalargearrayofpixels.The
theoreticalframeworkforhowDEPisusedtotransportcellsandvesiclesonachip
isdetailedinChapter2ofthisthesis.
6
Figure1.1(a)TheoriginalICbuiltbyJackKilbyatNationalInstrumentsin1958(Kilby,1976)(b)amicrographoftheIntelquad‐coreprocessor,whichhas820milliontransistos(IntelCorp.),(c)atwochannelglassmicrofluidicchip(MicronitCorp.),(d)anexampleofacomplexmicrofluidicdevicethatconsistsofhundredsofpneumaticallycontrolledgatesandvalvesandisusedtoperformgeneticanalysisonmicrobesinthehumanmouth,(Marcyetal.,2007)(e)aphotographofanIC/microfluidicchip,the‘DEPChip,’andamicrographofthearrayofpixelsthatareusedtotrapandmoveobjectswithdielectrophoresis(DEP).(Chapter3)
7
1.3OverviewofThesis
Thisthesisdescribesthedevelopmentofaversatilelab‐on‐a‐chipplatformusing
hybridIC/microfluidicchips.Thechipsperformawiderangeoffunctionsonliving
cellsandpLbiomimeticcontainersforbiologyandchemistryexperimentsonachip.
Previousworkhasbeendonedevelopingprogrammablelab‐on‐a‐chipplatformsto
controlsmallvolumesoffluidandcells.Pneumaticcontrolhasbeenusedtocreate
reconfigurablemicrofluidiccomponentssuchasvalves,latches,pumps,and
multiplexers.(Ungeretal.,2000)Recently,similarstructureshavebeendeveloped
thatreplacethecumbersomepneumaticlineswithelectronicallyactivated
componentsthataremadeofshapememoryalloys(SMAs)andwhichcanbebuilt
ontopofcommercialprintedcircuitboards(PCBs).(Vyawahareetal.,2008)
Dropletshavebeentrapped,moved,mixed,andseparatedusingboth
electrowetting(Leeetal.,2002andPollacketal.,2002)anddielectrophoresis
(DEP)(Vykoukaletal.,2001andPeteretal.,2004)withelectronicsthatareexternal
tothefluidicsystem.Hybridintegratedcircuit(IC)/microfluidicchipshavebeen
developedthatharnessthematuretechnologyofICstomakegeneralpurpose,
programmablefluid‐handlingsystems.(Leeetal.,2007,Gascyoneetal.,2004,Hunt
etal.,2008)Thehighlylocalizedelectricandmagneticfieldsthatcanbecreatedby
ICshavebeenusedtotrapandmovesmallvolumesofwatersuspendedinoil(Hunt
etal.,2008),livingsinglecellssuspendedinwater(Gascyoneetal.,2004,Huntetal.,
2008),andmagneticallytaggedbiologicalobjectssuspendinwater(Leeetal.,2007)
alongprogrammablepaths.Figure1.2showsahybridIC/microfluidicchip
8
simultaneouslypositionthousandsofyeastcellswithDEPtospell“LabonaChip”.
ThechipshowninFig.1.2isdescribedinfulldetailinChapter3ofthisthesis.
9
Figure1.2ThecoverofLabonaChipfeaturingourhybridIC/microfluidicchipsimultaneouslypositioningthousandsofyeastcellswithDEPtospell“LabonaChip.”
10
Thereareseveralkeyfunctionsthatarenecessarytoperformexperimentsonachip
thatareanalogoustotypicallaboratoryprocesses.Anyworkingmedicalor
researchlaboratoryhasbasicequipment:testtubestokeepssamplesseparate,
pipettestomovesamplesaroundthelaboratory,mixerstobringsamplesand
reagentstogether,andheaterstocontrolthetemperatureofexperiments.Inthis
thesisbasiclaboratoryfunctions,aswellasseveralfunctionsthatarenotpossiblein
amacro‐scalelaboratory,areperformedonIC/microfluidicchipsusingelectricand
magneticfieldscreatedabovetheIC’ssurface.InTable1.1thefunctionsperformed
onhybridIC/microfluidicchipsinthisthesisarelisted.
Reagentsandsamplesarekeptseparated,suchthattheycanbetransportedaround
the‘laboratory’andmixedatthepropertimeinanexperiment,bypackagingthem
inpLcontainersasisdemonstratedinChapter4.Inspirationistakenfromcellular
biologyandphospholipidbilayervesiclesareusedtopackagepLvolumesoffluidon
thechip.Vesiclesarecommonlyusedincellsforpackagingquantitiesofsubstances
forintercellulartransport,tostoreenzymes,andasareactionchamber.(Albertset
al.,2007)UnilamellarvesiclesmadewithelectroformationproviderobustpL
containersthatareimpermeableandstableforawiderangeofsalinity,pH,and
otherenvironmentalconditions.(Chiuetal.,1999)
Vesiclesandcellsaretransportedacrossthe‘laboratory’usingDEPwithelectric
fieldsatMHzfrequenciesabovethechip’ssurface,asisdemonstratedinChapters3,
4,and5.Thesmallfeature‐sizeofICsallowsmicrometer‐sizedvesiclesand
individuallivingcellstobeindependentlytransported.Thefastelectronicsand
11
complexcircuitryofICsallowthousandsoflivingcellsandvesiclestobe
simultaneouslytrappedandmovedonthechip,allowingmanyparallel,well‐
controlledbiologicalandchemicaloperationstobeperformedinparallel.The
theoreticalframeworkforDEPisintroducedinChapter2.
Vesiclesandcellsarecontrollablypermeabilized,fused,andreleasedusingkHz
frequencyelectricfieldsabovethechip’ssurface,asisdemonstratedinChapter4.
Thevesiclescanbetriggeredtoreleasetheircontentslocallyintothesolutionand
mixtheircontentswithothervesiclesusingelectricfieldscreatedbythechip.
ElectricfieldsatkHzfrequenciesinducevoltagesacrossthevesicle’smembrane
inducingelectroporationorelectrofusion.Electroporationcanalsobeperformed
onlivingcells,inducingthecellstotake‐upsubstancesfromthesolution.The
complexcircuitryofthechipallowsspecificvesiclesorcellstobetargetedfor
electroporationorelectrofusionwithoutharmingsurroundingcellsorvesicles.The
chipcantime‐multiplexthekHzfrequencyelectricfieldswiththeMHzfrequency
electricfieldsusedforDEP,suchthatobjectsremaintrappedinplace,astheyare
electroporatedorelectrofused.Thetheoreticalframeworkforelectroporationand
electrofusionisexplainedinChapter2.
AhybridchipthatcansimultaneouslyperformDEPandmagnetophoresisis
demonstratedinChapter5.Magnetophoresis,usingmagneticfieldscreatedonthe
chip,providesacomplimentarymethodtoDEPfortransportingsubstancesacross
the‘laboratory.’Theabilitytotrapandmovemagneticobjectsalongprogrammed
12
pathsisausefultooltocontrolthepositionofobjectsthatcannotbetrappedwith
DEP,butwhichcanbetaggedwithmagneticparticles.(Leeetal.,2007)
Themechanicalenvironmentofindividualcellsandvesiclescanbedefinedusing
thechip,afunctionalitythatisnotpossiblewithmacro‐scalelaboratorytools,asis
demonstratedinChapters4and5.In‐vitroexperimentsoftensufferfornot
controllingthemechanicalenvironmentofcells,anaspectthatplaysanimportant
rolein‐vivo.(Seifritz,1924,Curtisetal.,1964andWangetal.,1994)SeveralDEP
pixelscanbepatternedunderneathasinglevesicleorcelltocontrolitsmechanical
environmentbychangingtheshapeoftheDEPtrap,asisdemonstratedinChapter4
withunilamellarvesicles.Magneticfields,createdwithDCcurrent,areusedtotrap
andmovemagneticallypermeableobjectssuchasironoxidenanoparticles.These
particlescanbeimplantedintovesiclesorlivingcellstoapplylocalforces
selectivelytothelocationofthenanometersizedparticles.Thistechniqueis
demonstratedbycontrollablydeformingvesiclesimplantedwithironoxide
nanoparticleswithmagneticfieldscreatedbythechipinChapter5.
Thetemperatureofsmallcompartmentsoffluidcanbelocallyandrapidly
controlledusingdielectricheatingwithelectricfieldsatGHzfrequencies,asis
demonstratedinChapter6.Electricfieldswithafrequencyf=3GHzareusedto
heatthermallyisolatedpLdropswithdielectricheating.Changesintemperature
ΔT=0°–30°Careachievedinacharacteristictimeτs=15ms.
13
Table1.1AlistofthefunctionalitiesdemonstratedonthehybridIC/microfluidicchipsinthisthesis.
14
ThreedevicesaredescribedinthisthesisandareshowninFig.1.3.InChapter3a
customIC/microfluidicchipthatconsistsof128x256(32,768)11x11µm2pixels,
eachofwhichcanbeindividuallydrivenwith5Vpeak‐to‐peakradiofrequency(RF)
voltageswithfrequenciesfromDCto11MHz,(Hunt,etal.,2007)isdevelopedintoa
lab‐on‐a‐chipplatform.ThischipiscalledTheDEPChip(Fabutron1.0)anda
micrographofitisshowninFig.1.3a.
InChapter5asecondcustomIC/microfluidicchipispresentedthatcantrapand
moveobjectswithbothDEPandmagneticforces.Thechipsconsistsof
60x61(3,660)30x38µm2pixels,eachofwhichcanbedrivenwitha50Vpeak‐to‐
peakRFvoltagewithfrequenciesfromDCto10MHz.UnderneaththeDEPpixel
array,thereisamagneticgridthatconsistsof60horizontalwiresand60vertical
wiresrunningacrossthechip,eachofwhichcanbesourcedwith120mAtocreate
localmagneticfields.ThischipiscalledTheHV‐DEP/MagneticChip(Fabutron2.0)
andamicrographofitisshowninFig.1.3b.
InChapter6APDMSbasedmicrofluidicdevice,fabricatedwithsoft‐lithography,is
usedtodemonstraterapidandlocaldielectricheatingofdrops.Thedeviceconsists
ofanintegratedflow‐focusingdropmaker,dropsplitters,andmetallineelectrodes
tolocallydelivermicrowavepower.ThischipiscalledTheMicrowaveHeateranda
photographofitisshowninFig.1.3c.
15
Figure1.2(a)AmicrographoftheDEPChip(Fabutron1.0),(b)amicrographoftheHV‐DEP/MagneticChip(Fabutron2.0),(c)aphotographofthePDMSmicrofluidicdevice(MicrowaveHeater)usedtodemonstratemicrowavedielectricheatingofdrops.
16
Chapter2.Theory:DielectricandMagnetic
ControlofMicroscopicObjects
2.1Overview
Thischapterdescribeshowelectricandmagneticfieldsareusedtocontrol
microscopicobjectsonhybridintegratedcircuit(IC)/microfluidicchips.Electric
fieldsatkHzfrequenciesandbelowareusedtoinduceelectroporationand
electrofusion.ElectricfieldsatMHzfrequenciesareusedfor
dielectrophoresis(DEP).ElectricfieldsatGHzfrequenciesareusedfordielectric
heating.And,magneticfieldscreatedwithDCelectricalcurrentsareusedtotrap
andmoveobjectswithmagnetophoresis.
Thefrequencydependenceofthedielectricpropertiesofvesicles,cells,and
solutionsenablethedielectricphenomenonusedinthisthesistobeindependently
accessedwithelectricfieldsatdifferentfrequencies.Thefrequencydependenceof
DEP,electroporation,anddielectricheatingisshowninFig.2.1.Thestrengthof
eachoftheseeffectsisplottedinunitlessvaluesthataredescribedindetaillaterin
thischapter.AttheMHzfrequencieswhereDEPworksbest,bothheatingand
electroporationisminimal,enablingvesiclesandcellstobetrappedandmoved
withoutdamage.AttheGHzfrequencieswhereheatingworksbest,both
electroporationandDEPareminimal,enablingcellsandvesiclestobeheated
withoutinadvertenttrappingordamage.AtthekHzfrequencieswhere
electroporationandelectrofusionworkbest,heatingisminimalandtheDEPforceis
17
negative,enablingvesiclesandcellstobepermeabilizedandfusedwithout
significantheatingorinadvertenttrapping.
Figure2.1.Asemi‐logplotoftheefficacyofheatingP/Pmax(red),dielectrophoresisCM(ω)(green),andelectroporation
€
VTM /VTMmax (blue)versusthefrequencyfofthe
appliedelectricfield.Thethreefrequencydomainsthatareusedforelectroporation,DEP,anddielectricheatingarelabeled.
18
Inthischapterthevariousdielectricandmagneticphenomenonthatareusedto
controllivingcellsandvesiclesonourchipsareexplained.InSection2.1water’s
uniquedielectricproperties,whichplayanimportantroleinourabilitytocontrol
objectswithelectricfields,areintroduced.InSection2.2dielectrophoresis,the
forcethatisusedtotrapandmoveobjectswithelectricfields,isintroduced.In
Section2.3ageometriclumped‐circuitmodelisdevelopedtodescribethedielectric
propertiesofcellsandvesicles.InSection2.4thislumped‐circuitmodelisusedto
explaintheinducedvoltagethatformsacrossthemembranesofcellsandvesicles,
whichisutilizedinthisthesisforelectroporationandelectrofusion.InSection2.5
dielectricheatingwithmicrowavefrequencyelectricfieldsisintroduced.And
finally,inSection2.6magnetophoresisisintroducedasamethodtocompliment
DEPfortrappingandmovingobjects,utilizingmagneticdipolesinsteadofelectric
permittivity.
2.1TheDielectricPropertiesofWaterandSolutions
Tounderstandthedielectricpropertiesofcellsandvesiclesitisnecessarytofirst
understandthedielectricpropertiesoftheirmostimportantingredient,water.
Wateristheprimaryconstituentofcellsandisthemostcommonlyusedsolventin
chemicalandbiochemicalexperiments.TheDCdielectricconstantofwaterεs=78ε0
(atT=25°C)isverylargecomparedtothatofmostotherorganicandinorganic
solventsεs<10ε0,duetoitspermanentmoleculardipolemoment.Waterwill
typicallydominatethedielectricpropertiesofanyobjectthatismadeofit.(Murrell
etal.,1994)
19
Thedielectricresponseofwatervarieswiththefrequencyoftheappliedelectric
field.(Grant,etal.,1978)Thepermittivityisdescribedbyboththemagnitudeand
thephaseofthepolarizationrelativetotheappliedfieldandcanberepresentedasa
complexfunctionε(ω) =ε’(ω)+jε’’(ω),witharealcomponentε’andanimaginary
componentε’’.Figure2.2ashowsaplotoftherealε’andimaginaryε’’components
ofthepermittivityofwaterplottedversusfrequency.Therealcomponentofthe
permittivityofwaterε’hasaconstantvalueofεs=78ε0untilacornerfrequency
definedbythedielectricrelaxationofwater1/(2π∗τW)≈18GHz,atwhichpointthe
permittivitymonotonicallydrops.Theimaginarycomponentε’’approacheszero
everywhere,exceptataresonantfrequencydefinedbythedielectricrelaxationtime
ofwaterτW.ThepermittivityofwatercanbedescribedbyanequationwithaDebye
form:(A.Stogryn,1971)
€
εW = ε∞ +εs −ε∞1− jωτW
2.1
whereεs=78.4ε0isthelowfrequencydielectricconstantofwater,ε∞=1.78ε0isthe
opticaldielectricconstant,andτw=9.55psisthedielectricrelaxationtimeat
T=25°C.Thedielectricrelaxationtimeτwcanbeunderstoodasthecharacteristic
timethatittakesforwatermoleculestorealignthemselvestoaninstantaneous
appliedelectricfield.
Thedielectricresponseofwaterdependsontheconcentrationofionsinthe
solution.Dissolvedionschangetheconductivityofthesolutionσ byactingasfree
20
chargecarriers.Theeffectofionsinthesolutioncanbeincorporatedintothe
permittivityofthesolution:(A.Stogryn,1971)
€
εW = ε∞ +εs −ε∞1− jωτW
+ j σεoω
2.2
Figure2.2bshowsaplotoftheimaginarycomponentofthepermittivityε’’plotted
versusfrequencyonalog‐logscaleforsolutionswithseveraldifferent
conductivitiesσ.Forsolutionswithafiniteconductivity,theimaginarycomponent
ofthepermittivityε’’decreaseswithincreasingfrequencyuntilacharacteristic
frequency1/τI.Thecriticalfrequency1/τ1isdefinedbythedielectricrelaxation
timeτI=εs/σthatarisesfromconductivityofthesolutionσandthelowfrequency
dielectricconstantofthesystemεs.Astheconductivityofthesolutionσis
increased,thecharacteristicfrequency1/τIispushedtohighervalues,ascanbe
seeninFig.2.1b.Forlowfrequenciesω<1/τ1theimaginarycomponentofthe
permittivityε’’=σ/ωε0isafunctionofonlytheconductivityσ andnoothermaterial
propertiesofthesolution.Inthelimitofwaterhavingzeroconductivityσ=0the
imaginarycomponentofthepermittivityε’’increasesmonotonicallywithfrequency
untilthecharacteristicfrequencyofwaterτw.Therealpartofthedielectric
constantisslightlyreducedwiththeadditionofelectrolytes,aswaterboundtothe
ionshaveasmallerdielectricresponsethanfreewatermolecules.(A.Stogryn,
1971)
21
Figure2.2.(a)Therealε’andimaginarypartsε’’ofthepermittivityofwaterεWplottedversusfrequency,(b)theimaginarycomponentε’’ofthepermittivityofsolutionsofwaterplottedversusfrequencyforvariousconductivitiesσ.Theconductivityσ increasesgoingfromlefttoright(asisshownwiththeblackarrow)withvaluesof0S/m,0.03S/m,0.06S/m,0.14S/m,0.3S/m,0.62S/m.
22
2.2Dielectrophoresis
Dielectrophoresisistheinducedmotionofadielectricobjectinanon‐uniform
electricfield.Anyobjectwithapermittivitythatisdifferentthanthesurrounding
mediumcanbecontrolledwithDEP.TheDEPforceonasphericalobjectis:(Jones,
1995)
€
F DEP = 2πεma3CM(ω)∇
E RMS2 2.3
whereaistheradiusoftheparticle,εmisthedielectricconstantofthemedium,and
CM(ω)istheClausius‐Mosottifactor,arelationbetweenthefrequencydependent
complexpermittivityoftheparticleandthemedium.
2.4
whereεpisthecomplexdielectricconstantoftheparticle.WhenCM(ω)<0,the
mediumismorepolarizablethantheparticleandtheparticleispushedawayfrom
thelocalmaximumoftheelectricfieldandthisiscallednegativeDEP.PositiveDEP
occurswhentheparticleismorepolarizablethanthefluidCM(ω)>0andthe
particleispulledtowardthemaximumoftheelectricfield.TheClausius‐Mosotti
factorCM(ω)variesbetween‐0.5and1.
Themotionofmicroscopicobjectsisopposedbytheviscousdragofthesolutionon
theparticle.Atmicrometerlengthscalesinertiaisverysmallcomparedtoviscous
drag(theReynoldsnumberRe≈10‐3).Theviscousdragonaspherecanbe
describedwithaStokesDragForce
€
Fdrag = −6πηa v whereηisthedynamicviscosity
!
CM(") = Re#p $#m#p + 2#m
%
& '
(
) *
23
ofthemedium,aistheradiusofthesphere,and
€
v isthevelocityofthesphere
relativetothemedium.ThecombinationoftheexpressionfortheDEPforceona
sphere,Eq.2.3,withtheStoke’sDragforcegivesthevelocityofaparticle
€
v DEPmoved
withDEP,
€
v DEP =εma2CM(ω)∇
E RMS2
3η . 2.5
ThelowReynoldsnumberofthesystemallowstheaccelerationoftheparticletobe
ignoredbecausethesystemissufficientlyover‐dampedthattheparticlereachesits
terminalvelocityataratethatiseffectivelyinstantaneous.
2.3DielectricModelsforCellsandVesicles
Thedielectricpropertiesofcellsandvesiclescanbedescribedwithsimplemodels.
Thepermittivityofcellsandvesiclesaresetbytheintrinsicpropertiesofthe
object’sconstituentmaterialsandalsotheobject’sgeometry.Bothvesiclesandcells
canbemodeledasspheresofwaterwrappedinathin,impermeablemembrane,as
isshowninthemodelinFig.2.3a.(Jones,1995)Thesolutioninsidethevesiclehasa
permittivitythatisdefinedbytherealpartofthepermittivityεcanda
conductivityσc.Thethinshellsurroundingthevesiclehasacapacitanceperunit
areacmemandasurfaceconductivitygm.
Fromthemodeldescribedabove,alumped‐elementcircuitmodelisdevelopedto
describevesiclesandcells.Thecapacitanceacrossthemembraneisdefined
C=2cmema,whereaistheradiusofthevesicle,andthesurfaceconductivityis
24
assumedtobenegligible.(Jones,1995)Theinteriorofthevesicleisdefinedby
R=1/σcandC=εcinparallel.Bycombiningthelumpedcircuitelementsassuminga
sphericalvesicleandanelectricfieldinradialdirections,asisshowninFig.2.3a,an
effectivepermittivityfortheobjectisarrivedat
. 2.6
whereτm=cma/σcistherelaxationtimeofthechargesthatbuilduponthe
membraneandτc=εc/ σcisthedielectricrelaxationtimeofthesolutioninsidethe
object.Asimilarmodelcanbedevelopedtounderstandtheyeastcell,whereathin
dielectriclayerisaddedtothemodeltorepresentthecellwallthatisnotpresentin
mammaliancellsandvesicles.(Jones,1995andHunt,2007)
TheDEPforceonavesicleorcellsuspendedinasolutioncanbecalculatedusingthe
dielectricmodeldevelopedabove.Thesolutionhasarealpermittivityεsand
conductivityσs.TheeffectivepermittivityforthevesicleorcellεefffoundinEq.2.6
iscombinedwiththeexpressionfortheCMfactorinEq.2.4:
2.7
Therearenowtwomorecharacteristictimesinthesystem,thedielectricrelaxation
timeofthesolutionoutsideofthevesicleτs=εs/σsandtherelaxationtimeofthe
chargesontheoutsideofthevesiclebuildinguponthemembraneτm’=cma/σs.
!
"eff = cmaj#$ c +1
j#($m + $ c ) +1
%
& '
(
) *
!
CM(") = Re" 2(# s#m $ # c#m
') + j"(#m
' $ # s $ #m ) $1
" 2(# s#m
'+ 2# s#m ) $ j"(#m
'+ 2# s + #m ) $ 2
%
& '
(
) *
25
AvesiclecanbetrappedandmovedwithpositiveDEPatRFfrequenciesifthe
internalconductivityσcislargerthantheexternalconductivityσs.InFig.2.3bthe
CMfactor,Eq.2.7,isplottedversustheinteriorconductivityσcofavesicleorcell
suspendedinasolutionwithσs=10‐3S/m,εs’=εc’,a=5µm,andf=1MHz.Ifthe
conductivityinsidethevesicleσc>10‐3S/mthentheCMfactorispositiveandthe
vesicleexperiencesapositiveDEPforce.TheCMfactor,andasaresulttheDEP
force,plateaustoitsmaximumvalueaboveaconductivityσc=1S/m.
TheDEPforceonvesiclesandcellsdependsonthefrequencyoftheappliedelectric
field.InFig.2.3ctheCMfactor,Eq.2.7,isplottedversusfrequencyforavesiclewith
aninteriorconductivitygreaterthanthatofthesolutionσc>σsTheDEPforceis
negativeatlowfrequencies,positiveatintermediatefrequencies,andnegativeat
highfrequencies.
ThefrequencyresponseofCM(ω)canbeunderstoodheuristicallybyanalyzingthe
lumpedcircuitmodelinFig.2.3a.Forfrequenciesslowerthantheinterfacial
chargingtimeofthemembraneτmem=acmem(1/σc+1/2σs),theDEPforceis
negative(CM<0).Attheselowfrequenciesthemembranehasahighimpedance
andcausesthevesicletoactlikeaninsulatingsphere,makingitlesspolarizable
thanthemedium.Atintermediatefrequencies,abovethechargingtimeofthe
membraneτmemandbelowtheinterfacialdielectricrelaxationtimeofthe
26
vesicle τmw=(εp+2εm)/(σc+2σs),CM>0andtheDEPforceispositive.Atthese
intermediatefrequenciesthemembraneistransparenttotheelectricfieldandthe
solutioninsidethevesicleactslikeaconductor,makingthevesiclemorepolarizable
thanthemedium.Atfrequenciesabovetheinterfacialdielectricrelaxationtimeτmw
ofthevesicle,theDEPforceisnegative(CM<0).Atthesehighfrequenciesthe
vesicleistransparenttotheelectricfield,makingthevesiclelesspolarizablethan
themedium.
27
Figure2.3(a)Alumpedcircuitmodelforavesicleoralivingcellwitharadiusa,thatconsistsofaninternalsolutionwithaconductivityσcandadielectricconstantεc,wrappedinathinshellwithaconductivitygmemandacapacitanceperunitareacmem,(b)theClausiusMosottifactorCMplottedversustheinternalconductivityofavesiclesuspendedinasolutionwithanexternalconductivityσs=10‐2S/minafieldwithafrequencyω=1MHz,(c)CMofavesiclewithaninternalconductivityσc=0.1S/msuspendedinasolutionwithaconductivityσs=10‐2S/mfactorplottedversusthefrequencyωoftheappliedelectricfield.
28
2.4TransmembranePotentialDifferenceanditsApplications
InadditiontotheabilitytotrapandmoveobjectswithDEP,electricfieldscanbe
usedtoelectroporatevesiclestoreleasetheircontents,fusetwovesiclestogether,
orpermeabilizecellmembranes,asisdemonstratedinChapter4.Thesetasksare
accomplishedbyusinglowerfrequencyelectricfields(kHzfrequenciescomparedto
theMHzfrequenciesusedforDEP)toinducevoltagesacrossthemembranesof
vesiclesandcells.Alargetransmembranevoltagecancauseporestoformin
membranes(electroporation)orcausetwovesiclestofusetogether(electrofusion).
Theelectroporationofavesicleorcelloccurswhenthereisalargepotential
differenceacrossthemembraneofacellorvesicleVTM.Ingeneral,electroporation
andelectrofusionoccurwhenthemaximumtransmembranevoltage
€
VTMmax isgreater
than1V.(Sugaretal.,1987)Themaximumvoltage
€
VTMmax inducedacrossthe
membraneofasphericalvesicleinanexternalelectricfieldis:(Grosseetal.,1992)
€
VTMmax =
3 E a
2 1+ (ωτmem )2 2.8
whereτmem=acmem(1/σc+1/2σs)istheinterfacialchargingtimeofthemembrane,
asisdescribedintheprevioussection.Atypicalvesicleorcellhasacapacitanceper
unitareaCmem=10‐2F/m2.(Jones,1995)Foravesiclewitharadiusa=5μmwith
aninternalconductivityσc=0.1S/msuspendedinasolutionwithaconductivity
σs=10‐2S/m,thereisaninterfacialchargingtimeτmem=20μs.Thetransmembrane
voltageVTMisplottedversusfrequencyinFig.2.4foravesicleorcell,asisdescribed
insection2.3,inanelectricfield
€
E = 3 V /µm .Atthefrequenciesf≈1MHzusedfor
29
DEPthevesicleorcellwillhaveaninducedtransmembranevoltageofVTM≈1mV.
Thissmalltransmembranevoltagedoesnothaveasignificanteffectonthe
membraneofavesicleorcell.(Olofssonetal.,2003)Atfrequenciesslowerthanthe
interfacialmembranechargingtimeτmem,atransmembranevoltageontheorderof
VTM≈1Vforms.ThismuchlargertransmembranevoltageVTMcantrigger
electroporationorelectrofusion.
Ithasbeenshownintheliteraturethatelectricfieldpulsescantriggerthefusion
(electrofusion)betweenadheringvesiclesorcells.(Sugaretal.,1987andTressetet
al.,2007)Themodelforelectrofusionisamulti‐stepprocessthatisillustratedin
Fig.2.4b.Figure2.4b(i)showstwovesiclesthataretrappedandmovedtogether
withDEP.Figure2.4b(ii)showsthevesiclesbroughtintotightcontact,partially
squeezingoutthewaterlayerbetweenthetwovesicleswithDEP.Electricfield
pulseswithadurationlessthanτmemarethenappliedtocreatepores
(electroporation)inthetransmembranecontactarea,asisshowninFig.2.4b(iii).If
theporedensityislargeenoughthenporescanthennucleateintoastableholein
thecontactareaasisshowninFig.2.4b(iv).(Tressetetal.,2007)Onourchip
electricfieldsatMHzfrequenciesareusedtoholdvesiclesincontactwithDEPwhile
time‐multiplexedelectricfieldpulseswithfrequencieslessthanthemembrane
chargingtimeω<1/τmemtriggerthefusion,asisdemonstratedinChapter4.
Inducedvoltagesacrossacell’smembraneVTMcanaffectthecell’sviabilityand
physiologicalstate.Inalivingcell,ionpumpsmaintainatransmembranevoltageof
VTM=70mVacrossthecellmembrane.Itisageneralruleofthumbthataslongas
30
theinducedtransmembranevoltageismuchlessthanthenormalphysiological
transmembranevoltageVTM<<70mV,thenitwillhaveonlyasmalleffectoncell
physiology.(Olofssonetal.,2003)AcelltrappedinaDEPtrapwithanelectricfield
strength
€
E = 3V /µm andafrequencyf=1MHzwillhaveaninduced
transmembranevoltageVTM≈1mV.Therefore,acellinaDEPtrapshouldremain
healthyuntilthefrequencyoftheappliedelectricfieldispurposelyloweredto
induceelectroporationorelectrofusion.
31
Figure2.4(a)Thetransmembranepotentialofamodelofavesicleorcellwithaninternalconductivityσc=0.1S/m,suspendedinasolutionwithaconductivityσs=10‐2S/m,witharadiusa=5µmandacapacitanceofCmem=10‐2F/m2isplottedversusthefrequencyfofanappliedelectricfield
€
E = 3 V /µm ,(b)astep‐by‐step
illustrationofthefusionoftwovesicles.
32
2.5DielectricHeating
Dielectricobjectscanbeheatedwithtime‐varyingelectricfields.Inducedand
intrinsicdipolemomentswithinanobjectwillattempttoalignthemselveswitha
time‐varyingelectricfield.Theenergyassociatedwiththisalignmentisviscously
dissipatedasheatintothesurroundingsolution.ThepowerdensityPabsorbedby
adielectricmaterialisgivenbythefrequencyoftheappliedelectricfieldf,the
imaginarycomponentofthepermittivity(thelossfactor)ε’’ofthematerial,the
vacuumpermittivityε0,andtheelectricfieldstrength
€
E withthe
expression(Bengtssonetal.,1974):
€
P =ωεoε' ' E
2
2.9
Thelossfactorofthematerialdependsonthefrequencyoftheelectricfieldandthe
characteristictimeτWofthedielectricrelaxationofthematerial,withthe
expression:
€
ε' '= (εs −ε∞)1+ (ωτ )2
+σωεo 2.10
whereεs=78.4ε0isthelowfrequencydielectricconstantofwater,ε∞=1.78ε0isthe
opticaldielectricconstant,τW=9.55psisthecharacteristicrelaxationtimeofwater
atT=25°C(asisshowninFig.2.1),andσistheconductivityofthesolution.
(Murrelletal.,1994)
Thepowerthatamaterialabsorbsfromatime‐varyingelectricfielddependsonthe
frequencyofthefield.InFig.2.5thepowerdensityP[W/m3]absorbedbywaterin
33
anelectricfieldwithamagnitude
€
E =105 V /m isplottedversusfrequencyfor
severalsolutionswithdifferentconductivitiesσ.Fordeionizedwaterthepower
densityincreasesmonotonicallyuntilitplateausatf=18GHz,thefrequency
associatedwiththecharacteristicrelaxationtimeofwaterτw.Forsolutionswitha
finiteconductivityσ,thepowerdensityhasaconstantvalue
€
P = εo
E
2σ at
frequenciesbelowacriticalfrequencysetbythedielectricrelaxationtimeofthe
solutionτs= εs/σs.Atfrequenciesabovethedielectricrelaxationtimeofthe
solutionτsthepowerincreasesmonotonicallyuntilitplateausatf=18GHz.For
biologicalsolutionsσ≈0.5S/m,thedielectricrelaxationtimeofthesolutionis
τs≈1.4ns.
Duetowater’slargedielectriclossatGHzfrequencies,microwavepoweris
absorbedmuchmorestronglybywaterthanpolymers,glass,silicon,andmost
objectsthatmicrofluidicdevicescouldbeconstructedwith.Thisallowsmicrowave
powertobedeliveredtosolutionswithoutsignificantlyheatingthesurrounding
microfluidicdevice.Effectiveheaterscanbebuiltthatworkatf=3.0GHz,a
frequencyveryclosetothatofcommercialmicrowaveovens(2.45GHz),thatis
belowthefrequencyassociatedwiththerelaxationtimeofwaterbutwherewater
stillreadilyabsorbspower(asisdemonstratedinChapter6).
34
Figure2.5.ThepowerdensityP(W/m3)plottedversusthefrequencyfoftheappliedelectricfield.Severalsolutionswithdifferentconductivityσ areplotted,increasinggoingfromthebottomtothetopwithvaluesof0S/m,0.03S/m,0.06S/m,0.14S/m,0.3S/m,and0.62S/m.
2.6Magnetophoresis
Magnetophoresisprovideatechniquetotrapandmoveparticlesthatcompliments
DEP.Whereasalmostallmaterialshavesomedielectricresponse,mostobjectsare
completelytransparenttomagneticfields.Assuch,magnetophoresishasthe
benefitthatforcescanbeappliedspecificallytomagneticparticleswhilenot
affectingthesurroundingdielectricobjects.Thistechnique(asisdemonstratedin
Chapter5ofthisthesis)isusefulforapplyinglocalforcestomicroscopicobjects.
Aparticlewithamagneticmoment
€
m inamagneticfield
€
B experiencesaforce,
€
F MAG =
m • B . 2.11
35
Foraparamagneticparticlewithamagneticsusceptibilityχ,themoment
€
m = Vχ B /µoisproportionaltotheexternalmagneticfieldstrength
€
B ,thevolumeof
theparticleV,andthevacuumpermeabilityµ0.Theforceonaparamagneticparticle
is:(Leeetal.,2004)
€
F MAG =
−Vχµo
∇ B 2. 2.12
Intheworkpresentedinthisthesis,superparamagneticparticlesareused.
Superparamagnetismisaformofmagnetismwhereverysmallferromagnetic
particlesbehavelikeparamagnets.Theparticlesaresmallenoughthatthermal
fluctuationscanceltheirnetmomentwhenthereisnofieldapplied,butanapplied
fieldwillcausetheparticlestoalign.Assuch,superparamagneticparticlesmaybe
describedwithahighmagneticsusceptibilityχatfieldslowerthantheirsaturation
value.(Beanetal.,1959andLeeetal.,2005)
36
Chapter3.HybridIntegratedCircuit/
MicrofluidicChips
InthischapterhybridIC/microfluidicchipsandtheexperimentalapparatusthat
surroundthemarepresented.Twohybridchipsaredescribedinthisthesis.Inthis
chaptertheDielectrophoresis(DEP)Chip(TheFabutron1.0)isintroduced.(Huntet
al.,2008)TheHighVoltageDielectrophoresis/MagneticChip,(HV‐DEP/Magnetic
Chip,TheFabutron2.0)whichcombinesdielectricandmagnetictrappingontoa
singlechip,isdescribedinChapter5.Thedetailsoftheexperimentalapparatus
surroundingbothchipsarepresentedinthischapter,includingthefluidics,
electronics,optics,thermalmanagement,andcomputercontrol.
3.1Overview
HybridIC/microfluidicchipscombinetheinexpensivecomplexityandsmall
featuresizeofICswiththebiocompatibleenvironmentofmicrofluidicstoperform
programmablebiologicalandchemicalexperimentsonthemicrometerscale.(Leeet
al.,2007)Inthischapterachipisdescribedthattrapsandmovesindividual
microscopicobjectsalongprogrammablepathswithdielectrophoresis(DEP).The
DEPchipconsistofalargearrayofmicrometer‐scalepixelsthatcanbeindividually
addressedwithradiofrequency(RF)voltages,creatingelectricfieldsabovethe
chip’ssurface.Thechipcansimultaneouslyandindependentlycontrolthelocations
ofthousandsofdielectricobjectssuchaslivingcellsorpLdropsoffluid,allowing
thousandsofbiologicalandchemicalexperimentstobecontrolledinparallel.
37
3.2DielectrophoresisChip(Fabutron1.0)
TheDEPChipconsistsofacustomICandafluidcellthatholdsliquiddirectlyonthe
chip’ssurface,asisshowninFig.1.1e.ElectroniccircuitsintegratedintotheICare
usedtocreatelocalelectricfieldsabovethechip’ssurface.Theseelectricfieldscan
beusedtopositionobjectswithDEPorrelease,permeabilize,andfuseobjectswith
electroporationandelectrofusion.Thecomplexcircuitryandfastelectronicsofthe
DEPchipallowmanylivingcellsandvesiclestobecontrolled,allowingmany
parallel,well‐controlledbiologicalandchemicaloperationstobeperformed.
TheDEPchipconsistsofanarrayofelectrodes,eachofwhichcanbeindividually
drivenwithavoltage,tocreateanelectricfieldabovethechip’ssurfaceasisshown
intheinsetinFig.1.1e.Specifically,theDEPchipconsistsof128x256(32,768)
11x11μm2pixels,eachofwhichcanbeindividuallydrivenwitha5Vpeak‐to‐peak
radiofrequency(RF)voltagewithfrequenciesfromDCto11MHz.Underneatheach
pixelisastaticrandomaccessmemory(SRAM)element.ThestateoftheSRAM
elementdetermineswhetherthepixelisdrivenbytheexternalRFvoltagesource
(thepixelturnedoff)orbythelogicalinverseoftheRFvoltage(thepixelturnedon).
TheRFvoltagebetweenthepixelscreatesanelectricfieldabovethechip’ssurface
thatisusedtotrapandmoveobjectswithDEP.Theentirearrayofpixelscanbe
updatedhundredsoftimesinasecond.TheICisdesignedusingCadencedesign
softwareandisfabricatedwithacommercial0.35μmprocesswith4metallayers
throughMOSIS(process:TSMC35_P2).ThedetailedspecificationsoftheDEPChip
areoutlinedintheappendixintheformatofadatasheet.(AppendixA)
38
3.2.1OperatingPrincipals
TheDEPChipusespositiveDEPtotrapandmovedielectricobjects.Byshiftingthe
locationofthepixelsthatareturnedon,thelocationoflocalelectricfieldmaxima
aremovedaroundthearray.InFig.3.1aquasi‐staticfiniteelementsimulations
(Ansoft:Maxwell11)areshownoftheelectricfieldmagnitude
€
E 5μmabovethe
chip’ssurface.Twopixelsinthecenterofthearrayaresetto5Vandtherestofthe
pixelsaresetto0V.Adielectricobjectwitharadiusa=5µmisschematically
shownbeingpulledtowardsthemaximumofthefield.Thesimulationsshowthat
theobjectwillexperienceamaximumfieldof
€
E ≈ 5 V /µm .(Hunt,2007)
TheexpectedDEPforceonanobjectabovethechip’ssurfacecanbecalculatedby
combiningtheelectricfieldsimulationsshowninFig.3.1awiththedielectricmodels
presentedinChapter2.Theforceonadielectricobjectwitharadiusa=5µm,
describedbythemodelinsection2.3,suspendedinasolutionwithaconductivity
σs=10‐2S/m,inanelectricfieldwithafrequencyf=1MHzandanRMSmagnitude
showninFig.3.1a,isplottedinFig.3.1b.Thedielectricobjectistrappedatthe
interfaceoftwopixelsontheleftthatareturnedonandtwopixelsontherightthat
areturnedoff.ThemagnitudeofthetrappingforceisF~1nNandtheforceis
localizedwithintwopixellengthsofthetrap.Alineisfittotheforcecurveatthe
locationofthetrap,andtheeffectivespringconstantisfoundtobek=520pN/µm.
39
3.2.2CharacteristicTimes
Theratethatthedevicecanperformoperationsislimitedbythetimethatittakes
formicroscopicobjectssuspendedinsolutionabovethechip’ssurfacetoreactto
theelectricormagneticfields,notthespeedoftheelectronics.Therearetwo
importantcharacteristictimesthatdescribethemicroscopicobjectssuspendedin
solutiononourchips:thetimethatittakesforaparticletodiffuseawayifatrapis
turnedoffτdiffandthetimethatittakesforaparticletomovebetweentrapswhen
onepixelisturnedoffandthenextisturnedonτmove.Aparticlesuspendedina
solutiontakesatimeτdiff=L2/16Dtodiffusethedistanceofhalfofapixel
lengthL/2.Theself‐diffusionconstantDisdefinedbytheStokes‐Einstein
EquationD=kBT/6πηa,wherekBTisthethermalenergy,ηistheviscosityofthe
solution,andaistheradiusoftheparticle.Thetimethatittakesforana=1µm
particlesuspendedinwatertodiffuseL/2=5µmisτdiff≈1sec.Thediffusion
timeτdiffislongerforbiggerparticles.
Thetimethatittakesforaparticletomovefromonepixeltothenextτmoveis
calculatedusingtherelationshipbetweentheDEPforceandvelocityinEq.2.5and
theplotoftheDEPforceversusdistanceinFig.3.1b.Thetimethatittakesforan
a=1µmparticleontheDEPchiptomovefromonepixeltothenextisτmove≈10ms.
Forlargerparticlesthetimethatittakestomoveparticlesbetweenpixelsτmove
increases.TheICoperatesatsub‐mstimescales(asisdescribedbelow)suchthat
theICcanchangeitsstatemanytimesinthetimethatittakesforaparticletoreact
tothechangeinfield.
40
Figure3.1.(a)Afiniteelementsimulationoftheelectricfieldstrength5μmabovethechip’ssurface,(b)aplotoftheforceonadielectricobject(asmodeledinchapter2.3)attheinterfaceoftwopixelsontheleftthatareturnedon(inyellow)andtwopixelsontherightthatareturnedoff(inwhite).
41
3.2.3IntegratedCircuitDesign
ThissectionoutlinesthedetaileddescriptionoftheDEPchipgiveninTomHunt’s
Thesis.(Hunt,2007andHuntetal.,2008)Thechip’s128x256(32,768)pixelsare
addressedwithlogicandmemorybuiltintotheIC,suchthatonly8datalines(not
includingthetwoclocksandfourcontrolsignals)arerequiredtoupdatetheentire
array.AschematicoftheSRAMarrayandthelogicandmemorythatsurrounditare
showninFig.3.2a.TheSRAMmemoryisorganizedas128wordsx256bits.The
bitsofeachwordarereadinandoutofthechipwithasequentiallyloadedtwo‐
phaseclockedshiftregister.Eachwordisaddressedtoaspecificrowinthearray
usinga7bitrowdecoder.Therowdecodertakes7binaryinputsandusesthemto
choose1of27(128)rowsoftheSRAMarray.Amicrographofthechip,showinga
sectionofthearrayofDEPpixelssurroundedbythecontrolelectronics,isshownin
Fig.3.2c.
AschematicofthecircuitunderneatheachDEPpixelisshowninFig.3.2b.The
SRAMmemoryelementsunderneatheachpixelareaddressedwithaword‐lineand
itsvaluesetwithabit‐line.TheSRAMelementcontrolsamultiplexer(MUX)that
routesoneoftwosignals,producedoffofthechip,tothe11x11µm2pixelabove.
GenerallythetwosignalsareRFvoltagesthatare180°outofphase,butcanbeset
arbitrarily.TheoutputoftheMUXgoesthroughavoltage‐amplifierbeforedriving
thepixel.Thevoltage‐amplifierisatwotransistorinverter,appropriatelysizedto
drivethecapacitiveloadofthepixel.Thevoltage‐amplifiershaveanon‐resistance
ofRon≈10kΩ,drivingapixelcapacitanceoflessthanCload≈50fF,whichyieldsa
42
sub‐nsRCtime.Thevoltageonthepixelcanhaveapeak‐to‐peakvoltageof5Vand
abandwidthofDC–11MHz.Thebandwidthcouldhavebeenlarger(DC–GHz)if
greatercarewastakeninthedesignofthechiptobuffertheRFlinesthroughoutthe
circuit.
Thechip’saddressingarchitectureisaresultoftrade‐offsbetweenmaximizingthe
refreshrateoftheSRAMarray,minimizingthesizeoftheelectronicsneeded
underneatheachpixel,andminimizingthenumberofinputandoutputpinsonthe
chip.Therefreshrateofthechipisimprovedbyallowingregions‐of‐interest(ROI)
tobeupdatedwithoutupdatingtheentirearray.Onthechip,wordsthatare256
bitsinlengthareupdatedwithrandom‐access.Foreachpixeltobefullyrandom‐
accesswouldrequiretwoextratransistorsunderneatheachpixel,increasingthe
pixelsizeandcomplexityofthecircuit.Therefreshrateismaximizedbybreaking
thearrayintowordswiththesmallestnumberofbits.Inthelimitingcase,thechip
wouldbeasfastasitcouldbeifeachpixelhadawiretotheoutside‐world.
Conversely,thenumberofpinsisminimizedbyincreasingthesizeofeachword.In
thelimitingcase,thechipwouldhavetheminimumnumberofpinsiftheentire
arraywasupdatedinserialwithashiftregister.
Inourdesign,witha128wordx256bitarray,theshiftregisterisupdatedata
maximumrateof1bit/0.1µsmakingittake~26µstoupdateasinglewordonour
chipand~3.3mstoupdatetheentirearray.Thus,thechipcanrefreshitsentire
statefasterthanthetimeτmove>10msthatittakesfortheparticlesabovethe
chip’ssurfacetoreacttothefields.Theupdaterateofthechipiscurrentlylimited
43
bytheelectronicssurroundingthechip.Withthecurrentelectronicstherefresh
rateofthechipis400mstoupdatetheentirearray.Thechipisinterfacedtoa
computerandtheinterfaceisslowerthanthechipitself,asisexplainedinmore
detailbelow.Anewgenerationofgraduatestudentsiscurrentlydesigninga
microcontrollerinterfacetothechipthatcanrefreshthechipatitsmaximumrate.
Thedesignofthechip,usingtheCadencesoftwarepackage,ishierarchicaland
incorporatesdigitalandanalogtestingsuchthat(ifdonecorrectly)thelayoutis
guaranteedtomeetdesignspecificationsandthedesignrulesofthefabrication
process.Thechipdesignbeginswithbreakingthechipintoseparablemodules:the
pixel,theshiftregister,therowdecoder,asingleSRAMelement,etc…Theindividual
modulesaredesignedintermsofothermodules,continuingdownwardsinthe
hierarchyuntilreachingthetransistor‐level.Eachtransistorcorrespondstothe
layoutofmasks(dopingandmetallayers,vias,etc…).Themodulesarecombinedat
themask‐layoutlevelbymanuallyplacingthemodulesandmanuallyconnecting
themwithmetallayers.Thedesignsoftwarechecksthatthecircuitsatthemask‐
levelmatchesthecircuitsatthemodule‐level.Theelectricaltestingbeginsatthe
bottomofthehierarchyatthemodule‐level,usingSPICEmodelsforeachelement,
totestthedigitalandanalog(bothtimedomainandfrequencydomain)responseof
thecircuits.Oncethelayoutforamoduleisdrawn,additionalunintended
capacitancesinthesystemareextractedfromthelayoutandincorporatedintothe
testing.Thenewlayoutcanbemodifiedbasedontheresultsofthetesting,andthis
cyclecanberepeatediniterationsuntilthedesignspecificationsofthemoduleare
met.Themodulesarecombinedandthesystemistestedworkingfromthebottom
44
ofthehierarchytothetop,untiltheentirechipislaidoutandfullytested.Forthe
chipsdesignedinthisthesis,thedesignprocesstakesabouttwomonthsfromstart
tofinishandresultsinachipwithN≈300,000transistors.
45
Figure3.2.(a)AschematicofthearchitectureoftheSRAMarrayandthelogicandmemorythatareusedtoreadandwritetoitontheDEPchip,(b)aschematicofthecircuitunderneatheachDEPpixel,showingtheSRAMmemoryelement,amultiplexer(MUX)thatdirectseithertheRFsignaloritslogicalinversetoavoltagedriverthatconnectstotheelectrode,(c)amicrographofa250x200µm2sectionshowingacornerofthechipwithaportionoftheDEParrayintheupperleftcorner,therowcontrolcircuitsonthebottom,andthebitcontrolcircuits,includingtheshiftregister,ontheright.
46
3.2.4Capabilities
TheDEPchiphasbeenusedtotrapandmovedielectricobjectssuchasliving
cells,(Fig.3.1aandFig.3.1c)dropsoffluidimmersedinoil,(Fig.3.1b)andvesicles
usedasbiomimeticcontainers(Chapter6).Inadditiontotheabilitytotrapand
moveobjectswithDEP,electricfieldscanbeusedtoelectroporatevesiclesto
releasetheircontents,fusetwovesiclestogether,orallowcellstotakeup
substancesfromthesolution.(Chapter6)Thesetasksareaccomplishedbyusing
lowerfrequencyelectricfields(kHzfrequenciescomparedtotheMHzfrequencies
usedforDEP)toinducevoltagesacrossthemembranesofvesiclesandcells,asis
describedinChapter2.Inadditiontowhathasbeendemonstratedbyourgroup,
anyapplicationthatcouldbenefitfromaprogrammableelectricfieldcontrolledon
thelengthscaleofL=10µmwithanRFbandwidthcouldbeperformedontheDEP
chip,suchaselectro‐optics(Lopez,1970),electro‐wetting(Pollacketal,.2000),or
electro‐chemistry(Nyholm,2005).
47
Figure3.3(a)AtimesequenceofyeastandratalveolarmacrophagesbeingtrappedandmovedwithDEP.Theblackarrowsindicatethedirectionthatthecellsaremovedbetweenframes.Thecellsmoveatarateof~50µm/sec,(b)atimeseriesofdropsofcoloredwaterbetweenalayeroffluorocarbonoilandhydrocarbonoiltrapped,moved,split,andmergedwiththechip,(c)amicrographoftheentirechipbeingusedtopositionthousandsofyeastcellstospell“Harvard.”(Huntetal.,2008)
48
3.3ExperimentalApparatus
TheapparatusthatsurroundstheIC/microfluidicchipsisdescribedinthissection.
Theapparatusconsistsofaninterfacetocontrolthechipwithacomputerandan
opticalmicroscopewithavideocameratoobservewhathappensonthechip.A
blockdiagramoutliningtheorganizationoftheapparatussurroundingthechipsis
showninFig.3.4a.Fluidicsbringsamplesandreagentstothechip.Thechip
communicatestotheoutsideworldthroughelectricalconnectionsonacustom
printedcircuitboard.Theprintedcircuitboardconnectstoacomputerthrougha
digitalinput/output(I/O)card.Thecomputercanbothwriteandread‐outthe
stateofthechipthroughcustomcontrolsoftware.Afluorescencemicroscope
imagesthecontentsofthechip.Theimagesaresenttothecomputerthrougha
digitalcamera.Agraphicaluserinterface(GUI)providesanintuitiveplatformfor
theusertointeractwiththechip.Theoverallsystemisaimedatbeingrobust,easy,
andflexibleenoughtousethatnewexperimentscanbequicklyprototyped,with
minimumsetupandmaintenancetime.
3.3.1Fluidics
AfluidcellisbuiltdirectlyontopoftheICwithasiliconegasket(Invitrogen:p‐
24744)witha1.2mmholethatiscutwithaholepunch,asisshowninFig.3.4c.A
3x3mm2glasscoverslipisplacedontopofthefluidcelltosealit.Thefluidcell
canbefilledwithapipette.Directlypipettingfluidontothechip’ssurface,rather
thansettingupamicrofluidicnetwork,keepstheexperimentalapparatusflexible,
allowingmanyexperimentstobeattemptedinashorttime.
49
TheIC/microfluidicchipcanbeintegratedasacomponentinalargerPDMSor
glassbasedmicrofluidicsystem.Passivemicrofluidicscancoupletheoutsideworld,
anditsmacroscopicfluidsamples,tothechipinarolethatisanalogoustotherole
printedcircuitboardsplayforICsincomputers.TheIC/microfluidicchipcould
behaveasaprogrammablecomponenttoperformtaskssuchassortingor
combiningsamplesandreagents.Insuchasetup,thefluidoutputofthechipcould
befedtooutputchannelsofthepassivemicrofluidicdeviceforfurtherprocessingor
collection.
3.3.2Electronics
InthissectiontheelectronicssurroundingtheDEPchiparedescribed.
Electronicallythechipiseffectivelya32kbitSRAM;Assuch,theelectronics
surroundingthechipareidenticaltotheread/writeelectronicssurroundingany
SRAMarray.TheIC/microfluidicchipismountedonastandard84pinchipcarrier
asisshowninFig.3.4c.TheICismountedwithsilverpainttobothelectrically
groundthesubstrateoftheICandtocreateathermallyconductiveconnectiontoa
thermalreservoir.WirebondsconnecttheICtothepinsofthechipcarrier.Inall
thechiprequires24wirebonds,includingredundantpowerlines.Allofthewire
bondsareonthesamesideoftheICtomakeiteasiertoprotectthemfromthefluid.
Thewirebondscanbecoveredwithepoxytoprotectthemfurtherfromthenearby
fluid.Howevermostoftenthebondsareleftunprotected,asthefluidcellisa
sufficientbarrierbetweenthefluidandthewirebonds.
50
ThechipcarriersitsonacustomPCBwhichconnectstheICtothecomputer,
providespower,andconnectstheICtoanRFvoltagesource,asisshownin
Fig.3.4d.ThePCBhasRCfiltersoneachoftheinputsandoutputsofthechipwitha
cornerfrequencyf3dB=100MHztoprotectthechipfromvoltagespikes.Thedigital
linesconnectfromthePCBtothecomputerthroughaproprietaryNational
Instrumentscable(NI:SHC68‐68‐EPM)tothedigitalI/Ocard(NI:PCI‐6254).The
RFvoltageisproducedoffofthechipandisbroughtontotheboardwithaBNC
connector.TheDEPchiphasapowerconsumptionof0.5‐2W,dependingonhow
manypixelsareturnedonandthefrequencyoftheRFvoltage.
3.3.4Optics
TheIC/microfluidicchipsitsunderafluorescencemicroscope,asisshownin
Fig.3.4d.Fluorescencemicroscopyisapowerfulandwidelyusedtooltoimage
biologicalandchemicalsystems.(Pawley,2008)Couplingfluorescencemicroscopy
withthechipenablesaccesstoawelldevelopedtechniquetoimagethesamplesand
reagentsthatthechipcontrols.ThesiliconsubstrateoftheICisnotoptically
transparentandsothemicroscopeoperatesbymeasuringreflectedlight.TheICis
notfluorescentatopticalfrequenciesandsobehavesasablackbackground.The
microscopeviewsthesamplesthroughtheglasscoverslipofthefluidcell.
ThemicroscopeapparatusconsistsofapillarmountedOlympusBX‐52,hovering
abovetheIC/microfluidicchip,asisshowninFig.3.4d.Longworkingdistance
objectivesareusedtogiveenoughspaceforthefluidcell(Olympus:LMPLFLseries).
Thelightsourceisa100Wmercurylamp.Thedeviceismonitoredwithan
51
HamamatsuORCA‐ERcooledCCDcamera.ImagesaretakenwithMicroSuiteBasic
Edition(Olympus)andStreamPixdigitalvideorecordingsoftware(Norpix).
FuturedevicesthatutilizeIC/microfluidicchipsandhopetobeportablesurelycan
notincludealaboratory‐sizedfluorescencemicroscope.Recentworkhasshown
thatfluorescencemicroscopycanbeperformedinmicrofluidicdevicesusingsmall
LEDlightsourcesandphotodiodes.(Psaltisetal.,2006)Moreforwardlooking,a
CCDarraycouldbeflippedandbondedontopoftheICformingafluidchannel
betweenthetwochips.(Cuietal.,2008andHengaetal.,2006)
3.3.5ThermalManagement
Temperaturecontrolisessentialforexperimentsonbiologicalsystems.The
temperatureofthechipiscontrolledusingheat‐sinks.TypicalICshaveoperational
temperaturesbetween65°Cand85°C.(Leeetal.,1998)Formanybiologicaland
chemicalexperimentsthatmightbeperformedonthechip65°Cisfartoohot.As
such,stepsaretakentocontrolthetemperatureofthechip.
Inthefirstiterationofthermalmanagement,theceramicchipcarriersitsontopofa
machinedcopperblockwithathinlayerofthermalgrease(ArcticSilverInc.:Arctic
Silver).Airisblownwithafanoverthelargesurfaceareaofthecopperblock’s
bottomside.ThetemperatureoftheDEPchip’stopsurfacewiththechipturnedon
is40°C‐50°Ccomparedto90°Cwhennoheatsinkisattached.Thechip
temperatureismeasuredwithaninfrarednon‐contacttemperaturesensor(Control
Company4477).
52
TheseconditerationoftheheatsinkwasdesignedfortheHV‐DEP/Magnetic
Chip(Chapter5)andutilizesawatercooler.Withwatercoolingthetemperatureof
thechipcanbecontrolledbysettingtheflowrateandtemperatureofthewater
bath.Thewatercoolerisconstructedwithamachinedcopperpiecethatis
connectedwithathermallyconductiveepoxy(Loctite383and7387)toa
commercialCPUwatercooler(DangerDen,MC).Thetopofthecopperblockis
polishedandimmediatelycoveredwithathermallyevaporatedlayerof
8nmTi/100nmgoldtokeepcopperoxidefromgrowing.Thechipcarriersitson
topofthemachinedcopperblockwithathinlayerofthermalgrease(ArcticSilver).
Thewatercoolerisdrivenwithasubmersiblepump(BecketCorp,Versa)witha
flowrateof92gallons/hour.TheHV‐DEP/MagneticChip(Chapter5)has
temperaturesensorsintegratedintotheIC.Theanalogoutputofthesensorscanbe
placedintoafeedbackloopwiththewater‐coolertocreateacontrolsystemto
maintainthechiptemperature.
53
Figure3.4.(a)AschematicoftheexperimentalapparatussurroundingtheIC/microfluidicchip.Thegreenboxesrepresentcomponentsthatareelectrical,bluerepresentfluidic,orangerepresentoptic,andyellowrepresentswherethecomponentscometogether,(b)anopticalmicrographoftheIC(b)84pinchipcarrierholdingtheIC,ontopoftheICisaredfluidcell,(c)aphotographoftheexperimentalsetupofthehybridIC/microfluidicsystem.
54
3.3.6ComputerControl
ComputersoftwareisusedtoprogramandtointerfacetheIC/microfluidicchip
withauser.ThecomputercommunicateswiththechipthroughaNational
InstrumentsPCIboard(NI:PCI6254).ThePCIboardiscontrolledwithcustomLab
View(NI)software.MATLAB(TheMathWorks)codeisusedonthefront‐endto
interfacewiththeuserandtotranslateauser’sinputintothecommandsthatwillbe
senttothechip.AllMATLABandLabViewcodeisincludedinAppendixC.
TheGUIthatwedesignedisshowninFig.3.5.IntheGUIausercandrawobjects
ontoabit‐mapthatcorrespondstotheDEPpixelarrayofthechip.Objectscanbe
created,recognizedbythecomputer,andmovedaroundthescreenwithacursor.
Basicdefaultshapescanautomaticallybedrawnsuchashorizontal,vertical,and
diagonallinesandchecker‐boardpatterns.Inadditiontothestaticdrawingsthat
canbedrawnintheGUI,multi‐framemovies,whereeachframeisastateofthechip,
canbeimportedintoacompilerandplayedonthechip.(AppendixC)Tomaximize
therefreshrate,thecodecansendcommandstowriteonlyword‐linesintheSRAM
arraythathavechangedfromframetoframe.
Thedigitalcameraonthemicroscopeconnectstothecomputerandisdisplayed
withStreampix(Norpix).Currently,thecoordinatesystemonthevideo‐feedfrom
thedigitalcameraandtheGUIismanuallyalignedbytrappingandmovinganobject
andthenfindingitonthemicroscope.Thecombinationofimage‐recognition
softwareandautomatedcontrolofthemicroscopestagecouldbeusedtoremove
55
thehumanfromthecontrolloop,andallowcomplexautomatedtaskssuchas
sortingtobedoneinaclosed‐loop.
Figure3.5.ScreenshotsoftheGUI,(a)ThemainscreenfortheGUIwhereobjectscanbedrawnonabitmapthatrepresentsthestateofthechip.Theblackandwhitestripesarethearbitrarypatternthatisbeingwrittentothechipinthescreen‐shot,(b)the“advancedmode”oftheGUIwhereobjectsthatarecreatedinthemainscreencanbemovedwithacursor.
56
Chapter4.AHybridIntegratedCircuit/
MicrofluidicPlatformtoControlLivingCells
andpLBiomimeticContainers
4.1Overview
ThischapterdescribeshowthehybridDielectrophoresis(DEP)Chip,introducedin
Chapter3,canbeusedtoperformbiologyandchemistryexperiments.TheDEP
chipsimultaneouslycontrolsmanyindividuallivingcellsandsmallvolumesoffluid.
ThesmallvolumesoffluidarecontainedinpLsizedlipidvesicleswhichprovidea
stablecontainerforaqueoussolutionsthatcanbesuspendedinwater.Thevesicles
canbemovedaroundthechip,fusedtogether,andhavetheircontentsreleasedinto
thesolution.Livingcellscanalsobemovedaroundthechipandcanbe
electroporatedtoallowsubstancesfromthesolutiontoenterthecells.Inaddition,
thechipcancontrollablymechanicallydeformthecellsorvesicles.Thesebasic
functions,whicharedemonstratedinthischapter,canbestrungtogetherto
performcomplexbiologicalandchemicallaboratorytasks.
Previousworkhasbeendonetousehybridintegratedcircuit(IC)/microfluidic
chipsforbiologyandchemistryexperiments.TomHunt’sthesisdescribeshybrid
chipsbeingusedtotrapandmovedropsofwatersuspendedinoil.(Hunt,2007)
TheDEPChipcanpositionthedrops,splitsthedropsintwo,andmergestwodrops
together,demonstratingapotentialplatformforperformingprogrammable
chemistryexperiments.(Hunt,2007andHuntetal.,2008)Intheseemulsion‐based
57
systems,dropsofwateraresuspendedinoilandarestabilizedusingasurfactant,as
isshowninFig.4.1.(Holtzeetal.,2008)Dropbasedmicrofluidicchipshavebeen
showntobeimportanttechnologyforperforminghigh‐throughputbiologicaland
chemicalexperiments.(Kosteretal.,2008)Achallengeforthesesystems,however,
isthatcellsmustalsobeencapsulatedindropsbecausetheoil‐basedcontinuous
phaseisnotbiocompatible.Inaddition,moleculeswithhydrophobictailscanleak
frominsidethevesiclesintotheoil,thuslimitingthechemistrythatcanbedonein
thesesystems.(Hunt,2007andHoltzeetal.,2008)
Inthischapteraversatileplatformisdevelopedforbiologyandchemistry
experimentsontheDEPchip.Thisplatformcansimultaneouslycontrolbothliving
cellsandsmallisolatedvolumesoffluidsuspendedinwater.InSection4.2.1the
functionalityandcapabilitiesoftheDEPChiparereviewed.Topackagesmall
volumesoffluid,suchthattheycanbesuspendedinwater,inspirationistakenfrom
cellularbiology.Thepreparationofthevesiclesusedinthischapterisdescribedin
Section4.2.2.Lipidvesicles,dropsofwatersurroundedbyathinphospholipid
bilayermembrane,arecommonlyusedincellstopackagesubstancesfor
intercellulartransport,tostoreenzymes,andtocreateisolatedchemicalreaction
chambers.(Albertsetal.,2007)Artificiallyproducedunilamellarvesiclesmimic
naturalvesiclesandproviderobustpLcontainersthatareimpermeableandstable
forawiderangeofsalinity,pH,andotherenvironmentalconditions.(Tressetetal.,
2007)AschematicofaphospholipidvesicleisshowninFig.4.1.Thischapter
demonstratestheuseofunilamellarvesiclesasarobustpLcontainerforfluidson
theDEPChip.
58
Thefrequencydependenceofthedielectricpropertiesofvesiclesandcellsenable
thedielectricphenomenonusedinthischaptertobeindependentlyaccessedwith
electricfieldsatdifferentfrequencies.VoltagesatMHzfrequenciesareusedfor
DEP,totrap,position,anddeformcellsandvesiclesasisdescribedinSection4.2.3.
Voltagepulseswithmspulse‐widthsareusedforelectroporationandelectrofusion,
totriggerthereleaseofthecontentsofvesicles,fusetwovesiclestogether,and
permeablizethemembranesofcellsasisdescribedinSection4.2.4.
TheDEPChipisusedinthischapterdemonstratebasicfunctionsonlivingcellsand
unilamellarvesicles,whichcanbestrungtogethertoperformcomplexbiological
andchemicallaboratorytasks.InSection4.3.1theDEPChipisusedto
simultaneouslyandindependentlytrapandmovemanyindividualvesiclesand
livingcellswithdielectrophoresis(DEP).InSection4.3.2voltagespulsescreatedby
theDEPChipareusedtoselectivelyelectroporatevesiclestoreleasetheircontents,
fusetwovesiclestogether,andpermeabilizelivingcells.InSection4.3.3vesiclesare
deformedbychangingtheshapeoftheDEPtraps.Theplatformdemonstratedin
thischaptercanbeusedforawiderangeofchemicalandbiologicalapplicationsand
isastepforwardforthefieldofhybridIC/microfluidics.
59
Figure4.1.Aschematicrepresentationofawaterdropinoilemulsionstabilizedwithsurfactantsisshownontheleft.Aschematicrepresentationofaunilamellarvesicleisshownontheright.Thephospholipidmoleculesthatformthesurfactantandthevesiclemembraneareshownwithredcirclesforthehydrophilicheadsandblacklinesforthehydrophobictails.
60
4.2Methods
4.2.1TheHybridIntegratedCircuit/MicrofluidicPlatform
InthissectiontheDEPChip,whichisdescribedfullyinChapter3,isusedtocontrol
livingcellsandsmallvolumesoffluidusingspatiallypatternedelectricfields.Here,
theDEPchipanditscapabilitiesaresummarized.Thehybridchipconsistsofa
microfluidicchamberbuiltdirectlyontopofanIC.TheICconsistsofanarrayof
electrodes,eachofwhichcanbedrivenwithanRFvoltagetospatiallypatternthe
electricfieldmagnitudeabovethechip’ssurface.Underneatheachpixelisastatic
randomaccessmemory(SRAM)element.ThestateoftheSRAMelement
determineswhetherthepixelisdrivenbytheexternalRFvoltagesource(thepixel
turned‘off’)orbythelogicalinverseoftheRFvoltage(thepixelturned‘on’).The
arrayconsistsof128x256(32,568)11x11µm2pixels,eachofwhichcanbedriven
withanRFvoltagewithabandwidthfromDC–11MHz.Theentirearrayofpixels
canbeupdatedhundredsoftimesinasecond.AmicrographoftheIC/microfluidic
chipisshowninFig.1.1b.
4.2.2UnilamellarVesicles
Lipidvesiclesaredropsofwaterencapsulatedbyathinphospholipidbilayer
membrane.Aschematicofthecross‐sectionofaphosphlipidunilamellarvesicleis
showninFig.4.1.Thephospholipidmembraneconsistsofamphipathic
phospholipidsthatself‐assembleintobilayerssuchthattheirhydrophilicheadsface
theinsideandoutsideofthevesicle,providingarobustcontainerforaqueous
61
solutions.(Albertsetal.,2007)Aunilamellarvesicleconsistsofasinglebilayer
membrane,asopposedtoseveralassomevesiclesdo.
ThevesiclesaremadethroughcollaborationwithThomasFrankeatUniversityof
AugsburginGermany.Thevesiclesarepreparedwithliposomeelectroformation
usingamodificationoftheAngelovaMethod.(Angelovaetal.,1986)Therecipe
usedtocreatethevesiclesisasfollows.Briefly,asmallamountoflipidin
chlorophorm(ca.20microliterofa5mg/mlsolution)isdepositedontotwoindium‐
tin‐oxidecoatedglassslides.Afterevaporationoftheorganicsolventforatleast
6hourstheslidesareassembledinparallelwithaseparating2mmteflonspacerto
formthepreparationcellandincubatedforonehourinasaturatedwatervaporat
roomtemperatureforprehydration.Subsequently,theelectroformationcellisfilled
with200mOsmaqueoussolution(50mMsodiumchlorideandsucrose)andalow
frequencyelectricfieldof500Hzandamplitude1V/mmisappliedtotheITO‐
slides.Afterapproximately8hourslipidvesiclesofdiameterslargerthe20µmare
harvested.Theexternalsaltysolventisremovedbyrepeatedlywashingwithaniso‐
osmoticaqueousglucosesolutionandgentlecentrifugationatlowrotation
frequencies.ThisprocedureprovidesthedielectriccontrastnecessaryforDEP
actuationandstillensurestheintegrityofthevesiclecontainers.
4.2.3Dielectrophoresis(DEP)ofVesiclesSuspendedinWater
ThehybridchipusespositiveDEPtotrapandmovecellsandvesiclessuspendedin
water.Anyobjectthathasacomplexpermittivitythatisdifferentthanthe
surroundingmediumcanbecontrolledusingDEP,(Jones,1995)asisdescribedin
62
detailinChapter2.ByshiftingthelocationoftheDEPChip’spixelsthatareturned
on,thelocationoflocalelectricfieldmaximaaremovedaroundthearray.In
Fig.3.1aquasi‐staticfiniteelementsimulations(Ansoft:Maxwell3D)areshownof
theelectricfieldmagnitude
€
E 5µmabovethechip’ssurface.Twopixelsaresetto
5Vandtherestaresetto0V.Avesiclewitharadiusa=5µmisshownbeing
pulledtowardsthemaximumofthefield.Thesimulationsshowthatavesicle
experiencesamaximumelectricfieldstrength
€
E ≈ 0.5 V /µm .
Thedielectrophoreticresponseofvesiclescanbecontrolledbysettingthesalinityof
thesolutioninsidethevesicleσintrelativetotheconductivityofthesolutiononthe
outsideofthevesicleσsol,asisdescribedinfulldetailinChapter2.InFig.2.3ba
plotoftheClausius‐Mosotti(CM)factor,ameasureofthedifferenceinpermittivity
oftheobjectandthemedium,isshownversustheinteriorconductivityofan
a=5µmvesiclesuspendedinadeionizedsolutionwithaconductivityof
σsol=10‐3S/m.Iftheconductivityinsidethevesicleisgreaterthanσint>10‐2S/m
thentheCMfactorispositive,andthevesiclecanbetrappedandpositionedwith
positiveDEP.
Thedielectrophoreticresponseofcellsandvesiclesisafunctionofthefrequencyof
theappliedelectricfield.InFig.2.3caplotofCMversusfrequencyisshownfora
vesiclewithinteriorconductivityσint=50mS/m.Belowacut‐offfrequencyof
ωd=50MHzandaboveacut‐offfrequencyofωmem=2kHz,theCMfactorispositive
andinvariabletochangesinfrequency.Itisinthisfrequencybandthatpositive
DEPisusedinthischapter.Thehighfrequencycut‐offissetbythedielectric
63
relaxationtimeτd=εp/σintofthesolutioninsidethevesicle.Thelowfrequencycut‐
offissetbythechargingtimeofthemembraneofthevesicle
τmem=aCmem(1/σint+1/σsol).
4.2.4ElectroporationandElectrofusion
Voltagepulsescreatedbythechipcanbeusedtoelectroporateandelectrofuse
vesiclesandcells,asisexplainedinfulldetailinChapter2.Electroporation
generallyoccurswhenthereisatransmembranevoltageVTM>1V.(Sugaretal.,
1987)ThemaximumvoltageinducedacrossamembraneVTMofasphericalvesicle
orcellinanexternalelectricfieldisgivenbyEq.2.8.Thecharacteristiccharging
timeofthemembraneisgivenbytheexpression:(Jones,1995)
€
τmem = aCmem (1σ int
+1
2σ sol
) 4.1
whereCmem=10‐2F/m2isthespecificmembranecapacitanceofaunilamellar
vesicle.(Jones,1995)Foravesiclewitharadiusa=5µmandinternalconductivity
σint=0.1S/minasolutionwithconductivityσsol=10‐2S/m,thereisamembrane
chargingtimeτmem=20ms.Electricfieldswithfrequencies1/ω<τmemareusedfor
DEP.Atthef=1MHzfrequenciesusedforDEPthetransmembranevoltage
VTM≈10mVandnodamageiscausedtothemembrane.(Albertsetal.,2007and
Grosseetal.,1992)Electricfieldscreatedwithvoltagepulseswithapulsewidth
τ>τmemcreatetransmembranevoltagesVTM~1Vandareusedtoelectroporateand
electrofusevesiclesandcells.
64
Theabilitytoselectivelyelectroporatevesiclesenablesthechiptolocallyrelease
substancesintothesolution.Thislocalreleaseofsubstancescreatesagradientin
theconcentrationasafunctionofthedistancerfromthecenterofthevesicle.
Consideravesiclewitharadiusafilledwithaconcentrationn=nsofasubstance
suspendedinasolutionthathasnoneofthesubstancen=0.Thegradientinthe
concentrationiscalculatedbysolvingthediffusionequationinspherical
coordinatesaroundthevesiclewiththeboundaryconditionsthattheconcentration
n=nsadistancer=afromthecenterofthevesicleandn=0atadistancer=∞.
(DillandBromberg,2003)Thesolutiontothediffusionequationisplottedfora
vesiclewithradiusa=5µminFig.4.2aandisgivenbytheexpression:
€
n(r) =nSar . 4.2
Whenacellorvesicleiselectroporated,suchthatareactionoccursatthe
membrane’ssurface,aconcentrationgradientisalsocreated.Considera
reactionthatoccursatthemembranesurfacewithareagentinthesolutionthat
hasabulkconcentrationn∞.Inthiscase,theconcentrationatthesurface
disappearsbecauseitisconsumedbythereaction,andtheconcentrationatan
infinitedistanceawayisthatofthebulkconcentration.Theboundaryconditions
arethenthatn=n∞atadistancer=∞formthecenterofthevesiclesandn=0at
adistancer=a.Thesolutiontothediffusionequationisplottedforavesicle
withradiusa=5µminFig.4.2bandisgivenbytheexpression:
€
n(r) = n∞(1−ar) 4.3
65
Theconcentrationgradientsdependonlyontheconcentrationofthesubstances,
theradiusofthevesicle,andthedistancefromthevesicle.Theabilitytocreate
well‐controlledconcentrationgradientsinthesolutionisatoolthatcanbeusedfor
deliveringreagentsandsamplestocellsortootherreagentsforchemistryand
biologyexperimentsonthechip.
Thevoltagepulsesusedforelectroporationcanbetime‐multiplexedwiththe
voltagesusedforDEP.Thisispossiblebecauseduringthedurationofthems
voltagepulses,duringwhichtheDEPforceisturnedoff,cellsorvesiclesdonot
diffuseasubstantialamount.Aparticlesuspendedinasolutiontakesτdiff=L2/16D
todiffusethedistanceofhalfofapixellengthL/2,asisexplainedinChapter3.The
timethatittakesforaparticlewitharadiusa=1μmsuspendedinwatertodiffuse
L/2=5μmisτdiff≈1sec,whichismuchlongerthanthetimethattheDEPtrapis
turnedoff.Thediffusiontimeτdiffisevenlargerforbiggerparticles.
Electricfieldpulsescanalsobeusedtriggerthefusionofvesicles,asisexplained
infulldetailinChapter2.Themodelforelectrofusionisamultistepprocess.
TwovesiclesarebroughtintotightcontactwithDEP.Electricfieldpulsesare
thenusedtoinduceatransmembranevoltageacrossthecontactareaofthetwo
vesicles.Thetransmembranevoltagecauseselectroporationonthecontact‐area,
andiftheporedensityislargeenoughthentheporesnucleateandthevesicles
fuse.(Sugaretal.,1987)Onthechip,voltagesatMHzfrequencyareusedtohold
thevesiclesincontactwithDEPwhiletime‐multiplexedelectricfieldpulsesare
usedtotriggerthefusion.
66
Figure4.2(a)Aplotoftheconcentrationgradientcreatedaroundavesicle,shownastheorangecircle,thatisreleasingasubstanceatitssurface,(b)aplotoftheconcentrationgradientcreatedaroundavesicleorcell,shownasthepurplecircle,thatisreactingwithasubstanceinthesolutionatitssurface.
67
4.3Demonstrations
4.3.1TrappingandMoving
InthissectionitisshownhowthehybridIC/microfluidicchipscantrapandmove
individualvesiclesandcells,bothsuspendedinwater,alongarbitrarypaths.
Figure4.3ashowshowindividualvesiclescanbeindependentlytrappedandmoved
alongindependentpaths.Figure4.3a(i)showsthreevesiclesthatare
independentlytrappedonthechipwithDEP.InFig.4.3a(i‐iii)thevesicleontheleft
isbroughtbetweenthetwoothervesiclesat70µm/sec.InFig4.3a(iv‐vi)the
vesicleonthebottomismovedupwards,positioningthevesiclesintoatriangle.
ThisdemonstrationshowsthattheDEPChipcanindependentlycontrolseveral
objectsatonce,enablingcomplexexperimentstobeperformedinparallelonthe
hybridchip.
Figure4.3bshowshowthehybridIC/microfluidicchipcansimultaneouslytrap
andmovemanyvesicles.InFig.4.3bhundredsofvesiclesaresimultaneously
positionedintoan‘H’,demonstratingthatthechipcancontrolthepositionofmany
vesiclesatonce.Toaccomplishthis,manyDEPpixelsareturned‘on’tospell‘H’and
thevesiclessedimentontotheinterfaceofthepixelsthatareturned‘on’andthose
thatareturned‘off’.ThisdemonstrationshowsthattheDEPChipiscapableof
simultaneouslycontrollingmanyindividualobjects.
Figure4.3cshowshowthehybridIC/microfluidicchipcansimultaneouslytrapand
movebothvesiclesandlivingcellsthataresuspendedinwater.Yeastcellsare
68
culturedovernightinYPDbroth(BDInc.)at37°C,andthenresuspendedina
250mMglucosesolution.Figure4.3cshowsavesicleandabuddingyeastcell
trappedonthechip.Thefluorescenceimageoftherhodaminefilledvesicleis
superimposedontothebrightfieldimageoftheyeastcellsandiscoloredred.In
Fig.4.3c(ii)thevesicleismovedupwardswhilethecellistrappedinplace.In
Fig.4.3c(iii)theyeastcellismovedtotheleftwhilethevesicleisheldinplace.In
Fig.4.3c(iv)theyeastcellismoveddownwardsasthevesicleissimultaneously
movedtotheleft.ThisdemonstrationshowsthattheDEPChipcansimultaneously
controlcellsandsmallvolumesoffluidheldinvesicles,bothsuspendedinwater.
Thisenablesbiologicalandchemicalexperimentstobeperformedonthehybrid
chipthatusebothcellsandsmallcompartmentalizedvolumesoffluid.
69
Figure4.3(a)TimesequenceofvesiclespositionedwithDEP.Pixelsareturnedoninsequencetotrapandmovethevesicles.Thegreenlineshowsthedirectionthatthechipmovesthevesicle.Themaximumspeedofavesicleis70μm/sec.Eachframeisseparatedby~1sec,(b)amicrographofhundredsofvesiclessimultaneouslypositionedtospellan‘H,’(c)timesequenceofvesiclesandcellssimultaneouslytrappedandmovedonthechip.Thefluorescenceimageofthevesiclesissuperimposedontothebrightfieldimageofthecellsandiscoloredred.Thegreenlineshowsthedirectionthatthechipismovingthevesiclesorcells.Eachframeisseparatedby~1sec.
70
4.3.2TriggeredReleaseoftheContentsofVesicles
InthissectionitisdemonstratedthatthehybridIC/microfluidicchipscan
controllablyreleasethecontentsofvesicles.Figure4.4ashowshowthehybrid
IC/microfluidicchipcancontrollablyreleasethecontentsofavesicleintothe
surroundingsolutionwhileholdingitinplacewithaDEPtrap.Figure4.4ashowsa
vesiclefilledwith4mMNaClsolutionsuspendedinaconcentratedfluorescently
self‐quenchedfluorosceinsolution.A1mselectricfieldpulse,thatistime
multiplexedwiththeDEPfiel,andrepeatsevery5ms,isturnedonattimet=0sec.
Thevesicleiselectroporatedandtheconcentratedfluorosceinmixeswiththe
solutioninthevesiclecausingthevesicletofluoresce.AfterΔt=8secthepulse
sequenceisturnedoffandthevesicleisheldinaDEPtrapforΔt=2minutes.The
vesiclemaintainsitsfluorescence,demonstratingthatwhenthepulsesareturned
offthevesiclehealsitselfandstopsmixingwiththesolution.AfterΔt=2minutes
thepulsesequenceisturnedonuntiltheconcentrationoffluorosceininsidethe
vesiclematchesthatofthesolutionandthevesicleceasestofluoresce.This
demonstrationshowsthatthechipcanuseelectroporationtocontrollablymixthe
contentsofvesicleswiththesolution.Electroporationofvesiclescanbeusedto
formchemicalgradientsinthesolutionsurroundingthevesicleortoallow
substancesinthesolutiontomixwiththecontentsofthevesicle.
4.3.3ElectroporationofCells
InthissectionitisdemonstratedhowthehybridIC/microfluidicchipscan
permeabilizeacell’smembrane,allowingsubstancesinthesolutiontoenterthecell.
71
Figure4.4bshowsanindividualyeastcellheldinplacewithDEPandselectively
electroporated,allowingsubstancesinthesolutiontoenterthecell.Yeastcellsare
culturedovernightinYPDbroth(BDInc.)at37°Candresuspendedin250mM
glucosesolution.A0.4%solutionoftheviabilitystain,TrypanBlue,isaddedtothe
suspendedcells.WhenTrypanBlueentersacellitstainsitblue.InFig.4.4bayeast
cellisheldinplacewithDEP.Becausethecellishealthy,TrypanBluedoesnot
enterthecellandthecelldoesnotturnblue.A1mspulsethatrepeatsevery5ms,
thatistimemultiplexedwiththeDEPfield,isturnedonatt=0.AfterΔt=40sec
thecellisobservedtoturndarkblue,asisshowninagrayscaleimageinFig.4.4b.
Theabilitytoelectroporatecellsonthechipenablessubstancesinthesolutiontobe
controllablyintroducedintoselectedcells.Thisfunctionalitycouldbeusedto
introducesubstancessuchasmolecularprobes,DNA,ordrugsintoselectcells.
4.3.4TriggeredFusionofVesicles
InthissectionitisshownhowthehybridIC/microfluidicchipcanfusetwovesicles
togetherandmixtheircontents.Figure4.4cshowstwovesiclesthatarebrought
intocontactwithDEPandthenfusedtogetherwithelectrofusion.InFig.4.4c(i)
twovesiclesarebroughtintocontactwithDEP.InFig.4.4c(ii)thevesiclesare
broughtintotightcontactwithDEP,creatingalargecontactareabetweenthetwo
vesicles.InFig.4.4c(iii)a1mspulsethatrepeatsitselfevery5ms,multiplexed
withtheDEPfield,isturnedonfor0.5seccausingthetwovesiclestofuseintoone.
ThevesicleisstretchedintotheshapeoftheDEPtrap.InFig.4.4c(iv)thetrapis
turnedoffandthefusedvesiclerelaxesintoasphericalshape.
72
ThefusionoftwovesiclesallowspLvolumesofsamplestobecontrollablymixed.
Thevoltagepulsesusedtofusethevesiclesdonotleadtoanappreciablemixingof
thecontentsofthevesiclewiththesolution.Thevesiclesareobservedtofuseafter
0.5secofthepulsesequence.IntheexperimentshowninFig.4.4a,ittakes~4sec
forasimilarvesicletohaveappreciablemixingwiththeoutsidesolutionusingthe
samepulsesequence.Thereforeinthetimethatittakestofusethevesiclesthere
shouldnotbeappreciablemixingofthevesicleswiththesolution.Theabilityto
selectivelyfusetwovesiclestogetherenablessmallisolatedvolumesofsamplesto
bemixedonthechip,animportantfunctionforperformingchemistryandbiology
experiments.
73
Figure.4.4(a)TimesequenceofavesicleheldinplacewithDEPfieldswhileitscontentsarereleasedintothesolutionwithelectroporation.Thevesicleissuspendedinaconcentratedfluorosceinsolution.Theelectroporationcausethevesicletomixitscontentswiththesolution,makingthevesicletofluoresce.Inthefirstthreeframes,eachseparatedby4seconds,thevesicleiselectroporated.ThevesicleisheldinaDEPtrapfor2minutes.Inthenextthreeframes,separatedby4seconds,thevesicleiselectroporateduntilthecontentsofthevesiclefullymixeswiththesolutionandthevesiclestopsfluorescing,(b)acellisheldinplacewithDEPandelectroplatedinasolutioncontainingTrypanBluestain.Theleftandrightframeshowthecellbeforeelectroporationandafter40sec.ofelectroporation,respectively,(c)twovesiclesarebroughtintocontactwithDEPandthenfusedtogetherwithelectrofusion.Theschematiconthebottomshowwhichpixelsareturnedon.AgreenpixelindicatesapixelthatisturnedonforDEPandaredpixelindicatesapixelthatisturnedonwithvoltagepulsesmultiplexedwiththeMHzfrequencyvoltage.
74
4.3.5DeformingVesicleswithDielectrophoresis
InthissectionitisshownhowthehybridIC/microfluidicchipcancontrollably
deformobjects.Figure4.5showshowchangingtheshapeoftheDEPtrapscan
deformvesicles.SeveralDEPpixelsareturnedonunderneathavesicle,causingthe
vesicletobedeformedintotheshapeoftheDEPtrap.TheleftcolumnofFig.4.5
showsamapofwhichpixelsonthechipareturnedon.Themiddlecolumnshows
simulations(Ansoft:Maxwell)oftheelectricfieldgenerated5µmabovethechip’s
surface.TherightcolumnshowsmicrographsofvesiclesdeformedintheDEPtrap.
Thesamevesicleisusedforeachdemonstration.InFig.4.5athevesicleisheldina
simpletrapof4pixelsandthevesicle’scross‐sectioniscircular.InFig.4.5bthetrap
isspreadintotwodisplacedbars,andthevesicleispulledintoanoblongshape.
MorecomplicatedpixelpatternsleadtoshapessuchasdiamondsinFig.4.5c,
hexagonsinFig.4.5d,andsquaresinFig.4.5e.Theabilitytodeformvesiclesusing
DEPtrapsisanimportantproof‐of‐conceptforperformingrheologyandfor
controllingthemechanicalenvironmentofobjectsonthechip.(Riskeetal.,2006)
Thistechniquecouldbeespeciallyusefulinfuturechips,thathavesmallerpixel
sizes,forcontrollingthemechanicalenvironmentoflivingcells.(Wangetal.,1994)
75
Figure4.5VesiclesaredeformedintoavarietyofshapesbychangingtheshapeoftheDEPtrap.Theschematicsontheleftshowwhichpixelsareturnedon.Redandwhitepixelsindicatepixelsthatareturnedonoroff,respectively.Thecentergraphicsareplotsofthesimulatedelectricfieldstrength
€
E 5µmabovethechip’s
surface.Ontherightaremicrographsofthedeformedvesicles.
76
4.4Discussion
Theplatformthatispresentedinthischaptertakesadvantageofthecapabilitiesof
ICsandthepropertiesofunilamellarvesiclestomakeaversatileplatformfor
biologicalandchemicalexperimentsonachip.Thischapterhasdemonstrateda
platformthatcantrap,move,deform,fuse,andlocallyreleasethecontentsofpL
volumevesiclessuspendedinwater.Onthesamechip,atthesametimeandinthe
samesolution,theplatformcantrap,move,andpermeabilizelivingcellssuspended
inwater.Thisplatformprovidesanimportantstepforwardforperforming
biologicalandchemicalexperimentsonahybridIC/microfluidicchipby
demonstratingfunctionsthatareimportantbuildingblocksformorecomplextasks.
Tomovethislaboratory‐on‐a‐chipplatformfromthelaboratoryintoaportable,
inexpensivetoolforbiologicalandchemicaltesting,furtherfunctionalitymustbe
included.Forinstance,whiletheplatformcanperformcomplexoperationsonmany
vesiclesandcells,thesamplesthemselvesarepreparedonlaboratorybench
equipmentandthenpipettedontothechip.Integratingthehybridchipintoa
complexmicrofluidicnetwork,wheresamplepreparationcouldbeperformed,could
solvethisproblem.Anotherhurdleforthehybridchipisthatitisconnectedto
large,laboratorysizedequipment,suchasafluorescencemicroscopeandadesktop
computer.Furtherintegrationoftasksintothehybridchipssuchaslocal
temperaturecontrol,(Issadoreetal.,2009)NMRsensors,(Leeetal.,2008)
measurementofeletrogeniccells,(DeBusschereetal.,2001)andintegrated
77
optics(Cuietal.,2008)wouldopenupmoreapplicationsandleadtoamore
versatileandself‐sufficientplatform.(Leeetal.,2007)
78
Chapter5.HybridMagneticand
DielectrophoreticIC/MicrofluidicChip
5.1Overview
Thischapterdescribesthedevelopmentofahybridintegratedcircuit(IC)/
microfluidicchipthatusesbothdielectrophoresis(DEP)andmagneticforcesto
controllivingcellsandsmallvolumesoffluid.Thechipthatispresentediscalled
theHighVoltageDielectrophoresis/MagneticChip(HV‐DEPChip,Fabutron2.0).
ThehybridchipisfabricatedusingaspecialhighvoltageICprocessthatenables
DEPforcesthatareroughly100xasstrongasthosedemonstratedontheDEPChip
inChapter3.AmicrographoftheHV‐DEP/MagneticChipisshowninFig.5.1a.
ThecombinationofbothDEPandmagneticforcesonthesamechipexpandsthe
capabilitiesofhybridIC/microfluidictechnology,asisdemonstratedinthis
chapter.
TheHV‐DEP/MagneticChipissimilartotheDEPChipdescribedinChapter3,but
includesadditionalfunctionality.TheICincludesanarrayofpixelsthatcaneachbe
addressedwitharadiofrequency(RF)voltagetolocallyapplyDEPforces,amatrix
ofwiresthatrunsunderneaththeDEPpixelarraytoapplylocalmagneticforces,
andintegratedsensorstolocallyreportthetemperatureofthechip.Thelargearray
ofDEPpixels,magneticwires,andtemperaturesensorsarecontrolledusingstatic
randomaccessmemory(SRAM)andlogicthatarebuiltintotheIC.Figure5.1a
showsamicrographoftheIC.
79
Previoushybridchipshavebeendevelopedtocontrollivingcellsanddropsoffluid
usingelectricormagneticfields.Chipshavebeendevelopedtosimultaneously
controlthepositionofindividualpLdropsoffluid(Huntetal.,2008)andlivingcells
(Manaresietal.,2003andHuntetal.,2008)usingdielectrophoresis(DEP)for
biologicalandchemicalexperimentsonachipasisshowninChapter4.Chipshave
alsobeendevelopedtocreatelocal,programmablemagneticfieldsusingmatrixesof
wires(Leeetal.,2004)andarraysofcoils(Leeetal.,2006)tocontrolcellsand
biologicalobjectsimplantedwithmagneticnanoparticles.Magneticactuationof
nanoparticleshasbeenusedtoapplyprecisemechanicalstressesoncell
membranes,(Berryetal.,2003)manipulateandactivateindividualmechanically
sensitiveionchannelsandsurfacereceptorsonspecificcells,(Dobsonetal.,2008)
andtotrapandsortcellsandothermagneticallytaggedobjects.(Berryetal.,2003)
ThischapterdemonstratestheHV‐DEP/MagneticChipuseitsDEPpixelarrayto
positionpLvolumesoffluidencapsulatedinlipidvesiclesinSection5.3.1,useits
magneticmatrixtopositionmagneticbeadsinSection5.3.2,anduseitsDEPpixel
arrayandmagneticmatrixtogethertodeformmicroscopicobjectsimplantedwith
magneticnanoparticlesinSection5.3.3.Unilamellarvesiclesareusedasbiologically
inspiredpLcontainersforfluidsthatareimpermeableandstableforawiderangeof
salinity,pH,andotherenvironmentalconditions,asisdescribedinChapter4.(Chiu
etal.,1999andTressetetal.,2007)
80
5.2DescriptionoftheChip5.2.1FieldSimulations
Thechipcreateselectricfieldsaboveitssurfacewitha60x61pixelarrayof
30x38µm2electrodesthatcanbeindividuallyaddressedwith50Vpeak‐to‐peak
voltagesatfrequenciesfromDCto10MHz.Thepixelsareseparatedfromone
anotherby0.8µm.InFig.5.1btheresultsofquasi‐staticfiniteelementsimulations
(Maxwell3D,Ansoft)oftheelectricfieldareshown.Twopixelsareheldat50V
relativetothesurroundingpixels.Theelectricfieldisplotted5µmabovethechip’s
surface.Themaximummagnitudeoftheelectricfieldis
€
E = 3 V /µm .
Thechipuseselectricfieldstotrapandmovedielectricobjects,suchascellsorpL
volumesoffluid,withDEP.(Manaresietal.,2003andHuntetal.,2008)Aspherical
objectinanelectricfield
€
E willexperienceaforcegivenbyEq.2.3(Jonesetal.,
1995)Foranobjectwitharadiusa=5µmwiththedielectricpropertiesofaliving
cellorvesiclesuspendedindeionizedglucosesolution,a
€
F ≈1 nN forcepullsthe
objecttowardstheactivatedpixelsfromalocationonepixel‐lengthaway.Avesicle
filledwithsalinesolutionhasdielectricpropertiessimilartothatofalivingcellin
electricfieldsatMHzfrequencies,andthusbehavessimilarlyintheDEPfield.(Chiu
etal.,1999)
BeneaththeDEPpixelsisamagneticgridthatconsistsof60wiresthatrun
horizontallyand60wiresthatrunverticallyacrossthechip,whichareusedtoform
amagneticfieldabovetheIC’ssurface.Thewirescanbeindividuallysourcedwith
±120mAor0mA.Fig.5.1cshowstheresultsofquasi‐staticsimulations5µmabove
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thechip’ssurface.Twowiresthatruninperpendiculardirectionsacrossthechip
aredrivenwith120mAandallotherwiresarenotdrivenwithcurrent.The
maximummagnitudeofthemagneticfieldis
€
B =6mT.
Thechipusesmagneticfieldstotrapandmoveobjectswithmagneticsusceptibility
differentthanthesurroundingmedium.Asphericalobjectinamagneticfield
€
B
willexperienceaforcegivenbyEq.2.12.Onthechipanironoxidebeadwitha
radiusa=1µm(Bioclone:FF102)willexperiencea10pNtowardsthemaximumof
themagneticfield,whichexistswherethetwoactivatedwiresintersect.Objectscan
betrappedandmovedalongarbitrarypathsbychangingthelocationwherethe
activatedwirescross.Currentcanbedriventhroughseveralwiresinthearray
simultaneouslytocreatemorethanonefieldmaximum,allowingthechiptotrap
andmovemanymagneticobjectsindependently.(Leeetal.,2004)
82
Figure5.1.(a)Amicrographoftheintegratedcircuit,(b)themagnitudeoftheelectricfield|
€
E |,fromaquasi‐staticelectricfieldsimulation,plotted5µmabove
thechipsurface.Thepixelsareshownasbluetilesthatcoverthesurfaceofthechip.Twopixelsareheldat50Vrelativetothesurroundingpixels,(c)themagnitudeofthemagneticfield|
€
B |,fromsimulation,plotted5µmabovethechip’ssurface.The
wiresareshownasbluestripesrunningacrossthesurfaceofthechip.Twowiresaresourcedwith120mAandallsurroundingwiressetto0mA.
83
5.2.2ChipArchitectureThechip’slargearrayof60x61(3,660)DEPpixels,60horizontaland60
vertical(120)magneticwires,and16temperaturesensorsareaddressedwithlogic
andmemorybuiltintotheIC,suchthatonly6datalines(notincludingtwoclocks
andthe4controllines)arerequiredtoupdatetheentirechip.Figure5.2ashowsa
micrographofasectionofthechipthatshowstheDEPpixelarray,thecurrent
driversforthemagneticmatrix,andthedigitallogicthatisusedtoupdateandread
fromthearray.ThestateofthechipisstoredinanintegratedSRAMmemorythat
controlstheDEPpixels,magneticwires,andthetemperaturesensors.
TheSRAMmemoryisorganizedinto32wordsthathave128bits.ThereisanSRAM
memoryelementunderneatheachDEPpixel,temperaturesensor,andcurrent
driverforthemagneticwires.Thememoryarrayisupdatedbyselectingaword‐line
andthenloadingitsstateusinga128bitshiftregister.Theshiftregisterusesatwo‐
phaseclockingscheme.Wordselectionisdonewithastandard5bitrowdecoder.
TheentireSRAMarraycanbeupdatedatarateof~50Hzandindividualwordsin
theSRAMcanbeupdatedatarateof~1.5kHz.Thelogic,memory,andsurrounding
electronicsthatareusedtoupdatethechiparesimilartothatusedfortheDEPchip
describedinChapter3.
AschematicforthecircuitunderneatheachDEPpixelisshowninFig.5.2b.The
stateoftheSRAMmemoryelementcontrolsthe2:1multiplexer(MUX)thatdirects
eithertheRFsignaloritslogicalinversetothevoltagedrivingcircuitthatconnects
tothepixel.AschematicforeachmagneticlineisshowninFig.5.2c.Themagnetic
84
lineshavedrivingcircuitryonbothends.Bysettingthememoryelementsoneither
sideofthewireacurrentof±120mAor0mAflowsthroughthewire.Interspersed
throughoutthearrayare16distributedtemperaturesensorsthatcanbe
individuallyaddressedtoberoutedtoananalogvoltageoutput.
ThehybridchipisconstructedbybuildingasimplefluidcelldirectlyontopoftheIC
withasiliconegasket.Thefluidcellispreparedbycuttinga1.2mmholeoutof
Press‐to‐SealTM0.5mmthicksiliconesheetsfromInvitrogen(Invitrogen:p‐24744)
withahole‐punch.ThesiliconegasketisplacedontotheICunderastereoscope
withtweezers.A3x3mm2glasscoverslipsealsthefluidcell.Theintegrated
circuitwasdesignedatHarvardusingCadenceDesignSoftware(Cadence)and
fabricatedinacommercialfoundryonahighvoltage0.6µmprocess(X‐Fab–XC06
MIDOX).
TheexperimentalsetupsurroundingthehybridIC/microfluidicchipissimilarto
thatoftheDEPChipdescribedinChapter3.Thedevicesitsonachipcarrierona
customprintedcircuitboard(PCB)connectedtoacomputerthroughaPCI
card(NationalInstruments:PCI‐6254).Thepatternssenttothechiparecreated
usingaGUIwritteninMATLAB(TheMathWorks)andsenttothePCIcardwitha
customLabview(NationalInstruments)program.Thedevicesitsunderan
OlympusfluorescencemicroscopewithanOrca‐ER(Hammamatsu)digitalcamera
thatconnectstothecomputer.
85
Figure5.2.(a)AmicrographofapartoftheintegratedcircuitshowingtheDEParray,themagneticcurrentdrivers,andthelogicandmemoryusedtoupdatetheSRAMarray,(b)aschematicofthecircuitryunderneatheachDEPpixel,showingtheSRAMmemoryelement,amultiplexer(MUX)thatdirectseithertheRFsignaloritslogicalinversetoavoltagedriverthatconnectstotheelectrode,(c)aschematicofthecircuitryforeachmagneticwireinthematrix,showingtheSRAMmemoryelements,andtheMUXsthatconnecttothecurrentdriverstodrivethewireswitheither±120mAor0mA.
86
5.3Demonstrations
ThissectiondemonstratestheHV‐DEP/MagneticchipusingDEPtotrapandmove
dielectricobjects,magneticforcestotrapandmovemagneticallytaggedobjects,and
magneticandDEPforcesusedtogethertodeformobjectsimplantedwithmagnetic
nanoparticles.Section5.3.1showshowthehybridchipcanbeusedtotrapand
moveobjectsalongprogrammablepathswithDEP.Section5.3.2showshowobjects
thataretaggedwithmagneticbeadscanbetrappedandmovedalong
programmablepathsusingthemagneticmatrix.Section5.3.3demonstratesthe
utilityofhavingbothelectricandmagneticforcesonthesamechipbyholdinga
unilamellarvesicleembeddedwithironoxidenanoparticleinplacewithDEPand
controllablypullingathintetherfromthevesicle’smembraneusingthemagnetic
matrix.
Thevesiclesarepreparedwithliposomeelectroformationusingamodificationof
theAngelovaMethod,asisdescribedindetailinChapter4.(Angelovaetal.,1986)
Theunilamellarvesiclesareloadedwith4mMNaClandsuspendedinaglucose
solutionwithmatchedosmolarity,whichprovidescontrastinthecomplexdielectric
responsetofacilitateDEPforces.(Huntetal.,2008)Thevesicles’membranesare
stainedwithrhodaminesuchthatthevesiclesareeasilyobservableundera
fluorescencemicroscope.
5.3.1Dielectrophoresis: Trapping and Positioning Vesicles
ThissectiondemonstrateshowtheHV‐DEP/MagneticChipsimultaneouslyand
independentlycontrolsthepositionofobjectswithDEP.Figure5.3shows
87
unilamellarvesiclesthataretrappedandmovedalongprogrammedpathsusingthe
DEPpixelarray.Figure5.3ashowstwovesiclesthatareindependentlytrappedon
thechipwithtwosetsofDEPpixels.InFig.5.3(a‐d)thevesiclesaremovedat
speedsupto80μm/secbysequentiallychangingthepixelsthatareturnedon.In
Fig.5.3athevesicleontherightisindependentlymoveddownwardswhilethe
vesicleontheleftisheldinplace.InFig.5.3bboththeleftandrightvesicleare
moveddownwardssimultaneously.InFig.5.3cthevesicleontheleftis
simultaneouslymovedtotherightwhilethevesicletotherightismovedtotheleft.
Thechiphas3,660pixelsandtheentirearraycanberefreshedat~50Hz,enabling
manyindividualvesiclestobecontrolledsimultaneously.Theabilitytotrapand
moveobjectswithDEPisausefultooltocontrolthepositionofpLvolumesoffluid
andlivingcellsthatarenottaggedormodified.
88
Figure5.3AtimesequenceoftheDEPpositioningofvesicles.Theframes(a‐d)arefluorescenceimagesoftherhodaminestainedmembranesofthevesicles.TheDEPpixelsareturnedoninsequencetopositionthevesicles.Thedashedgreenlineshowsthedirectionthatthechipismovingthevesicles,theyellowsquareshowstheDEPpixelthatisactivated.Themaximumspeedofavesicleis80μm/sec.Eachframeisseparatedbyroughly1sec.
89
5.3.2Magnetophoresis: Trapping and Positioning Magnetic Beads
ThissectiondemonstrateshowtheHV‐DEP/MagneticChipcancontroltheposition
ofobjectswithmagnetophoresis.Theabilitytotrapandmovemagneticobjects
alongprogrammedpathsisausefultooltocontrolthepositionofsamplesthat
cannotbetrappedwithDEPforces,butwhichcanbetaggedwithmagnetic
particles.(Leeetal.,2007)Figure5.4showsthemagneticmatrixtrappingand
movinganironoxidebeadwitharadiusa=1µm(Bioclone:FF102)alongan
arbitrarypath.InFig.5.4atwoperpindicularwiresaredrivenwith120mA,such
thatthebeadistrappedatthefieldmaximumthatoccursatthewire’sintersection.
InFig.5.4(b‐d)differentintersectingwiresareturnedontomovethebeadalonga
programmedpathatspeedsof2µm/s.Themagneticforceisnotaslocalizedasthe
DEPforce.Alongthelengthofeachintersectingwire,particlesareattractedtothe
activatedwire.
90
Figure5.4Timesequenceofmagnetictrappingandpositioningofamagneticbead.Magneticwiresinthematrixareturnedoninsequencetopositionthebead.Thepositionoftheparticleismarkedwithagreencircle.Thedirectionthatthebeadisbeingpulledismarkedwithagreenarrow.Theredlinesshowswhichmagneticwiresinthematrixhavebeenturnedon,theactualwiresareburiedundertheDEPpixelsandarenotvisible.Eachframe(a‐d)isseparatedbyroughly2sec.
91
5.3.3DielectrophoresisandMagnetophoresis:DeformingVesicles
ThissectiondemonstrateshowtheHV‐DEP/MagneticChipcanusemagneticand
DEPforcestogether.Figure5.5showthechipholdingavesicle,thatisimplanted
withironoxidenanoparticles,inplacewithDEPbodyforceswhilepullingathin
tetherfromthevesicle’smembranebyapplyingpointforcesonthemagnetic
nanoparticleswithmagnetophoresis.ThisdemonsrationshowsthatDEPand
Magnetophoresiscanbeusedtogethertoapplypointforceswithnmresolutionto
micrometersizedobjects.(Waughetal.,1992)InFig.5.5aavesicleispositioned
withDEPpixels.TwomagneticwiresthatcrossneartheDEPpixelaresourcedwith
120mAandtheironoxidenanoparticlesinsidethevesiclearepulledtowardsthe
magneticfieldmaximum.Thevesicle’smembraneislocallydeformedintoathin
tether,asvesicleshavebeenshowntodointhepresenceofapointforce.(Waughet
al.,1992)IntheproceedingframesFig.5.5(b‐f)themagneticfieldisturnedoffand
tensioninthemembranepullsthetetherbackintothevesicle.(Waughetal.,1992)
TheabilitytolocallydeformmicroscopicobjectsusingDEPasabodyforceand
magnetophoresisasapointforceisausefultooltoapplyprecisemechanical
stressesonvesiclesandlivingcells.(Berryetal.,2003,Dobsonetal.,2008,and
Tressetetal.,2007)
92
Figure5.5AvesicleisheldinplacewithaDEPpixelwhileatetherispulledusingthemagneticmatrix.TheyellowsquareshowstheDEPpixelthatisactivated,andtheredlinesshowthemagneticwiresthatareturnedon.Thevesicleisimplantedwithironoxidenanoparticles.Afterthefirstframe(a)themagneticfieldisturnedoffandthetetherispulledbackintothevesicle.Eachframe(a‐f)isseparatedby0.2seconds.
93
5.4Discussion
HybridIC/microfluidicchipsprovideaversatileplatformtocontrolsmallvolumes
offluidandlivingcells.Thecombinationofbothmagnetic(Leeetal.,2004andLee
etal.,2007)andDEPforces(Manaresietal.,2003andHuntetal.,2008)ontoa
singlechipfurtherexpandsthecapabilitiesandgeneralityoftheplatform.
Dielectrophoresiscanbeusedtotrapandpositionuntaggedlivingcellsanddropsof
fluidandmagneticforcescanbeusedtotrapandpositionobjectstaggedwith
magneticnanoparticles.Dielectrophoresisandmagnetophoresiscanbeused
togethertoapplyprecisenm‐resolutionpointforcestomicrometer‐sizedobjects.
Magneticactuationofnanoparticlesembeddedincellsorvesicleshasmanyexciting
scientificapplications.Thetechniquedemonstratedinthischapter,pullingathin
tetherfromaunilamellarvesicleandobservingthevesiclepullthetetherbackinto
itself,canbeusedasatooltostudythecomplexmechanicalpropertiesoflipid
bilayers.(Waughetal.,1992)Anumberofexcitingapplicationsexistfor
magneticallyactuatingcellstaggedwithmagneticnanoparticles,suchasapplying
precisemechanicalstressesoncellmembranes(Berryetal.,2003)ormanipulating
andactivatingindividualionchannelsandsurfacereceptorsonspecificcells.
(Dobsonetal.,2008)
Thetechniquesdemonstratedinthischaptercanbecombinedwiththe‘laboratory‐
on‐a‐chip’functionsdemonstratedinChapter4,tocreateageneral‐purpose
platformforbiologyandchemistryexperimentsonindividuallivingcells.The
techniquesdemonstratedinChapter4canbeusedtomixsmallvolumesofreagents
94
andsamplestogetherandtocontrolthechemicalenvironmentofindividualcells.
Themagneticactuationofnanoparticles,demonstratedinthischapter,canbeused
tocontrolcellsembeddedwithmagneticnanoparticlestoactivateordeactivate
specificsurfacereceptorsorionchannels.Thehybridchipscansimultaneously
performmanyoftheseexperimentsinparallel,allowingastatisticalnumberof
independentlycontrolledsingle‐cellexperimentstobecarriedout.
95
Chapter6.MicrowaveDielectricHeatingof
DropsinMicrofluidicDevices
6.1Overview
Inthischapteratechniqueispresentedtolocallyandrapidlyheatdropsofwater
withmicrowavedielectricheating.Thistechniqueaddsakeyfunctionalitytothe
listoffunctionsthataredemonstratedinChapters3,4,and5,theabilitytocontrol
temperature.Waterabsorbsmicrowavepowermoreefficientlythanpolymers,
glass,silicon,oroils,duetoitspermanentmoleculardipolemomentthathasalarge
dielectriclossatGHzfrequencies.Thisselectiveabsorptionofmicrowavepowerby
waterallowsthermalenergytobedirectlyinsertedintosmallvolumesoffluidina
microfluidicdevicewithoutsignificantlyheatingthesurroundings.Therelevant
heatcapacityofsuchasystemisthatofasinglethermallyisolatedpicoliter‐scale
dropofwater,enablingveryfastthermalcycling.
Inthischaptermicrowavedielectricheatingisdemonstratedinamicrofluidic
devicethatintegratesaflow‐focusingdropmaker,dropsplitters,andmetal
electrodestolocallydelivermicrowavepowerfromaninexpensive,commercially
available3.0GHzsourceandamplifier.Thetemperaturechangeofthedropsis
measuredbyobservingthetemperaturedependentfluorescenceintensityof
cadmiumselenidenanocrystalssuspendedinthewaterdrops.Characteristic
heatingtimesaredemonstratedthatareasshortas15mstosteady‐state
temperaturechangesaslargeas30°Cabovethebasetemperatureofthe
96
microfluidicdevice.Manycommonbiologicalandchemicalapplicationsrequire
rapidandlocalcontroloftemperatureandcanbenefitfromthisnewtechnique.
Microwavedielectricheatingiswellsuitedtobeintegratedwiththehybrid
integratedcircuit(IC)/microfluidicchipsdescribedinChapters3,4,and5.Using
modernICtechnologyanddesigningthecircuitswithappropriateradiofrequency
designprincipals,pixelscanbedrivenwithGHzfrequencyvoltages.Ahybrid
IC/microfluidicchipwithelectrodesthatcanbedrivenwithvoltagesatGHz
frequencies,couldindividuallycontrolthetemperatureofthermallyisolated,small
volumesoffluidusingthetechniqueoutlinedinthischapter.
Agrowinglibraryofelementsformicrofluidicchipshavebeendevelopedinrecent
yearsfortaskssuchasthemixingofreagents,detectingandcountingcells,sorting
cells,geneticanalysis,andproteindetection.(Whitesidesetal.,2001,Stoneetal.,
2004,Tabelingetal.,2005,Yageretal.,2006,Martinezetal.,2008,Leeetal.,2007,
Huntetal.,2008,Maltezosetal.,2005)Thereisonefunction,however,thatis
crucialtomanyapplicationsandwhichhasremainedachallenge:thelocalcontrol
oftemperature.Thelargesurfaceareatovolumeratiosfoundinmicrometer‐scale
channelsandthecloseproximityofmicrofluidicelementsmaketemperature
controlinsuchsystemsauniquechallenge.(Maltezosetal.,2005,Leeetal.,2005)
Muchworkhasbeendoneinthelastdecadetoimprovelocaltemperaturecontrolin
microfluidicsystems.ThemostcommontechniqueusesJouleheatedmetalwires
andthinfilmstoconductheatintofluidchannels.(Nakanoetal.,1994,Lagallyetal.,
2000,Liuetal.,2002,Khandurinaetal.,2000)Thethermalconductivityof
97
microfluidicdevicescontrolthelocalizationofthetemperaturechangeandtendsto
beontheorderofcentimeters.(Nakanoetal.,1994andKhandurinaetal.,2000)
Temporalcontrolislimitedbytheheatcapacityandthermalcouplingofthe
microfluidicdevicetotheenvironmentandthermalrelaxationtimestendtobeon
theorderofseconds.(Nakanoetal.,1994andKhandurinaetal.,2000)Alternative
techniquestoimprovethelocalizationandresponsetimehavebeendeveloped,
suchasthosethatusenon‐contactinfraredheatingofwateringlassmicrofluidic
systems(Odaetal.,1998)andintegratedmicrometersizePeltierJunctionsto
transferheatbetweentwochannelscontainingfluidatdifferenttemperatures.
(Maltezosetal.,2005)Fluidshavealsobeencooledonmillisecondtimescaleswith
evaporativecoolingbypumpinggassesintothefluidchannels.(Maltezosetal.,
2006)
Thefocusoftheresearchdescribedinthisthesisistointegrateelectronicswith
microfluidicstobringnewcapabilitiestolab‐on‐a‐chipsystems.Thischapter
presentsatechniquetolocallyandrapidlyheatwaterindropbasedmicrofluidic
systemswithmicrowavedielectricheating.Thedevicesarefabricatedusingsoft
lithographyandareconnectedtoinexpensivecommerciallyavailablemicrowave
electronics.Thisworkbuildsonpreviousworkinwhichmicrowaveshavebeen
usedtoheatliquidinmicrofluidicdevices(Shahetal.,2007,Sundaresanetal.,2005,
Sklavounosetal.,2006,andGeistetal.,2007)byachievingsignificantlyfaster
thermalresponsetimesandagreatertemperaturerange.Inourdevice,dropsof
waterarethermallyisolatedfromthebulkofthedeviceandthisallows
exceptionallyfastheatingandcoolingtimesτs=15mstobeattainedandthedrops’
98
temperaturetobeincreasedby30°C.Thecouplingofmicrowaveelectronicswith
microfluidicstechnologyoffersaninexpensiveandeasilyintegratedtechniqueto
locallyandrapidlycontroltemperature.
6.2ModelofDielectricHeatingofDrops
Duetowater’slargedielectriclossatGHzfrequencies,microwavepoweris
absorbedmuchmorestronglybywaterratherthanPDMSorglass.Thedevice
describedinthischapteroperatesat3.0GHz,afrequencyveryclosetothatof
commercialmicrowaveovens(2.45GHz),thatisbelowthefrequencyassociated
withtherelaxationtimeofwaterbutwherewaterstillreadilyabsorbspower.Itis
inexpensivetoengineerelectronicstoproduceanddeliver3.0GHzfrequencies
becauseitisnearthewell‐developedfrequenciesofthetelecommunications
industry.
Twoindependentfiguresofmeritdescribetheheater,thesteady‐statechangein
temperatureΔTssthatthedropsattainandthecharacteristictimeτsthatittakesto
changethetemperature.Thesteady‐statetemperatureoccurswhenthemicrowave
powerenteringthedropequalstheratethatheatleavesthedropintothethermal
bath.Thethermalrelaxationtimedependsonlyonthegeometryandthethermal
propertiesofthedropsandthechannelandisindependentofthemicrowave
power.
Todescribetheheaterasimplifiedmodelisusedinwhichthetemperatureofthe
channelwallsdonotchange.ThethermalconductivityoftheglassandPDMS
channelwallsismuchlargerthanthatofthefluorocarbon(FC)oilinwhichthe
99
dropsaresuspended,whichallowtheglassandPDMSmoldtoactasalargethermal
reservoirthatkeepthechannelwallspinnedtothebasetemperature.
Thedropismodeledashavingaheatcapacitythatconnectstothethermalreservoir
throughathermalresistance.ThedrophasaheatcapacityC=VCwthatconnectsto
thethermalreservoirwithathermalresistanceR=LD/Akoil,whereVisthevolume
andAisthesurfaceareaofthedrop,Cwistheheatcapacitypervolumeofwater,LD
isthecharacteristiclengthbetweenthedropandthechannelwall,andkoilisthe
thermalconductivityoftheoilsurroundingthedrop.Asteady‐statetemperature
ΔTssisreachedwhenthemicrowavepowerPVenteringthedropisequaltothe
powerleavingthedropΔTsskoilA/LD.Thesteady‐statetemperatureΔTss=PVRis:
€
ΔTSS =VALDkoil
P. 6.1
Thesystemhasacharacteristictimescaleτs=RCthatdescribesthethermal
responsetimeofthesystem,
€
τ S =VALDkoil
CW. 6.2
Thecharacteristictimescaleτsdescribesthethermalrelaxationtime,thetimeit
takesforthedroptoreachanequilibriumtemperaturewhenthemicrowavepower
isturnedonandthetimethatittakesforthedroptoreturntothebasetemperature
ofthedevicewhenthemicrowavesareturnedoff.
100
Thissimplifiedmodeldescribesseveralkeyfeaturesofthemicrowaveheater.The
steady‐statetemperatureislinearlyproportionaltothemicrowavepower,whereas
thecharacteristicthermalrelaxationtimeisindependentofthemicrowavepower.
Thecharacteristictimeandthesteady‐statetemperaturearebothproportionalto
thevolumetosurfaceratioofthedrops.Atrade‐offrelationexistsbetweentherate
ofheating1/τsandthesteady‐statetemperature,wherebyalargervolumeto
surfaceratioreducesthethermalrelaxationtimeoftheheaterbutdecreasesits
steady‐statetemperatureforagivenmicrowavepower,andviceversa.Similarlyan
increaseintheratioofthecharacteristiclengthbetweenthedropandthechannel
wallandthethermalconductivityoftheoilLD/koilincreasesthethermalrelaxation
timeandincreasesthesteady‐statetemperature.
6.3TheMicrofluidicDevice
Thedevicesarefabricatedusingpoly(dimethylsiloxane)(PDMS)‐on‐glassdrop‐
basedmicrofluidics.Microwavepowerislocallydeliveredviametalelectrodesthat
aredirectlyintegratedintothemicrofluidicdeviceandthatrunparalleltothefluid
channel,asisshowninFig.6.1a.Thedropsarethermallyinsulatedfromthebulkby
beingsuspendedinlowthermalconductivityoil.Aschematicofthedeviceisshown
inFig.6.2a.Syringepumpsprovidetheoilandwateratconstantflowratestothe
microfluidicdevice.Adropmakerandtwodropsplittersinseriescreatedropsthat
areproperlysizedforthemicrowaveheater.Themicrowavepoweriscreatedoff
chipusinginexpensivecommercialcomponents.
101
Dropsarecreatedusingaflow‐focusinggeometry(Annaetal.,2003)asisshownin
Fig.6.2b.Afluorocarbonoil(FluorinertFC‐40,3M)isusedasthecontinuousphase
andtheresultingdropscontain0.1μMofcarboxylcoatedCdSenanocrystal
(Invitrogen)suspendedinaphosphatebufferedsaline(PBS)solution.Asurfactant
comprisedofapolyethyleneglycol(PEG)headgroupandafluorocarbontail
(RainDanceTechnologies)isusedtostabilizethedrops.(Holtzeetal.,2008)The
wallsofthemicrofluidicchannelsarecoatedwithAquapel®(PPGIndustries)to
ensurethattheyarepreferentiallywetbythefluorocarbonoil.Fluidflowis
controlledviasyringepumps.
102
Figure6.1(a)Aschematicofthemicrowaveheater.Theblacklinesrepresentthemetallineswhichareconnectedtothemicrowavesource,thecenterfluidchannelcarriesdropsofwaterimmersedinfluorocarbon(FC)oil,(b)acrosssectionofthemicrowaveheaterwithaquasi‐staticelectricfieldsimulationsuperimposedisshown,theelectricfieldisplottedinlogscale,(c)thecalibrationcurveoftheCdSenanoparticleswhichareusedastemperaturesensors,wherethecirclesaredatapointsandtheredlineisthefit.
103
Tomakedropssmallerthanthechannelheight,andthusseparatedfromthewalls
ofthechanneltoensureadequatethermalisolation,dropsplittersareused(Linket
al.,2004)asisshowninFig.6.2c.Thedropsplittersaredesignedtobreakeach
dropintotwodropsofequalvolume.Passingasphericaldropthroughtwodrop
splittersinseriesdecreasestheradiusofadropbyafactorof(½)⅔=0.63.Drop
splittersallowthedevicetobemadeinasinglefabricationstep,becausethey
removethenecessityofmakingthedropmakerwithachannelheightsmallerthan
therestofthedevice.(Annaetal.,2003)
ThemetalelectrodesaredirectlyintegratedintothePDMSdeviceusingalow‐melt
solderfilltechnique.(Siegeletal.,2006)Themasksforthesoftlithographyprocess
aredesignedtoincludechannelsforfluidflowandasettobefilledwithmetalto
formelectrodes.AfterinletholeshavebeenpunchedintothePDMSandthePDMSis
bondedtoaglassslide,themicrofluidicdeviceisplacedonahotplatesetto80°C.A
0.02inchdiameterindiumalloywire(Indalloy19;52%Indium,32.5%Bismuth,
16.5%TinfromIndiumCorporation)isinsertedintotheelectrodechannelinlet
holesand,asthewiremelts,theelectrodechannelsfillwithmetalviacapillary
action.Theresultingelectrodechannelsrunalongeithersideofthefluidasis
showninFig.6.2d.Tokeepthedropsfromheatingfromthefringeelectricfields
beforethedropenterstheheaterthefluidchannelisconstrictedtopressthedrops
againstthePDMSwall,whichkeepsthedropsatthesametemperatureasthebase
temperatureofthemicrofluidicdevice.
104
Theelectronicsthatcreatethemicrowavepowerareassembledusinginexpensive
modules.Themicrowavesaregeneratedwithavoltagecontrolledoscillator(ZX95‐
3146‐S+,Mini‐Circuits)andamplifiedtoamaximumof11.7Vpeak‐to‐peakwitha
maximumpowerof26dBmwithapoweramplifier(ZRL‐3500+,Mini‐Circuits).The
microwaveamplifierconnectswithacabletoasubminiatureassembly(SMA)
connectormountednexttothemicrowavedeviceasisshowninFig.6.2e.Copper
wiresapproximately2mminlengthconnecttheSMAconnectortothemetal
electrodesinthePDMSdevice.Ourelectronicsoperateat3.0GHzwherewater’s
microwavepowerabsorptionisroughly1/3asefficientasatthefrequency
associatedwithwater’srelaxationtime(~18GHz)butwhereelectronicsare
inexpensiveandcommerciallyavailable.Theelectronicsusedinoursystemcosts
lessthan$US200andareeasytosetup.
Finiteelementsimulationsareperformedtodeterminetheelectricfieldstrengthin
themicrowaveheaterwhichisusedtocalculatethemicrowavepowerabsorbedby
thedrops.Figure6.1bshowsaschematiccross‐sectionofthemicrofluidicdevice
wherethedropspassbetweenthemetalelectrodes.Thechannelcross‐sectionhas
dimensions50x50μm2.Paralleltoand20μmawayfromeachsideofthefluid
channelaremetallinesthatare100μmwideand50μmhigh.Superimposedonthe
schematicinFig.6.1bisaquasi‐staticelectricfieldsimulationoftheelectricfield
(Maxwell,Ansoft).Fora12Vpeak‐to‐peaksignalacrossthemetallines,theRMS
electricfieldwithinadropwitha15μmradiussuspendedinfluorocarbonoilis
|E|~8×103V/m.Theelectricfieldlinearlyscaleswiththevoltageacrossthemetal
lineswhichallowsustocalculatethefieldwithinthedropforanyvoltage.The
105
simulatedelectricfieldiscombinedwithEq.2.1andEq.2.2tocalculatethe
microwavepowerthatentersthedropswhichmaybecombinedwithEq.6.1to
predictsteady‐statetemperaturechanges.
Thetemperaturechangeofthedropsismeasuredremotelybyobservingthe
temperature‐dependentfluorescenceofCdSenanocrystalssuspendedinthe
drops.(Maoetal.,2002)Tocalibratethisthermometer,themicrowavepoweris
turnedoffandahotplateisusedtosetthetemperatureofthemicrofluidicdevice.
ThefluidchannelisfilledwithCdSenanocrystalssuspendedinwaterandthe
temperatureofthehotplateisslowlyincreasedfrom25°Cto58°Cwhilethe
fluorescenceintensityoftheCdSequantumdotsismeasured.Themeasured
fluorescenceintensityisplottedasafunctionoftemperatureinFig.6.1c.Alineisfit
withaslope0.69%/°C±0.03%/°Ctothedataandthisslopeisusedtoconvert
fluorescencemeasurementsintomeasurementsofthechangeintemperature.A
lineisfittothedatausingaleast‐squarestechniqueandtheerroristheuncertainty
inthecoefficientofthefit.Acalibrationcurveistakenimmediatelybeforean
experiment.ThereisnoevidencethattheCdSenanoparticlesleakfromthedrops
intotheoilorprecipitateontothemicrofluidicchannel.Themicrofluidicchannel
andtheoilinthewastelinearecheckedaftertheexperimentsandthereisno
measuredfluorescencesignal.ThedeviceismonitoredwithanHamamatsuORCA‐
ERcooledCCDcameraattachedtoaBX‐52Olympusmicroscope.Imagesaretaken
withMicroSuiteBasicEditionbyOlympusandanalyzedinMATLAB(The
MathWorks,Inc.).ThemicrofluidicdeviceisconnectedwithanSMAtothe
106
microwaveamplifierandsitsontopofahotplateunderneaththemicroscopeasis
showninFig.6.2d.
Thedevicesaretestedbymeasuringthetemperaturechangeofwaterdropsasthey
travelthroughthemicrowaveheater.Along‐exposurefluorescenceimageofmany
dropstravelingthroughthemicrowaveheatershowstheensembleaverageofthe
temperaturechangeofdropsateachpointinthechannel.Aplotofthedropheating
intimemaybeextractedfromthisimageusingthemeasuredflowrateofthedrops
throughthemicrofluidicsystem.Anexperimentisperformedwiththeconstant
volumetricflowrateofthewaterat15μL/hrandtheoilat165μL/hr.Abright
field,shortshutterspeedimageistakenofthedropstravelingthroughthe
microwaveheaterandthedrops’averagediameterismeasuredtobe35μm.The
microwaveheateristurnedonwithafrequencyof3.0GHzandapeaktopeak
voltageof11V.Alongexposure(2seconds)fluorescenceimageistakenofthe
microwaveheaterthatisnormalizedagainstimagestakenwiththemicrowaves
turnedofftoremoveartifactsthatarisefromirregularitiesinthegeometryofthe
channel,thelightsource,andthecamera.
107
Figure6.2(a)Aschematicofthemicrowaveheatingdevice,showingtheoilinorange,thewaterinblue,andtheelectronicconnectionsinred,(b)amicrographoftheflow‐focusingdropmaker,(c)thetwosetsofdropsplittersinseries,(d)amicrographofthemicrowaveheater,thedarkregionsthatrunparalleltothefluidchannelarethemetallines,(e)aphotographofthemicrofluidicdevice,connectedtothemicrowaveamplifierwithacablewithanSMAconnector,ontopofahotplate,underneaththefluorescencemicroscope.
108
6.4Demonstration
Thedropsareheatedtoasteady‐statetemperaturechangeΔTssastheypass
throughthemicrowaveheater.Figure6.3ashowsthenormalizedfluorescence
intensityofthedropsastheyenterthemicrowaveheatersuperimposedontoa
bright‐fieldimageofthedevice.Itcanbeseenthatasthedropsenterthechannel
theiraveragefluorescenceintensitydropswhichshowsthattheyarebeingheated.
Alineaverageofthenormalizedimageistakeninthedirectionperpendiculartothe
fluidflowandisplottedagainstthelengthofthechannel,asinFig.6.3b.Asthe
dropsareheatedtheaveragefluorescenceintensityofthedropsfallsexponentially
withdistanceto85%ofitsinitialintensityafterapathlengthof300μm.The
fluorescenceintensitymeasuresthetemperaturechangeofthedrops,andsothe
dropsareheatedinacharacteristiclengthof300μm.
Thedropsareheatedtosteady‐statetemperaturechangesΔTssaslargeas30°C
abovethebasetemperatureofthemicrofluidicdeviceinonlyτs=15ms.The
averagetemperaturechangeofthedropsasafunctionoftimeanddistancetraveled
isplottedinFig.6.3c.Forthisheatingpower,thetemperaturerisestoasteady‐
statevalueof26°Cabovethebasetemperatureof21°Cin15ms.Thecurvein
Fig.6.3cisarrivedatbyusingthecalibrationcurveoftheCdSenanocrystals,
Fig.6.1c,toconvertthefluorescenceintensityinFig.6.3bintoachangein
temperatureΔT.Thesumoftheflowratesoftheoilandwaterareusedtocalculate
thespeedofthedropsthroughthechannel,whichmaybeusedtotransformthe
109
lengthinFig.6.3aintothetimethatthedropshavespentintheheater.Eachdata
pointconsistsoftheaverageof20independentlytaken2secexposures.
110
Figure6.3(a)A2secondexposureofthefluorescencesignalnormalizedtoanimagetakenwiththemicrowavesourceturnedoffsuperimposedontoabrightfieldimageofthedevice,(b)thelineaverageofthenormalizedintensityplottedversusdistancedownthechannel,(c)thetemperaturechangeΔTplottedversustimeandversusthedistancethedropshavetraveleddownthechannel.
111
Thecombinedstatisticalerrorofthemeasurementoftheheatingisalsoplottedin
Fig.6.3c.Theaverageerroris~1.4°C.Thestatisticalerrorinthesteady‐state
temperatureisfoundtocomeprimarilyfromvariationsinthesizeofthedrops.The
steady‐statetemperaturechangeofeachdropislinearlyrelatedtothevolume‐to‐
arearatioofthedropandtothedistanceofthedroptothechannelwalls,asis
describedinEq.6.1.Byobservingthedropsexitthemicrowaveheatingregionof
thechip,thedropsarefoundtocoolwithacharacteristictimesimilartotheheating
time.Inaddition,bynarrowingthechannelattheexitofthemicrowaveheater,the
dropsarebroughtclosertothechannelwallandthedropsreturntothebase
temperatureoftheoilinlessthan1ms.
Thesteady‐statetemperaturechangeΔTssofthedropsmaybesetfrom0°Cto30°C
byvaryingtheappliedmicrowavepowerasisdescribedinEq.6.1.Themicrowave
poweriscontrolledbyexperimentallyvaryingthepeak‐to‐peakvoltageofthe
appliedmicrowavevoltagewhichvariesthestrengthoftheelectricfieldinsidethe
dropsasisdescribedbyoursimulations.Aseriesofplotsofthechangein
temperatureversustimefordifferentappliedpowersisshownintheinsetof
Fig.6.4a,andshowssteady‐statetemperaturechangesrangingfrom2.8°Cto30.1°C,
withanaverageerrorof1.5°C.Itisnoteworthythatalloftheheatingcurveshave
anexponentialformandhavethesamecharacteristicrisetimeτs=15ms.
Goodagreementisfoundbetweenthesteady‐statetemperaturechangesobserved
inFig.6.4afordifferentappliedmicrowavepowersandthemodelofmicrowave
heatingoutlinedinthebeginningofthischapter.Thesteady‐statetemperature
112
changeisplottedversusthemicrowavepowerdensityinFig.6.4aandisfitwitha
line.AsisexpectedfromEq.6.1thesteady‐statetemperaturechangeriseslinearly
withappliedmicrowavepower.Themicrowavepowerdensityiscalculatedusing
theelectricfieldvaluesdeterminedfromsimulations(Fig.6.1b).Theonlyvariable
inEq.6.1thatisnotmeasuredorthatisnotamaterialpropertyisthecharacteristic
lengthscalebetweenthedropandthechannelwallLD.Thischaracteristiclength
LD=28μmisestimatedusingthemeasuredsteady‐statetemperaturechange
(Fig.6.4a)andtheknownmaterialproperties,usingEq.6.1.
Goodagreementisfoundbetweentheobservationthatthetemperaturechange
approachessteady‐stateexponentiallyintimeinFig.6.4bandthemodelfor
microwaveheatingoutlinedinthebeginningofthischapter.Tocomparethe
model’spredictionthatthedropsapproachequilibriumexponentiallyintimewitha
singlerelaxationtimeconstant(Eq.6.2)withourobservations,thechangein
temperatureΔTsubtractedfromthesteady‐statechangeintemperatureΔTssis
plottedversustimeonasemi‐logplotandfitwithaline.Asispredictedbythe
modelthedropsapproachequilibriumexponentiallywithasingletimeconstant.
Thethermalrelaxationtimeconstantismeasuredtobeτs=14.7±0.6ms.Theonly
variableinEq.6.2thatisnotmeasuredisthecharacteristiclengthLDbetweenthe
dropandthechannelwall.ThischaracteristiclengthLD=35μmisestimatedusing
themeasuredcharacteristictimeconstantandtheknownmaterialproperties,using
Eq.6.2.Theindependentmeasurementsofthethermalrelaxationtimeandthe
steady‐statetemperaturechangeversuspowergivetwoindependentmeasuresof
thelengthLDthatarewithin20%ofeachother.Thedifferenceinthetwo
113
predictionsofLDcanbeatleastpartiallyexplainedbynotingthattheheatcapacity
inEq.6.2islargerthaniscalculated,duetothefactthatthechannelwallsarenota
perfectheatsinkasthemodelassumes.Theagreementbetweenthetwo
independentmeasurementssupportsthesimplemodelformicrowaveheatingof
dropsoutlinedinthebeginningofthischapter.
114
Figure6.4(a)Thesteady‐statetemperaturechangeofthedropsiscontrolledbyvaryingtheamplitudeofthemicrowavevoltage.Intheinsetthegreencurveshowsthedropheatingversustimeformicrowaveswithanamplitudeof11.7V,thered11.0V,theyellow10.3V,thelightblue9.3V,thepurple8.5V,thegrey7.5V,andtheblue4.5V.Thesteady‐statetemperaturechangeisplottedversusthepowerdensity,ascalculatedbyEq.2.1,inthemainplot,(b)themagnitudeofthechangeintemperatureΔTsubtractedfromthemaximumchangeintemperatureΔTSSversustimeisplottedonalog‐linearscale.Thetemperaturerisesasasingleexponentialwithacharacteristictime,τs=14.7±0.56ms.Theinsetshowsthetemperatureversustimeplotfromwhichthelog‐linearplotistaken.
115
6.5Discussion
Inthischapteranintegratedmicrofluidicmicrowavedielectricheateris
demonstratedthatlocallyandrapidlyincreasesthetemperatureofdropsofwater
suspendedinoil.Thelargeabsorptionofmicrowavepowerbywaterrelativetooil,
glass,andPDMSallowslocalandrapidheatinginmicrofluidicdeviceswithout
difficultfabrication.Bothimprovingtheinsulationofdropsfromthechannelwalls
andincreasingthevolumetosurfacearearatioofthedropswouldallowforlarger
temperaturechanges.Thestatisticalerrorinthesteady‐statetemperatureofthe
dropscanbeimprovedbyreducingvariationsinthedropsizethatarisefrom
fluctuationsintheflowfromthesyringepumps.
Microwavedielectricheatingofdropsiswellsuitedforintegrationwiththehybrid
integratedcircuit(IC)/microfluidicchipsdescribedinChapters3,4,and5.(Leeet
al.,2007)IfelectrodesonthechiparedrivenwithvoltagesatGHzfrequencies,then
onecanusethechiptolocallyheatsmallvolumesoffluidusingdielectricheating.
Theadditionoflocallyaddressabletemperaturecontroltothelab‐on‐a‐chip
functionsdemonstratedinChapters3,4,and5wouldfurtherexpandthe
capabilitiesofthehybridchipplatformandwouldbeavaluabletoolforanumberof
applications,includingDNAanalysis.(Leeetal.,2007)
Microwavedielectricheatinghasmanyexcitingscientificandtechnological
applications.Onenoteworthypotentialapplicationforrapid,localizedheatingin
microfluidicdevicesisPCR.(Lagallyetal.,2000,Liuetal.,2002,Khandurinaetal.,
2000,Odaetal.,1998,Maltezosetal.,2005,Maltezosetal.,2006)Themicrowave
116
microfluidicheatercanraisethetemperatureofdropsupto30°Cabovethebase
temperatureoftheoilinwhichthedropsaresuspended.Bysettingthebase
temperatureoftheoilinourdeviceto65°Candappropriatelysettingthe
microwavepower,a30°Cchangeintemperaturecouldcyclethetemperaturefrom
65°Cto95°CasrequiredforPCR.Drop‐basedPCR,whichwouldbeespeciallywell
suitedforthistechnique,allowsfortherapidanalysisoflargepopulationsofgenes
andenzymes.Themicrowaveheatingtechniquemightalsobeusedtoset
temperaturesrapidlyandcontrollablyinbiologicalandchemicalassays,suchasfor
proteindenaturingstudies(Arataetal.,2008)andenzymeoptimizationassays
(Robertsonetal.,2004),whereobservationsofthermalresponsesaremadeonthe
millisecondtimescale.
117
Chapter7.Conclusions
7.1Summary
Thisthesisdescribesthedevelopmentofaversatileplatformforperformingbiology
andchemistryexperimentsonachip,usingtheintegratedcircuit(IC)technologyof
thecommercialelectronicsindustry.Thisworkisanimportantsteptowards
developingautomated,portable,andinexpensivedevicestoperformcomplex
chemicalandbiologicaltasks.Suchadevicewouldrevolutionizethewaythat
biologicalandchemicalinformationiscollectedforapplicationssuchasmedical
diagnostics,environmentaltesting,andscientificresearch.(Ahnetal.,2004,Chinet
al.,2007,andMartinezetal.,2008)
ThehybridIC/microfluidicchipsdevelopedinthisthesiscontrollivingcellsand
smallvolumesoffluid.Takinginspirationfromcellularbiology,phospholipid
bilayervesiclesareusedtopackagepLvolumesoffluidonthesechips.Table7.1
summarizesthebasiclab‐on‐a‐chipfunctionsthatthehybridchipscanperformon
thelivingcellsandvesicles.Thechipscanbeprogrammedtotrapandposition,
deform,setthetemperatureof,electroporate,andelectrofuselivingcellsand
vesicles.Thesebasicfunctionscanbestrungtogethertoperformcomplexchemical
andbiologicaltasks.ThefastelectronicsandcomplexcircuitryofICsenable
thousandsoflivingcellsandvesiclestobesimultaneouslycontrolled,allowingmany
parallel,well‐controlledbiologicalandchemicaloperationstobeperformedin
parallel.
118
Table7.1Alistofthelab‐on‐a‐chipfunctionsperformedinthisthesisusinghybridIC/microfluidicchips.
119
7.1FutureDirections
ThisthesisdevelopshybridIC/microfluidicchipsanddemonstratesthattheycan
performbasicfunctionsthatarenecessarybuildingblocksforbiologicaland
chemicalexperimentsonachip.However,thegoalofaportabledevicethatcan
performbiologicalandchemicaltasksremainsachallenge.Forthisgoaltobefully
realized,amoreself‐sufficientplatformmustbedeveloped.Althoughahybrid
IC/microfluidicchipcanbepackagedintoaportabledevice,thecurrentchip
requiresalaboratory‐sizedfluorescencemicroscope,lightsource,andcomputerto
fullyfunction.TocompletelyrealizethepotentialofhybridIC/microfluidicchips,
additionalfunctionsmustbeaddedtothechipssuchthatthelarge,external
componentsarenotrequired.
Thehybridchipsdescribedinthisthesiscouldbemademoreself‐sufficientby
integratingsensorsontotheICs.AhybridIC/microfluidicchipthatincludedboth
sensorsandthefunctionalitydemonstratedinthisthesiscouldperformcomplex
biologicalandchemicalexperimentsandmeasuretheresults.Theseexperiments
couldincludemultiplestepssuchas:mixingalibraryofcellswithalibraryof
chemicals,measuringtheoutcomeoftheexperimentswithsensorsbuiltintothe
chip,analyzingtheresultsoftheexperiment,anddesigningthenextsetof
experimentsbasedontheresultsofthefirst,andrepeat,untilfinallyreportingthe
results.Thissortofautomatedlab‐on‐a‐chipplatformcouldperformthecomplex,
multi‐stepexperimentsthatarecurrentlyperformedinlaboratoriesbytrainedstaff
usinglargeandexpensiveequipment.
120
SeveralsensorshavepreviouslybeendevelopedusinghybridIC/microfluidic
chips.SomeexamplesofthesesensorsareNMRsensorsforchemicaldetection,(Lee
etal.,2008)capacitivesensorsformeasuringeletrogeniccells,(DeBusschereetal.,
2001)electricalsensorsforDNAdetection,(Thewesetal.,2002)andcharge
coupleddevices(CCD)foropticalimaging.(Cuietal.,2008)Thesesensorsarebuilt
usingstandardcommercialICprocesses,similartotheprocessesusedtocreatethe
chipsdescribedinthisthesis.AsingleICcouldbebuilt,usingacommercialfoundry,
thatincludesthefunctionalitydemonstratedinthisthesisandthatofthesensors
describedabove.SensorsthatrequirespecializedICprocessesorthataremade
withe‐beamlithographycanalsobeincludedusingflip‐chipbonding.(Leeetal.,
2007)
AhybridIC/microfluidicchipthatcontrols,performsexperimentson,and
measuresmanylivingcellsandsmallvolumesoffluidwouldhaveabigimpact.
Taskssuchasthedetectionandcountingofcells,sortingcells,geneticanalysis,
proteindetection,andcombinatorialchemistrycouldbeperformedontheselow‐
cost,automateddevices.(Whitesidesetal.,2001,Stoneetal.,2004,Tabelingetal.,
2005,Yageretal.,2006,Martinezetal.,2008,Leeetal.,2007,Huntetal.,2008,
Maltezosetal.,2005)Thechipscouldbeprogrammedtoperformcomplex,multi‐
steptasks,andreacttotheresultsofexperimentsinreal‐time.Thedevicescouldbe
usedforapplicationssuchaspoint‐of‐carediseasedetectionordetectingtoxic
substancesorparasitesindrinkingwater.Suchadevicewouldrealizetheinitial
goalofmicrofluidics,tobringcomplexbiologicalandchemicaltestsfrom
laboratoriesoutintotheclinicandthefield.(Whitesides,2001)
121
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AppendixA.DataSheetfortheDielectrophoresisChip(DEPChip)
WedocumentthespecificationsfortheFabutron1.0,showthepinlayoutanddetail
theread/writeprotocolsuchthatanyuserwhoreadsthisdocumentshouldhave
alloftheinformationnecessarytomaketheFabutron1.0workintheirexperiment.
SummaryoftheFabutron1.0
TheFabutron1.0isacustomintegratedcircuitdesignedintheWesterveltlab.The
chipconsistsofanarrayof128x256pixels,11x11μm2insize,controlledbybuilt‐in
SRAMmemory;eachpixelcanbeindividuallyaddressedwitharadio
frequency(RF)voltageupto5Vpp,withfrequenciesfromDC‐11MHz.TheICwas
builtinacommercialfoundryandthemicrofluidicchamberwasfabricatedonits
topsurfaceatHarvard.
TableA1SpecificationsfortheDEPchip
Feature DescriptionBandwidth DC‐11MHzProcess 0.35µm,CMOSMOSISTSMCPixels 128x256(32,768)
11x11µm2ChipSize 2.32x3.27mm2Addressing 8‐bitwordlinedecoder
128bitshiftregister,twophaseclocked
DataLineBandwidth DC‐20MHzPixelVoltage Vp‐p=3‐5VPixelBandwidth DC–5MHzOperatingVoltage 5VOperatingCurrent 30mA–300mA
127
TableA2.AlistofthepinsfortheDEPChip
Pin DescriptionGND GroundVDD OperatingVoltage(5V)W(0:7)** WordAddressDecoderLinesφ1 PixelVoltage1φ2 PixelVoltage2c2 (0)decouplesSRAMelementsfromthebitline,(1)connects
SRAMelementsonactivatedrowtothebitlinec1 (0)Prechargethebitlines,(1)disconnectprechargeWR ControlLine:(1)enablestheshiftregistertowriteitsvalueto
thebitlines,(0)disablesshiftregisterfromwritingtobitlinesPASS Controlline:(1)Shiftregisterelementslookstoprevious
registerforinput,(0)registerslooktobitlineforinputDATA DatainputlineforshiftregistersCLK2 ClockforshiftregisterCLK1 ClockforshiftregisterOUT Dataoutputforshiftregister
FigureA1PinlayoutfortheDEPchip
128
Protocols
FigureA2.LoadDataIntoShiftRegisterandWritetoArray.
129
FigureA3.ReadDatafromSRAMArrayintoShiftRegisterandReadOutArray.
**WordAddressDecoderLinesareincorrectlylabeledonthechip.Notethatgoing
fromMSBtoLSBthecorrectorderingis:w3,w2,w1,w0,w7,w6,w5**
***Alsonotethattherewasanerrorinthedesignoftheshiftregisteranditactually
functionsasa64bitshiftregister,withneighboringregisterstiedtogether.Thefull
128bitsareaccessiblebyloadingin64bitsofdataatatime,anduseclock1toshift
thedataintotheproperposition.***
130
AppendixB.DataSheetfortheHighVoltageDielectrophoresis/MagneticChip(HVDEP/MagneticChip)
WedocumentthespecificationsfortheFabutron2.0,showthepinlayout,anddetail
theread/writeprotocolsuchthatanyuserwhoreadsthisdocumentshouldhave
alloftheinformationnecessarytomaketheFabutron2.0workintheirexperiment.
SummaryoftheFabutron2.0
TheFabutron2.0isacustomintegratedcircuitdesignedintheWesterveltlab.The
chipconsistsofanarrayof60x61pixels,30x38μm2insize,controlledbybuilt‐in
SRAMmemory;eachpixelcanbeindividuallyaddressedwitharadio
frequency(RF)voltageupto50Vpp,withfrequenciesfromDC‐10MHz.Interlaced
withtheDEPpixelsareamatrixof60x61wiresthatmaybeaddressablysourced
with100mAtoapplyforcesonmagneticallypolarizableobjects.TheICwasbuilt
inacommercialfoundryandthemicrofluidicchamberwasfabricatedonitstop
surfaceatHarvard.Thechipsuffersfromlocalheatingproblemsduetothe
magneticwires.Animprovedversionwouldswitchtopulsedcurrentstominimize
heating.Thechipalsohasissueswiththemagneticpowerlinesandthedigital
powerlinesbeingcoupled,whichleadstopowerlineissuesonthechip.Anupdated
versionwouldtakeextracaretomakesurethatpowerlinesaredecoupled.
TableB1.SpecificationsfortheDEPchip
Feature DescriptionBandwidth DC‐10MHzProcess 0.6µm,CMOSXFABXC06Pixels 60x61(3,660)
131
30x38µm2ChipSize 3.4x3.7mm2Addressing 5‐bitwordlinedecoder
128bitshiftregister,twophaseclocked
DataLineBandwidth DC‐20MHzPixelVoltage Vp‐p=20‐50VPixelBandwidth DC–1MHzOperatingVoltage(Logic)
5V
OperatingCurrent(logic)
20mA
OperatingMagneticWireVoltage
5V
OperatingMagneticWireCurrent
0–800mA*
OperatingHighVoltage
50V
OperatingHighVoltageCurrent
20‐60mA**
TableB2.AlistofthepinsfortheDEPChip
Pin DescriptionGND GroundVDD OperatingVoltage(5V)HV HighVoltage(50V)HVg HighVoltageGate(45‐50V),controlstheactiveimpedanceintheRF
voltagedrivingcircuitunderneatheachcircuitVmag PowerSupplyfortheMagneticLines(5V)W(0:5) WordAddressDecoderLinesTHERM AnalogOutputfortheThermometer(0‐5V)φ1 PixelVoltage1φ2 PixelVoltage2c2 (0)decouplesSRAMelementsfromthebitline,(1)connectsSRAM
elementsonactivatedrowtothebitlinec1 (0)Prechargethebitlines,(1)disconnectprechargeWR ControlLine:(1)enablestheshiftregistertowriteitsvaluetothe
bitlines,(0)disablesshiftregisterfromwritingtobitlinesPASS Controlline:(1)Shiftregisterelementslookstopreviousregisterfor
input,(0)registerslooktobitlineforinputDATA DatainputlineforshiftregistersCLK2 ClockforshiftregisterCLK1 ClockforshiftregisterOUT Dataoutputforshiftregister
132
FigureB1PinlayoutfortheDEPchip
Protocols
TheprotocoltoupdatetheSRAMarrayisidenticalasfortheFabutron1.0
TheSRAMaddressesoftheDEPpixels,magneticwires,andthermometersareas
follows:
TableB3.Amapofthefeaturesofthe50V/magneticchipontheSRAMarray
Array SRAMAddress(word,bit)DEPArray(1:60,1:161) SRAM(3:32,5:126)***MagneticMatrixXdirection(1:2,1:122) SRAM(1:2,5:126)***MagneticMatrixYdirection(1:2,1:122) SRAM(1:30,1:4)***Thermometers(1:16) SRAM(1:8,127:128)***
*Notethatspecialcaremustbetakenwhenthechipisfirstturnedon.TheSRAM
willbesettoarandomstateandmorethanafewmagneticlineswillbeturnedon.
ItisadvantageoustoleavetheVmagturnedoffuntilthestatesofthewirescanbeset
suchthatnoneorfewofthewiresareturnedon.
133
**Notethataspecialfuseandvoltageconditioning/currentlimitingcircuitrymust
bebuiltintothePCBthathousestheIC.SpikesintheHVlinecandestroythechip.
***FormoredetailonhowtheSRAMarraymapsontotheDEPandMagneticarray
pleaseseelinetheattachedMATLABcodewherethedetailedmappingislaidout.
134
AppendixC.FabutronControlSoftware
LabviewProgramsusedtoCommunicatewiththeChips
FigureC1.Labviewcodeusedtoreaddatacomingfromthechips.
135
FigureC2.Labviewcodeusedtowritedatatothechips
FigureC3.BlockdiagramofLabviewcodeusedtoreaddatafromthechips
136
FigureC4.BlockdiagramofLabviewcodeusedtowritetothechip
137
MatlabCodeusedtoGenerateandReadPatternstotheChip
ProgramtowritedatafromamatrixcreatedintheGUIandpackageitintoalineofcommandssenttothechipwiththeLabviewprogram
functionfabwrite(image,protocol)
%functionfabwrite(image,protocol)
%
%1.30.2008
%
%CreateaTextfilethatLabViewcanreadwhichdirectsittowriteafile
%totheFabutronandreadfromitatthesametime.theoutputfilez.txt
%WrittenbyDavidIssadoreandKeithBrown
%
%Changedon2.4.2008toalsoworkwith5and50voltfabutrons
%Changedon2.24.2008toaccomidatemultiplelayerimages
%changedon81508toswitchclock1andclock2online90
%
%%%
%%addedbydavetorearrangethedataforthe50Vchip.fornowwewill
%%justconcernourselveswiththeDEParrayandnotmagneticlinesor
%%thermometers
ifprotocol==50
%image=arrange50V(image);
end
%%SetupVariables
frames=size(image,3);
bits_per_row=size(image,2);
rows=size(image,1);
138
ecorr=10;
bcorr=10;
icorr=10;
command_lines=13+bcorr+ecorr+icorr;
cycles_per_bit=4;
commands_per_row=command_lines+bits_per_row*cycles_per_bit;
%%DefineRowWriteCommand
%Loadsdatafromdataportormemoryintoshiftregister
clock1=zeros(1,commands_per_row);
clock1(linspace(2,commands_per_rowcommand_lines2,bits_per_row))=ones(1,bits_per_row);
%clock1(bits_per_row*cycles_per_bit+12)=1;
%Advancestheshiftregister
clock2=zeros(1,commands_per_row);
clock2(linspace(4,commands_per_rowcommand_lines,bits_per_row))=ones(1,bits_per_row);
clock2(bits_per_row*cycles_per_bit+10+bcorr+icorr)=1;
%Modulatesthedataimagesothatithastherightform
data=zeros(commands_per_row,bits_per_row);
fori=0:bits_per_row1
data(i*cycles_per_bit+1:(i+1)*cycles_per_bit,i+1)=[1;1;1;1];
139
end
%Writeenablesdatatobewrittenintomemoryfromtheshiftregister
write=zeros(1,commands_per_row);
write(bits_per_row*cycles_per_bit+2+bcorr:bits_per_row*cycles_per_bit+5+bcorr)=ones(1,4);
%Passtellsitwhentolookatthepreviosregister/data(1)andwhento
%lookatmemory(0)
pass=ones(1,commands_per_row);
pass(bits_per_row*cycles_per_bit+8+bcorr+icorr:bits_per_row*cycles_per_bit+11+bcorr+icorr)=zeros(1,4);
%Tellswhenthewordlinesshouldbeenegaged
%words=zeros(1,commands_per_row);
%words(bits_per_row*cycles_per_bit10:commands_per_row1)=ones(1,command_lines+10);
words=ones(1,commands_per_row);
%Clock_C1Loadsdatato/frommemoryto/fromthebitline
clock_c1=zeros(1,commands_per_row);
clock_c1(bits_per_row*cycles_per_bit+3+bcorr:bits_per_row*cycles_per_bit+4+bcorr)=ones(1,2);
clock_c1(bits_per_row*cycles_per_bit+7+bcorr+icorr:bits_per_row*cycles_per_bit+11+bcorr+icorr)=ones(1,5);
%clock_c1(bits_per_row*cycles_per_bit+3:bits_per_row*cycles_per_bit+11)=ones(1,9);
%Clock_C2Turnsoffprechargingofpullupcircuitry
clock_c2=zeros(1,commands_per_row);
clock_c2(bits_per_row*cycles_per_bit+bcorr:bits_per_row*cycles_per_bit+6+bcorr)=ones(1,7);
clock_c2(bits_per_row*cycles_per_bit+6+bcorr+icorr:bits_per_row*cycles_per_bit+12+bcorr+icorr)=ones(1,7);
140
%outputtriggertellsthecodewhentostartrecording
output_trigger=zeros(1,commands_per_row);
output_trigger(bits_per_row*cycles_per_bit10)=1;
%output_trigger(1)=1;
%%
temp=clock1;
clock1=clock2;
clock2=temp;
%%
%plotthecontrolsingalstomakesuretheylookok
%Testcodetoberemoved
%
%figure
%plot(clock_c11.5,'b')
%holdon
%plot(clock_c2+0,'r')
%plot(words+1.5,'g')
%plot(write+3,'k')
%plot(pass+4.5,'y')
%plot(clock1+6,'bo','MarkerSize',3)
%plot(clock2+7.5,'ro','MarkerSize',3)
%plot(data*ones(bits_per_row,1)+9,'ko','MarkerSize',3);
%plot(output_trigger+10.5,'go','MarkerSize',3);
%legend('clockc1','clockc2','wordlines','write','pass','clock1','clock2','data','outputtrigger');
%%BuildCommand
141
temp=[];
forcf=1:frames
ifprotocol==5
k=linspace(0,255,256);
k=dec2bin(k,8);
%Thisistheorderthatweoriginallysuspected
%changed62708,foundinTom'sbinder.
w0=str2num(k(:,4));
w1=str2num(k(:,3));
w2=str2num(k(:,2));
w3=str2num(k(:,1));
w4=str2num(k(:,8));
w5=str2num(k(:,7));
w6=str2num(k(:,6));
w7=str2num(k(:,5));
%Thisistheorderthatistheoppositeoftheonethatwe
%origniallysuspected.pshaw.
%w0=str2num(k(:,1));
%w1=str2num(k(:,2));
%w2=str2num(k(:,3));
%w3=str2num(k(:,4));
%w4=str2num(k(:,5));
%w5=str2num(k(:,6));
%w6=str2num(k(:,7));
%w7=str2num(k(:,8));
%%buildbitmap
bit_map=zeros(32,(rows+1).*commands_per_row);
142
fori=0:rows1
%0.10.8notset
%0.9<>CLK1
bit_map(9,i*commands_per_row+1:(i+1)*commands_per_row)=clock1;
%0.100.13notset
%0.14<>CLK2
bit_map(14,i*commands_per_row+1:(i+1)*commands_per_row)=clock2;
%0.15<>OUT_TRIGGERnotsetinthisloop
%0.16<>DATA
%bit_map(16,i*commands_per_row+1:(i+1)*commands_per_row)=0;
bit_map(16,i*commands_per_row+1:(i+1)*commands_per_row)=(data*image(i+1,:,cf)')';
%0.17<>PASS
bit_map(17,i*commands_per_row+1:(i+1)*commands_per_row)=pass;
%0.18notset
%0.19<>W0
bit_map(19,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w0(i+1);
%0.20<>W1
bit_map(20,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w1(i+1);
%0.21<>WR
bit_map(21,i*commands_per_row+1:(i+1)*commands_per_row)=write;
%0.22<>C1
bit_map(22,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c1;
%0.23<>W4
bit_map(23,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w4(i+1);
%0.24<>W5
bit_map(24,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w5(i+1);
%0.25<>C2
bit_map(25,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c2;
143
%0.260.27notset
%0.28<>W6
bit_map(28,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w6(i+1);
%0.29<>W2
bit_map(29,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w2(i+1);
%0.30<>W7
bit_map(30,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w7(i+1);
%0.31<>W3
bit_map(31,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w3(i+1);
%0.32notset
end
%Settheoutputtriggertogooffonlyonce
bit_map(15,1:commands_per_row)=output_trigger;
%Setclock1,clock2andpassforthefinaldataout
bit_map(9,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock1;
bit_map(14,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock2;
bit_map(17,rows*commands_per_row+1:(rows+1)*commands_per_row)=pass;
elseifprotocol==50
%%preparewordlines
%preparewordlines
k=1:32;
k=dec2bin(k,5);
w0=str2num(k(:,5));
w1=str2num(k(:,4));
w2=str2num(k(:,3));
144
w3=str2num(k(:,2));
w4=str2num(k(:,1));
%%buildbitmap
bit_map=zeros(32,(rows+1).*commands_per_row);
fori=0:rows1
%0.10.16notset
%0.17<>PASS
bit_map(17,i*commands_per_row+1:(i+1)*commands_per_row)=pass;
%0.18<>C1
bit_map(18,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c1;
%0.19<>C2
bit_map(19,i*commands_per_row+1:(i+1)*commands_per_row)=clock_c2;
%0.20<>W4
bit_map(20,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w4(i+1);
%0.21<>DATA
bit_map(21,i*commands_per_row+1:(i+1)*commands_per_row)=(data*image(i+1,:)')';
%0.22<>OUT
%0.23<>W1
bit_map(23,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w1(i+1);
%0.24<>W0
bit_map(24,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w0(i+1);
%0.25<>WR
bit_map(25,i*commands_per_row+1:(i+1)*commands_per_row)=write;
%0.26and0.27notset
%0.28<>CLK1
bit_map(28,i*commands_per_row+1:(i+1)*commands_per_row)=clock1;
%0.29<>W3
145
bit_map(29,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w3(i+1);
%0.30<>CLK2
bit_map(30,i*commands_per_row+1:(i+1)*commands_per_row)=clock2;
%0.31<>W2
bit_map(31,i*commands_per_row+1:(i+1)*commands_per_row)=words.*w2(i+1);
end
%Settheoutputtriggertogooffonlyonce
bit_map(15,1:commands_per_row)=output_trigger;
%Setclock1,clock2andpassforthefinaldataout
bit_map(28,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock1;
bit_map(30,rows*commands_per_row+1:(rows+1)*commands_per_row)=clock2;
bit_map(17,rows*commands_per_row+1:(rows+1)*commands_per_row)=pass;
end
%convertto32bitnumber
exponent=(1:32)'*ones(1,size(bit_map,2));
temp=[temp,sum(bit_map.*2.^exponent,1)];
end
globalcommand;
command=temp;
%tellittowrite
globalstatus;
status=1;
Programtoreaddatafromthechipandrepackageitasamatrix
Function[pixel_array]=fabload(sizes,protocol)
%function[pixel_array]=fabload(sizes)
%CreatedbyKeithBrown1.30.2008toreadfilescreatedbytheLabView
%scriptthatreadsthefabutron
%2.12.2008Workswith5Vchip
146
%commentedoutbydavidissadoretoletlabviewpassthedatawithout
%savingtotheharddrive
%[headerdata]=hdrload('out.txt');
%%
globalouts
data=outs;
size(data)
%%
ifprotocol==5
%fabwrite
icorr=10;
bcorr=10;
ecorr=10;
overhead_per_row=13+icorr+bcorr+ecorr;
offset=26+icorr+bcorr+ecorr;
%%fabverify
%icorr=0;
%bcorr=0;
%ecorr=0;
%overhead_per_row=13+icorr+bcorr+ecorr;
%offset=21+icorr+bcorr+ecorr;
pixel_array=zeros(sizes(1),sizes(2));
bits_per_row=4.*sizes(2)+overhead_per_row;
temp=zeros(sizes(1),sizes(2)*4);
forj=1:sizes(1);
temp(j,:)=data(offset+bits_per_row*(j1):offset+bits_per_row*(j1)+sizes(2)*41);
end
pixel_array=temp(:,4*(1:sizes(2))1);elseifprotocol==50
147
icorr=20;
bcorr=20;
ecorr=20;
overhead_per_row=(15+icorr+bcorr+ecorr).*1;
pixel_array=zeros(sizes(1),sizes(2));
bits_per_row=8.*sizes(2)+overhead_per_row;
%bits_per_row=4.*sizes(2)+overhead_per_row;
%offset=45
offset=11+icorr+bcorr+ecorr;
temp=zeros(sizes(1)./2,sizes(2)*8)';
%temp=zeros(sizes(1),sizes(2)*4);
size(temp)
forj=1:sizes(1)./2;
%forj=1:sizes(1);
temp(:,j)=data(offset+bits_per_row*(j1):offset+bits_per_row*(j1)+sizes(2)*81);
%temp(j,:)=data(offset+bits_per_row*(j1):offset+bits_per_row*(j1)+sizes(2)*41);
end
%pixels(1:60,1:30)=DEP_array(1:60,1:30)
%pixels(61:120,1:30)=DEP_array(1:60,31:60);
pixel_array(1:60,1:30)=temp(4*(1:60)3,1:30);
pixel_array(1:60,31:60)=temp(4*(61:120)3,1:30);
%pixel_array=temp(:,4*(1:sizes(2))1);
size(pixel_array)
end