Microfluidic bio-particle for manipulation biotechnology Biochemical Engineering Journal

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Biochemical Engineering Journal

j ou rn a l h o m ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / b e j

Microfluidic bio-particle manipulation for biotechnology

Barbaros C¸etin

^a,∗

, Mehmet Bülent Özer

^b

, Mehmet Ertu˘grul Solmaz

^c,d

aMechanicalEngineeringDepartment,Microfluidics&Lab-on-a-ChipResearchGroup, ˙IhsanDo˜gramacıBilkentUniversity,Ankara06800,Turkey

bDepartmentofMechanicalEngineering,TOBBUniversityofEconomicsandTechnology,Ankara06560,Turkey

cDepartmentofElectricalandElectronicsEngineering, ˙IzmirKatipC¸elebiUniversity, ˙Izmir35620,Turkey

dNationalNanotechnologyResearchCenter,Ankara06800,Turkey

a r t i c l e i n f o

Articlehistory:

Received23March2014

Receivedinrevisedform8July2014 Accepted14July2014

Availableonline21July2014

Keywords:

Biomedical Bioprocessdesign Bioseparations Fluidmechanics Microfluidics

Bio-particlemanipulation

a b s t r a c t

Microfluidicsandlab-on-a-chiptechnologyoffersuniqueadvantagesforthenextgenerationdevices fordiagnostictherapeuticapplications.Forchemical,biologicalandbiomedicalanalysisinmicrofluidic systems,therearesomefundamentaloperationssuchasseparation,focusing,filtering,concentration, trapping,detection,sorting,counting,washing,lysisofbio-particles,andPCR-likereactions.Thecombi- nationoftheseoperationsledtothecompleteanalysissystemsforspecificapplications.Manipulationof thebio-particlesisthekeyingredientfortheseapplications.Therefore,microfluidicbio-particlemanip- ulationhasattractedasignificantattention fromtheacademiccommunity.Consideringthesizeof thebio-particlesandthethroughputofthepracticalapplications,manipulationofthebio-particles isachallengingproblem.Differenttechniques areavailableforthemanipulationofbio-particlesin microfluidicsystems.Inthisreview,someofthetechniquesforthemanipulationofbio-particles;namely hydrodynamicbased,electrokinetic-based,acoustic-based,magnetic-basedandoptical-basedmethods havebeendiscussed.Thecomparisonofdifferenttechniquesandtherecentapplicationsregardingthe microfluidicbio-particlemanipulationfordifferentbiotechnologyapplicationsarepresented.Finally, challengesandthefutureresearchdirectionsformicrofluidicbio-particlemanipulationareaddressed.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction

Theminiaturizationtrendofintegratedcircuitssince1970s,and thedevelopmentofadvancedfabricationtechniquesformicroand nano-scaledevices[1]since1980sledtotheusageofdeviceshav- ingthedimensionsofmicrometersandnanometersinmanyfields.

Thistrendhashelpedmicrofluidics,whichistheflowphysicsat micro scale,becomean activeresearchareaat theintersection ofchemistry,physics,biologyandengineering.Thisintersection eliminatedtheboundariesbetweenthesedisciplines.Theelimi- nationoftheseboundarieshasposedmanychallengesandnew directionsfororganizationsofeducationandresearch.Oneofthe importantchallengesistherapiddevelopmentofbiochips,minia- turizedanalysissystemsorlab-on-a-chip(LOC)deviceswhichare microfluidicplatformsonwhichonecanhandlechemicalandbio- logicalanalyses,point-of-caretesting,clinicalandforensicanalysis, molecularandmedicaldiagnosticsforbiological,biomedicaland

∗ Correspondingauthor.Tel.:+903122902108;fax:+903122664126.

E-mailaddresses:barbaros.cetin@bilkent.edu.tr,barbaroscetin@gmail.com (B.C¸etin),bulent.ozer@gmail.com(M.B.Özer),mehmete.solmaz@ikc.edu.tr (M.E.Solmaz).

chemical applications. LOC devices can perform the same spe- cializedfunctionsastheirbench-topcounterparts.Theycanalso perform clinical diagnoses, scan DNA, run electrophoretic sep- arations, act as microreactors, detect cancer cells and identify bacteriaandviruses[2].Onasinglechip,hundredsofdifferentreac- tionsand/oranalysescanbeperformedatthesametimethrough hundredsofparallelmicrochannels.Originallyitwasthoughtthat themostsignificantbenefitoftheseLOCdeviceswouldhavebeen theanalyticalimprovementsassociatedwiththescalingdownof thesize.Furtherdevelopmentsrevealedothersignificantadvan- tagessuchas:(i)smallamountofsample(inthenanotopicoliter range,openingthedoortothepossibilityofanalyzingcomponents fromsinglecells),(ii)smallamountofreagents,(iii)veryshortreac- tionandanalysistimecomparedtobench-topcounterparts,(iv) reducedmanufacturingcosts,(v)increasedautomation,(vi)high portability,and(vii)opportunityformassivelyparallelchemical analyseseitheronthesameormultiplesamples[3].

Forchemical,biologicalandbiomedicalanalysesinmicroflu- idic systems, there are some fundamental operations such as separation, focusing, filtering, concentration, trapping, sorting, detection,counting,washing,lysis ofbio-particles,andPCR-like reactions. Thecombination oftheseoperations ledtothecom- plete analysis system or LOC system for a certain application.

http://dx.doi.org/10.1016/j.bej.2014.07.013 1369-703X/©2014ElsevierB.V.Allrightsreserved.

Manipulation of the bio-particles is the key ingredient for the aforementioned operations. Therefore, microfluidic bio-particle manipulation has attracted significant attention from the aca- demiccommunity.Consideringthesizeofthebio-particlesandthe requiredthroughputforthepracticalapplications,manipulationof thebio-particlesisachallengingproblem.Manyresearchgroups andscientistshaveproposeddifferenttechniquestomanipulate bio-particlessuch ashydrodynamic-based, electrokinetic-based, acoustic-based,magnetic-based,optical-basedetc.Inthisreview, thesedifferenttechniquesarediscussed.Moreover,thecomparison ofdifferenttechniquesandtherecentbiotechnologyapplications regardingthemicrofluidicbio-particlemanipulationarepresented.

Finally, challenges and the future research directions are also addressed.

2. Manipulationmethods

Manipulationmethodscanbecategorizedaspassiveoractive methods dependingon thepresence of an external force field.

Passivesystemsutilizetheflowfield togetherwiththechannel geometryortopologychangestomanipulatethemotionofpar- ticles.Ontheotherhand,activesystemsutilizeanexternalforce fieldsuchaselectric,acoustic,magneticandoptictomanipulatethe motionofparticles.Thesemethodscanalsobecategorizedaslabel- basedorlabel-freemethodsdependingontheneedforanylabeling (ortags)forthebio-particles.Thelabel-freemethodsutilizethe intrinsicpropertiesofthebio-particlessuchassize,shape,den- sity,dielectricproperties,acousticpropertiesandrefractiveindex.

Ontheotherhand,thelabel-basedtechniquesrequireadditional labelstomanipulatebio-particles.Asanexample,twoconventional cellsortingtechniquenamelyfluorescence-activatedcellsorting (FACS)andmagnetic-activated cellsorting(MACS) requirecell- specificlabelingthroughfluorophore-conjugatedantibodiesand magneticbeadsconjugatedwithantibodies,respectively[4].

Consideringthemanipulationofabio-particleinamicrofluidic system,dependingonthemethodstheremayexistmultipleforces onabio-particle,someofwhich canbedominantornegligible.

Therefore,theorderofmagnitudeestimateofthevariousforces experiencedbyabio-particleis crucialformicrofluidic applica- tionstopredicttheresultantmotionofbio-particles.Asanexample, Brownianmotionistherandommovementofparticlesduetothe thermaleffects;however,Brownianmotionisnegligibleforthe particleswithasizelargerthan1␮mformicrofluidicapplications [5].

There are several techniques to manipulate bio-particles in microfluidicsystems.Severalofthosemethodsarereviewedwithin thispaper.Stand-alonereviewpapersarepresentforeachofthese methods[6–14]sincetherehasbeena vastamountofresearch effortonthesetechniquesformicrofluidicplatformsforthelast twodecades.Inthisreview,ourobjectiveistogivethebasicsof eachmethod.Moreworkisdedicatedforthecomparisonofthe techniquesinterms ofassociated samplepreparation, through- put,channelgeometry,materialandfabrication,andtherequired hardware.Webelievethatsuchacomparisonwillprovidevaluable helpfortheresearchersfrommanydisciplineswhowouldliketo applymicrofluidictechnologytobio-particlerelatedbiotechnology applications.

2.1. Hydrodynamic-based(HD)

Inmicrofluidicapplications,theflowcanbeinducedbypressure difference(pressure-drivenflow)and/orbyelectricalfield(electro- osmoticflow).Sinceelectricfieldisintroducedforelectro-osmotic flow,otherforces(whichwillbediscussedinthefollowingsub- section)otherthandragforcegeneratedontheparticlecomeinto

picture.Inthecaseofpressure-drivenflow,pressuredifferenceis themainparameterwhichcontroltheincompressiblefluidflow inmicrochannels.Thedragforceis theonlyforcegenerated on theparticlesasaresultoftheinteractionoftheparticlewiththe flowfield.Hydrodynamic-basedmethodsarepassivemethodsin whichthebio-particlemanipulationisperformedbyuseofthedrag forcegeneratedontheparticlesthroughspeciallydesignedchan- nelgeometriesandtopologies.Thedimensionlessnumberswhich characterizetheparticleflowinamicrochannelarethechannel Reynoldsnumber(Re)andtheparticleReynoldsnumber(Rep)[15]:

Re= UmaxD_h

 , Rep= Umaxd²

D_h =Re



_d

D_h



2

, (1)

whereUmax isthemaximumvelocity inthemicrochannel,is thefluiddensity,isthedynamicfluidviscosity,distheparticle diameter,andD_histhehydraulicdiameterofthechannel.Typi- cally,flowswithinmicrochannelsareinStoke’sflowregime(lowRe flows)whichmeanstheflowfollowstheboundariesofthedomain.

Whenparticlesarepresentwithinthechannel,theyalsofollowthe streamlinesoftheflowfieldinadeterministicmanner.However, whenanobstacleand/orflowcontraction/expansionispresented withinthechannel,theparticletrajectoriesrevealsizedependence.

Therefore,byspeciallydesignedchannelgeometries,bio-particles canbemanipulatedaccordingtotheirsizeanddeformability.

Introducing obstacles and posts with a critical spacing can be utilized as filter structure to capture (trap) or isolate spe- cific bio-particle of interest witha size largerthan the critical size[6].However,pore-based filtrationmaybeineffective with deformablebio-particlesand/orbio-particleswithuniqueshapes.

Byintroducingseriesofposts,asizedependentlateraldisplace- ment of bio-particles canalso beachieved, which is knownas deterministiclateraldisplacement(DLD)(seeFig1a)[16–20].DLD canbeutilizedforbio-particleseparation,sortingandfocusing.The presenceofslantedoranisotropicobstacleswithinthemicrochan- nelcanalsoinducesize-basedmotionoftheparticlesduetothe particle-obstacle interaction induced rotational flows, which is knownashydrophoresis (see Fig.1b) andcan beimplemented forbio-particleseparation,sortingandfocusing[21–26].Withthe introductionofcontraction/expansion(pinchsegment)withinthe microchannelnetworktogetherwiththelaminarflowprofile,bio- particlescanalsobemanipulatedtoflowatdifferentstreamlines, whichisknownaspinch-flowfractionation(PFF)(seeFig1c)and canbeimplementedforbio-particleseparation,sortingandfocus- ing[27–31].

TheStoke’sflowregimeisvaliduptoRe∼1.WhenRereaches unityandbeyond,theinertialeffectsbecomesignificantandmod- ifytheflowcharacteristics,whichisknownasinertialmicrofluidics.

Inthisregime,particlesdonotfollowthestreamlinesoftheflow field.Whentheinertialeffectscomeintopicture,twoinertiallift forcesareinducedontheparticle:(i)asheargradientliftforce and(ii)awall-effectliftforce[15].Awall-effectliftforceinduces arepellingforceaway formthewall.Ontheotherhand,shear- gradientliftforceinducesanattractiveforcetowardsthewall[15].

Whenthechannelgeometrybecomes curved,a secondaryrota- tionalflowbeginstobeobserveddue totheinertiaofthefluid whichisknownasDeanflow.Thedimensionlessnumberswhich characterizesthissecondaryflowaretheDeannumber(De)and thecurvatureratio(ı)[15,32]:

De=Re



_D

h

2r



1/2

, ı=D_h

2r, (2)

wherer istheradiusofcurvature ofthechannel.Deand ıare twoimportantparameterswhichaffectthemotionoftheparticles withincurvedchannels.Inertialmicrofluidicscanbeutilizedfor separation,sorting,focusing,andisolationofbio-particles[33–39].

Fig.1. Basicprinciplesofhydrodynamic-basedmethods:(a)DLD,(b)hyrdophoresis,(c)pinchedflowfractionation,(d)inertialmicrofluidics.

Aschematicsofaninertialmicrofluidicsbasedsortingcanbeseen in(seeFig1d).

TheHD manipulationcanbealsoutilized for theseparation byshape,sincethetrajectoryoftheparticleswithinamicroflu- idicchannelmayalsopossessshapedependencedependingonthe channelgeometryandcharacteristicsofflow.Morespecifically,the flowofasphericalparticleandnon-sphericalparticlemaydiffer.

Separationandsortingofsphericalandnon-sphericalparticlesis importantforclinicalapplicationssuchasseparationofyeastcells atdifferentcellstageandseparationofparasitesformblood.More recently,someresearcheffortshavebeenfocusedontheimple- mentationofHDapplicationsonseparationbyshape usingDLD [19,18,20],PFF[28]andinertialmicrofluidics[40].

2.2. Electrokinetic-based(EK)

Electricalforceslikeelectrophoresis(EP)anddielectrophoresis (DEP)arethesubtlesolutionstomanipulateparticlesinLOCdevices duetotheirfavorablescalingforthereducedsizeofthesystem [41].EPisthemovementoftheelectrically-chargedparticlesinan electricalfieldduetotheCoulombicbodyforce(electrophoretic force)actingontheparticlesbecauseoftheirsurfacecharge.For theutilizationoftheEP,theparticleneedstobechargedandthe appliedelectricfieldneedstobeconstantordirectcurrent(DC).

TheEPforceonaparticlesubjectedtoanelectricfieldofEcan bewrittenas:

FEP=qE, (3)

whereqisthenetchargeoftheparticle[2].EPiscommonlyused inconventionalandwell-developedseparationtechniquessuchas capillaryelectrophoresistoseparateDNAandproteins.

DEPisthemovementofparticlesinanon-uniformelectricfield duetotheinteractionoftheparticle’sdipoleandspatialgradientof theelectricfield.DEPisapplicableevenfornon-conductingparti- clesandcanbegeneratedeitherbyusingDCoralternatingcurrent (AC)field.

TheDEPforceonasphericalparticlesubjectedtoaDCfieldofE canbewrittenas[8]:

FDEP=2εmfCMR³∇(E·E)=2εmfCMR³∇



E



², (4) whereEistheelectricfieldvector,ε_mistheabsolutepermittivity ofthesuspendingmedium,andRistheparticleradius.f_CMisthe Clausius-Mossotti(CM)factor,whichisgivenby

fCM= εp−εm

εp+2εm, (5)

whereεisthepermittivity,andsubscriptspandmstandforthe particleandthemedium,respectively.CMfactor hasnumerical limitsfrom−0.5to1.0.FornegativeCM,negative-DEP(nDEP)force (whichisinthedirectionofminimaofthegradientoftheelec- tricfieldstrength)isgeneratedontheparticle.ForpositiveCM, positive-DEP (pDEP)force(whichisin thedirectionof maxima ofthegradientoftheelectricfieldstrength)isgeneratedonthe particle.

Similarly, for a spherical particle in an AC-field, the time- averagedDEPforcecanbeexpressedas[8]

FDEP(t)=2εmRe[fCM]R³∇E²_rms, (6) whereErmsistheroot-mean-squareoftheAC-field,Re[fCM]isthe realpartoftheClausius-Mossottifactorwhichisdefinedas f_CM(˜εp, ˜εm)= ˜ε_p− ˜εm

˜εp+2˜εm

, (7)

where ˜ε isthecomplexpermittivityanddefinedas

˜ε=ε−j



_

ω



. (8)

Time-averagedDEPforce,Eq.(6),isvalidforastationaryAC- field.IfthephaseoftheAC-fieldhasaspatialvariation,Eq.(6)needs tobemodifiedtoincludethiseffect.Ingeneralsense,time-averaged DEPforcecanbewrittenas[8]:

FDEP(t)=2εmRe[fCM]R³∇^E2rms+4εmIm[f_CM]R³



E²_rms,i∇^ϕi



, (9)

whereϕisthephaseoftheAC-field.Subscriptireferstoeachcom- ponentoftheelectricfieldandthephasegradient.Thelasttermin theparenthesisisatensornotationandreferstothesummation ofthecomponentsofthevectorquantitiesinsidethebracket.Im[·]

referstotheimaginarypartofacomplexquantity.Thefirstterm dependsonthenon-uniformityintheelectricfieldstrength,and thesecondtermdependsonthenon-uniformityinthephaseof theelectricfieldwhichisthedrivingforceforthetraveling-wave DEP(twDEP)applications.Inthecaseofseriesofplanarelectrodes patternedatthebottomsubstrateofaLOCdevicewhichareexcited withdifferentphases,thefirsttermleadstolevitationofparticles withn-DEPresponse,andthesecondtermleadstoanaxialmotion oftheparticlesovertheelectrodes.Directionoftheaxialmotion dependsonthesignoftheimaginarypartoftheCM.

DEPforce depends ontheparticlesize, dielectric properties oftheparticleandthemedium.Moreover,inthecaseofanAC field,DEPforce(throughCMfactor)alsobecomesfunctionofthe frequencyoftheACfield.Dependingonthedielectricproperties ofthe medium and particle, DEPresponse of a particle canbe switchedfromnDEPtopDEP.Thefrequencyatwhichthistran- sitionoccurs(i.e.thefrequencyatwhichDEPforcebecomeszero) iscalledthecross-overfrequency.Actually,theremayexistmulti- plecross-overfrequenciesforbio-particles[4].SinceDEPdepends onthebio-particles’intrinsicelectricalproperties,EKmanipulation techniquesdonotrequireanylabeling,andarelabel-free.Dielec- tricproperties ofa bio-particledependonthemorphology and chemicalcomposition oftheinternal matrixofthebio-particle.

Therefore,eachbio-particlehasitsowndielectricsignature[41].

Thisissueintroducesabio-particlespecificselectivity;however, alsointroducesachallenge.Sincethebiologicalbasisofthedielec- tricsignatureofthebio-particlesisnotwell-known,theprediction ofthedielectrophoreticmotionofthebio-particlesinanelectric fieldis notstraightforward.For EKmanipulation,both negative andpositiveforcescanbegeneratedwithdifferentconfiguration oftheelectrodesand/orthemicrofluidicchannelstructures,and switchingthepolarityand/orthefrequencyoftheelectricfield.In additiontothat,DEPhasafavorablescalingeffectwhichmakes itperfectcandidateforthemanipulationofmicro/nano-sizedpar- ticles[8].OnerequirementfortheDEPforcetobeinducedisthe non-uniformelectricfieldgeneratedwithinthemicrofluidicdevice.

Non-uniformelectricfield caneitherbegenerated bymeansof (i)insulatorstructuresor(ii)byspeciallydesignedmicroelectrode arrays.

2.2.1. Insulator-basedDEP(iDEP)

Thenon-uniformelectricfieldcanbegenerated bymeansof the specially designed microchannel network (such as serpen- tinechannelandspiralchannel)orspeciallydesignedstructures inside themicrochannelnetwork (suchas electricallyinsulated hurdlesandobstacles). Typically,theelectricfield isappliedby usingexternalelectrodesthataresubmergedintothereservoirs, andtheflowisalsoinducedbytheelectricfield(i.e.electro-osmotic flow).Highelectricvoltageisrequiredtogeneratethesufficient electrokineticforcewithinthemicrochannel networkforthese applicationswhichmakestheuseofDC field(orDC-biased AC) feasible.Therefore,commonlyiDEPapplicationsareDC-DEPappli- cations.However,specialcareisneededsincethehighelectrical voltagemayleadtoaseriousJouleheatingeffectinsidethechan- nel.Thisseveretemperatureincreaseinside thechanneldueto Jouleheatingmayleadtoabubbleformationwhichcanseverely interferewiththeoperationofthedevice[42].Ontheotherhand, duetotheabsenceoftheelectrodesinsidethedevice,iDEPdevices arerobust,chemicallyinertanddonotrequireanyphotolithogra- phy,thin-filmdepositionandlift-offand/oretchingforelectrode fabrication.

2.2.2. Electrode-basedDEP(eDEP)

Thenon-uniformelectricfieldcanbegeneratedbymeansofthe speciallydesignedmicroelectrodearrays(i.e.interiorelectrodes) patternedwithinthemicrochannels.Toavoidtheadverseeffects ofDCfieldsuchasmigrationofchargedparticlestowardstheelec- trodes,ACfieldisappliedforeDEPapplications.Mostofthetime, embeddedinternal electrodesareplanar(2D) (i.e.heightofthe electrodesareintheorderofhundrednanometers),andarefabri- catedwithinthedevicebymeansofcomplex,timeconsumingand relativelyexpensivemanufacturingtechniquessuchasthin-film deposition,sputtering,chemicalvapordeposition,etc.Moreover, foulingoftheelectrodesmaydistorttheoperationofthedevice whenworkingwithbio-particles[43].However,withanappropri- atedesign(i.e.closelyspacedelectrodes),operatingvoltagecanbe lowered(whichpreventsanysufferingfromJouleheating)foreDEP applications.Moreover,lowvoltagessimplifytheequipmentand circuitry.ForeDEPapplications,theinterfacialeffectsmayoccur attheinterfacebetweenthefluidmediumandtheelectrodesur- face,andmayleadtoadverseeffectssuchaselectrodepolarization, localheatingaroundtheelectrodes(whichmayresultinACelec- troconvection),bubbleformationanddissolutionoftheelectrodes [8].Therefore,someconstraintsneedtobeconsideredinthedesign ofsuchsystems.

2.3. Acoustic-based(ACT)

Theuseofultrasonicstandingwavesforbio-particlemanipu- lationreliesonthecreationofultrasonicstandingwaveswithina channel.Thegenerationofacousticradiationforcehasbeenstud- iedforalongtime.Theformulationforacousticradiationforceon inelastic[44]andelasticsphereswasderived[45]andlatergeneral- izedbyGorkov[46].Ingeneral,equationsofacousticsconsiderthe compressibleNavier–Stokesequationandthefirstorderharmonic variationsonthestaticmeanofacousticproperties.However,the time-averagevaluesofthesevariablesleadtozeroforharmonic inputs.Realistically,havingzeromeanfortheacousticvariables overonecyclecannotbethecasesincefromthetestsitisknown thatthereisnetdisplacementofparticlesovertimewhichmeans thattheaverageoftheacousticforceover onecyclecannotbe zero.Therefore,forthecalculationofacousticradiationforcetime averagedsecondorderradiationforcesneedtobeused[47].The gradientoftheacousticradiationpotentialcanberelatedtothe acousticradiationforceas:

F_rad=−∇^Urad, (10)

whereU_radis theradiation potential.Under theassumptions of particlesbeingsmallwithrespecttothewavelengthoftheacous- ticwavesandnotconsideringacousticwavescatteringfromother particles(i.e.smallparticleswithlowconcentration),theacoustic radiationforcecanbeobtainedas:

U_rad= 4

3 R³



f₁ 1

2_fc_f²p²_in−f₂3 4_fv²_in



, (11)

f1=1−_fc_f²

pc²_p, f2=2(p−_f)

2p+_f . (12)

Eq.(12)containsthevariablesrelatedtotheacousticproperties ofthefluidmediumaswellastheparticlestobemanipulated.

p_inandv_inaretheincidentacousticpressureandacousticparticle velocity,and_fandc_findicatesthedensityandthespeedofsound ofthesuspensionfluid,respectively.pandcpindicatesthedensity andthespeedofsoundofparticles,Ristheradiusoftheparticle.

Theaboveexpressionsaretrueforanyacousticfield.Forthecase ofanacousticstandingwavewhereacousticpressureismaximum, theparticlevelocityisminimumatthechannelwalls.Thefollowing

Fig.2.Arepresentationofacousticbasedbio-particle(a)washingand(b)separation.

equationshowstheacousticradiationforceduetoa1Dstanding waveasfollows:

F_rad=4R²(kR)Eac sin(2ky)ˆj, (13) wherekisthewavenumber,yisthelocationdirectionalongwhich theacousticpressurewavechanges,andistheacoustophoretic contrastfactorwhichcreatesdifferentamountofforcingonparti- clesduetotheiracousticproperties

=p+2/3(p−_f) 2p−_f − _fc²_f

3pc²_p, Eac= 1

4_fu²_y· (14) ThesignofEq.(14)alsodeterminesthedirectionoftheparti- cles’motionwhentheyarepushedbytheacousticradiationforce.

Ifthecontrastfactorispositive,theparticleswillmovetowards thenodalpointsand ifthecontrastfactor isnegative,particles movetotheantinodesoftheacousticpressurewaves.Hence,Eqs.

(13)and(14)canbeusedtodeterminetheacousticradiationforce appliedonabio-particlesolutionwithlowconcentrationsuchthat theacousticpressurefieldisnotdistortedduetoexistenceofthe particles.Moreover,thederivationalsoassumesthattheradiusof eachparticleissmallcomparedtotheacousticwavelength.

Theacousticstandingwavefieldcreatesanacousticradiation forceonbio-particleswhosemagnitudedependsonthesizeand acousticpropertiesofthebio-particle.Thedifferenceinthemag- nitudeof theacousticradiationforcecausestheparticles tobe manipulatedintothedifferentlocationswithinthemicrochannel basedontheirproperties.Propertydependentparticlemanipula- tioncanbeexploitedfordifferentapplicationsinbiotechnology.

Bio-particlewashingisoneoftheseapplicationsinwhichdeflection ofbio-particlesisusedtomovethebio-particlesfromonecarrying mediumtoanother.Forthistooccur,thereneedtobetwodifferent typesof carrying medium(buffer) flowing inthe microchannel andtheReynoldsnumberoftheflowneedstobelowtosatisfy

minimalmixingofthetwobuffersolutions.Theideabehindthe bio-particlewashingistomovetheparticlesfromonebufferto anotherthroughtheuseofacousticradiationforceinducedbythe ultrasonicstandingwavesinsidethemicrofluidicchannel.Thepro- cesscanbeseeninFig.2(a).Ultrasonicwavescanalsobeutilized totrapbio-particles(acoustictrapping)tocertainlocations(nodal planes)ofamicrochannel.Theacousticradiationforcemaybeused toovercomethedragforceonthebio-particlesandholdthemsta- tionary(i.e.trappedparticles).Thesameprinciplecanbeusedto cleanthemediumfrombio-particlesorincreasethenumbercon- centrationofthebio-particlesinthemedium.Sincethemagnitude oftheacousticradiationforceinducedonabio-particledepends onthesizeandacousticpropertiesofthebio-particle,standing ultrasonicwavescanbeusedinbio-particleseparation,whichis alsoknownasacoustophoresis.Bio-particlescanbemanipulated toarriveatcertainlocationsinsidethemicrochannelatdifferent instants.Forinstance,whenbio-particlesaredirectedtothecenter ofthechannelbytheacousticradiationforce,thetimetoreachthe centerofthechannelwillbedifferentfordifferentbio-particles,and thisdifferenceintimecanenabletheseparationofbio-particles.

Ifseparationintermsofsizeisdesired,thenthelargestdiameter particleswillreachthecenterchannelfirstwheretheycanbechan- neledoutfromthecenter,whereasthesmallerdiameterparticles canbechanneledoutfromlocationsthatareclosertothechannel sidewalls.ThisprocessisschematicallyshowninFig.2(b).

2.4. Magnetic-based(MG)

Magneticfieldcanbeusedasanexternalforcetomanipulate bio-particles.Theforceappliedbythemagneticfieldonaparticle canbewrittenas[48]:

FMG=Vp−m

₀ (B·∇)B, (15)

Fig.3.Theprinciplestepsofimmunomagneticbio-particleseparation.

whereprepresentsmagneticsusceptibilityoftheparticle,mis themagneticsusceptibilityofthemedium,₀isthemagneticper- meabilityofthefreespace,Visthevolumeoftheparticle,andBis themagneticfluxdensity.Theimportantparameterswhichaffect themagneticforceonaparticlearethedifferencebetweenthe magneticsusceptibilitiesoftheparticleandthemedium,aswell asthemagnitudeandthegradientofthemagneticflux.Moreover, themagneticforcedependsonthevolumeoftheparticle(i.e.the magnitudeoftheforcehassizedependence).

Typically, it is not possible to generate large enough mag- neticforceonabio-particlesuspendinginaconventionalaqueous solution. To be able to create large enough force, the differ- encebetween the magnetic susceptibilities of the particle and themediumneedsto belarge. Thisdifferencemayberealized eitherby increasingthemagnetic susceptibilityof bio-particles orthesuspensionmedium.Itisdifficulttoincreasetheinherent magneticsusceptibilityofbio-particles;however,anengineered paramagnetic micro-particle with high magnetic susceptibility canbeattachedtobio-particleswhichcausesbio-particlestobe manipulatedbytheparamagneticmicro-particle.Forthebinding processtooccur,thesurfaceoftheparamagneticmicro-particle iscoveredwithantibodieswhichhaveaffinitytobindontothe bio-particle(see Fig. 3). The magnetic manipulation using this processiscalledasimmunomagneticbio-particlemanipulation.

Alternatively, a ferrofluid or a paramagnetic suspension rather thanatypicalsuspensionmedium suchas salineorPBS (phos- phatebufferedsaline)canbeusedtogeneratealargedifferencein magneticsusceptibilitiesofthesuspensionfluidandbio-particle.

The use of such suspension medium eliminates the need for attachingamagneticmicro-particlesontothebio-particles.Once the field is introduced, the bio-particles can be pushed away fromthe magnetic field whereas in the immunomagneticcase theparamagneticparticles (hencethebinded bio-particles)are attractedtothemagneticfield. Therepulsionofbio-particles is duetosignchangeinEq. (15)sincethemagneticsusceptibility ofthemediumbecomes largerthantheparticles.Themagnetic

manipulationinwhichthebio-particlesaresuspendedinaparam- agneticorferrofluidsuspensionmediumisalsocalleddiamagnetic bio-particlemanipulation.The diamagneticcellmanipulation is commonlyusedforfocusingpurposes.However,thediamagnetic cell manipulation approach can also beused for concentration and separation of particles of different sizes as illustrated in Fig.4.

2.5. Optic-based(OP)

The use of radiation pressure of light to displace and trap micron-sized dielectric particles was first hypothesized and demonstratedin1970sbyAshkin[49].Itwasnotuntil1980sthat thepracticalapplicationsofopticalforcesinphysics[50]andbiol- ogy[51]haveshownitstruepotential.Thescientificcommunity hassincebeenusingthetermopticaltweezerstoidentifytheuseof opticalforcesasameanstomanipulatenanometertomicrometer- sizedobjects.Someofthesignificantachievementsusingoptical trapsarestudyingmolecularmotorswithsubnanometerresolution [52],fundamentalpropertiesofcolloidsandinterfacescience[53], mechanicalpropertiesoflivingcells[54],andpolymerelasticity [55].Theabilitytopreciselycontrolsmallobjectswithnomechan- icalcontactisdefinitelyadvantageousformicrofluidicapplications.

Recentadvancesonmicrofluidicdevicefabricationbroughtadif- ferent perspective to the utilization of optical forces. Practical applications such as sorting, separation, and self-assembly are nowpossibleusinglight-matterinteractionand basicprinciples ofmicrofluidics.

The basic instrumentation behind an optical trap is a high numericalaperture(NA)microscopeobjectivethatisabletotightly focusalightbeam.Thedielectricparticlenearthefocus,withhigher refractiveindexcomparedtoitssurroundings,receivestheinci- dentphotons,whicharescatteredand/orabsorbed.Scatteringof incidentphotonscreatesamomentumtransfer,hencetheoptical forcecomponent.Thephysicalprinciplebehindopticaltrapscan beexplainedbythebalanceoftwoforces:(i)scatteringforcesin

Fig.4.Diamagneticbio-particlemanipulationtypes.

Fig.5. (a)Thelightrayswithdifferentintensitiesresultindifferentforcevectors,thevectorsumoftheforcespullstheparticletothebeamaxiswhilealsopushingitinthe directionofbeampropagation.(b)Therayparalleltoz−axishitstheintersectionplanewithananglewithrespecttox–zplane.

thedirectionofpropagationand(ii)gradientforcesinthedirec- tionofopticalfieldgradient.Scatteringforcegeneratesradiation pressurethat pushesthedielectricparticleawayfromthelight source.Gradientforce,ontheotherhand,actstheoppositeway andattractstheparticletowardsthepeakspatiallightintensity.

Inthecaseoftightlyfocusedbeam,stabletrappingoccursifthe gradientforceexceedsthescatteringforce.Trappingdependson factorslikeparticlesize,refractiveindex,laserpowerandspatial characteristics.Theoreticaltreatmentofopticaltrappingcatego- rizestheforcesonparticlesdependingontheirsizes.Thecondition wherethesizeoftheparticleismuchlargerthanthewavelength oflight(d)iscalledtheMieregime.Inthisregime,theforces actingonaparticlecanbecomputedbyRayopticsprinciples,i.e.

reflectionand refraction. Reflection and refraction onthe front andbacksurfacescreatesperpendicularmomentumchangeona sphereasseeinFig.5(a).Inthefigure,adashedcirclerepresents crosssectionofthesphereandwaschosenfordemonstrationpur- poses.

AssumingaGaussianintensityprofile,therayclosertobeam axiscreatesmorepointforcethantheraysituatedfurtheraway.

Thenetforcepullstheobjecttowardsthebeamaxis,andpushesthe objectduetoscattering.Gradientforceonlydominatesandpulls theobjectwhenthereisasteepfieldgradientachievedbytightly focusingthebeam.InMieregime,theforceactingonasphereat anincidenceangleof andrefractionangleofˇisduetoasingle rayofpowerPis(n₁QP/c).Therefractiveindexofthemediumsur- roundingtheparticleisn1andtheparticlerefractiveindexisn2.Q describestheamountofmomentumtransferatthefrontandback surfacesformultiplebounces,andisafunctionofFresnelreflec- tion(R)andtransmission(T)coefficients.Qvalueforalltherays actingonacircleweregivenbyAshkin[56],andthetotalscatter- ingandgradientforcesactingonadielectricparticleweregivenby Roosenandcoworkers[57].ThecircleonFig.5(a)canbetreatedas theintersectionofaplaneandasphereonFig.5(b),andthetotal scatteringandgradientforcescanbeintegratedovereverypoint onthesphereas:

Fs=−

/2 0

d

2

0

d

E²r²

oc²sin cos



×

Rcos2 +1−T²cos2( −ˇ)+Rcos2 1+R²+2Rcos2ˇ



, (16)

Fg =−

/2 0

d

2

0

d

E²r²

oc²sin cos sin



×

Rcos2 −T²sin2( −ˇ)+Rsin2 1+R²+2Rcos2ˇ



, (17)

whereoisthefreespacepermittivity,cisthespeedoflight,Eis theincidentelectricfieldandincludestheopticalbeaminformation suchasitsradius,ristheradiusofthesphere,andistheangle betweentheplanethattheraypropagatesandthex–yplane.

Thecondition (d) iscalled theRayleighregime, describ- ing the diffraction-limited conditions. In this regime, particles are treated as point dipoles, and the gradient and scattering forcesareproportionaltotherealpartandimaginarypartofthe polarizabilityoftheparticle,respectively.Scatteringforce,which isinthedirectionofincidentpowerandthegradientforce,which isinthedirectionofintensitygradientcanbewrittenas[58]:

Fs=I_on₁ c

128⁵r⁶ 3⁴

m²−1 m²+2



2

, (18)

F_g=−n³₁r³ 2

m²−1 m²+2



∇^{E2, (19)}

wheremistheindexcontrastratio,n2/n1,andIoistheintensity oflight.Thescatteringforceisdependentonthelightintensity, theparticlesize,thewavelengthofthelight,andrefractiveindex contrast.Thegradientforceisdependentonthespatialvariation ofelectricfield,theparticlesizeandtherefractiveindex.Unlike scattering,gradientforcescaleswiththethirdpowerofparticle size.Hence,itiseasiertoachievetrappingforasmallerparticle thanalargeparticle.

Sincetheopticaltrappingtheoryissizeandgeometrydepend- ent, researchers depend on empirical determination of optical forces.Theprocedureiscalledopticalforcecalibration(standard methodswiththeiradvantagesanddisadvantagescanbefound elsewhere[13]).Themainobjective oftheopticalforcecalibra- tionistoextractthetrapstiffness(k)oftheopticaltrapthatis treatedasaHookeanspringwheretheforceislinearlyproportional todisplacement(F=−kx).Onecommonapproachisthedragforce calibrationwheretheparticleisheldstationaryusingthetrap,and thechambercontainingthefluidismovedatavelocitysothatthe particleescapesfromthetrap.Thevelocityinformationcanthen beusedtocalculatethedragforce,whichiscorrectedbasedon

thedistancetotheclosestsurface.Alternatively,Brownianmotion calibrationcanalsobeused.Inthisapproach,thefluctuationsin thepositionoftheparticlearerecorded,andapowerspectrum is formed and fitted to extract thetrap stiffness. This method alsorequiresknowingthedragontheparticle.Inbothmethods, accuratemeasurementsofdisplacementarecrucialforthecalcu- lationofappliedforceandeitheraquadrantphotodetectororvideo trackingisnecessary.

3. Assessmentofthemethods

3.1. Samplepreparationandselectivity

TheselectivityoftheHDmethodsisbasedonbio-particlesize anddeformability.Ifthereisanappreciablesizedifferenceamong bio-particles,theycanbemanipulatedtodifferentlocationswithin themicrofluidicnetwork.Onetypicalaspectofbio-particlesisthat theydonotpossessspecificsize,insteadtheypossesasizedis- tribution.Ifthere isasizeoverlapfortwo differentbio-particle populations,theselectivityoftheHD methodsdiminishes.One majoradvantageofHDmethodsisthattheydonotneedanylabel- ing.Althoughlabelingisnotnecessary,samplepreparationmay stillbeneeded,sincetheconcentrationoftheparticlesmayaffect theflowfield,andresultinaundesiredflowpatterns.Forexam- pleinthecaseofwholeblood,dilutionofthewholebloodmaybe usefuldependingontheapplication.

TheselectivityoftheEKapplicationsrelyonthedielectricprop- ertiesofbio-particles.Thedielectricstateofabio-particledepends onphysical(size,shape,surfacemorphology)andchemicalstate of the bio-particle.For DC-DEP applications, DEP response can bepredictedeasilysinceitonlydependsontheconductivityof thebio-particleandthe medium.For AC-DEPapplications,DEP responsealsodependsonpermittivityofthemediumandthebio- particle.Moreover,DEPresponseisalsoafunctionoffrequency oftheAC-field. Tomanipulate bio-particleseffectively, theDEP responseneedstobepre-determined.Althoughthere aremany studiesregarding DEP-based manipulationof bio-particles, it is stillchallenging topredicttheDEPresponse ofthebio-particle ofinterest.ThebestwayistomeasuretheDEPresponseofthe targetbio-particles.Therearesomeproposedmethodsinthelit- eratureforthemeasurementofDEPresponse[8,59,60],however theprocessisnotstraightforward.Astand-alonemicrofluidicplat- formwithsomespecialcircuitryand/oropticaldetectionsystemis neededtodeterminetheDEPresponseofabio-particlepopulation.

Ifthischallengeishandled,theselectivityofDEPcanbeutilizedto manipulatedifferentbio-particles.

Thepresence ofthe electricfield in EK applicationsinduces Jouleheatingwithinthefluid.Dependingontheconductivityof thebuffersolutionandthestrengthoftheelectricfield,theJoule heatingeffectcanbesevere,andcausedeteriorationofthesystem performance[8,42].Moreover,Jouleheatingmayalsocauseunde- siredelectrothermaleffectswhichmayaffecttheflowfieldwithin themicrochannel[8].Sincethephysiologicalbuffersolutionsfor bio-particlestypicallyhavehighelectricalconductivity,dilutionof thebuffersolutionmaybenecessaryforEKapplications.Thisissue ismorecriticalforiDEPapplicationsinwhichDCelectricfieldis presentthroughoutthedevice.

ForACTapplications,nospecialsamplepreparationisneeded.

Thebufferfluidcanbeanykindofbufferorsalinesincealmostall havesimilaracousticproperties.Particleswithhighcontrastfactors canbemanipulatedmoreeffectively.Asignorasignificantvalue differencein acoustic contrastfactor of bio-particles are desir- ableforeffectivedifferentiationofbio-particles.Whentheacoustic propertiesandsizesofparticlesthatneedtobeseparatedareclose toeachother,acousticpropertiesofthesuspensionmediumcan

beadjustedsuchthattheacousticcontrastfactorshowninEq.(14) ispositiveforonetypeofparticlesandnegativefortheothertype.

Utilizingthisidea,redbloodcells(RBCs)andplateletshavebeen separatedsuccessfully[61].Although,nobio-particlepreparation (otherthandilution)andlabelingisnecessaryforacousticmanip- ulation,labelingofparticleswereutilizedtohavebetterselectivity in a very limited number of studies where bio-particles were taggedwithbeadswhichhavenegativeacousticcontrastfactors [62].Thiswaywhentheacousticfieldwasemployed,thelabeled bio-particlescanmoveintheoppositedirectionoftheremaining particles.Moreover,thebio-particlemaybelabeledwithfluores- centdyetoimprovethevisibilityoftheparticlesmovement[63].

Considering the MG applications, both the diamagnetic and immunomagneticmanipulationmethodsrequiresampleprepara- tionpriortothebio-particlemanipulation.Inimmunomagnetic bio-particlemanipulation, theparticles need toincubated with thebio-particlesamplesothat thetargetcells arelabeled. The labelingoccurs dueto biochemicalreactionsbetween theanti- gensandantibodiesonthebio-particlesandthemagneticparticles.

The duration of the incubation is in between 10 and 30min dependingonthemixing,temperatureandbead-cellconcentra- tions.Moreover,ifthemanipulatedbio-particlesaretobeinfused backtothehost,themagneticparticlesneedtobewashedaway fromtheselectedbio-particles.However,thesamplepreparation stepallowstheimmunomagneticprocesstobehighlyselective.

It is possibletoseparate targetedcells which do not have sig- nificantphysical differences fromother cell groups even ifthe number of total totarget cellratio is in theorder of 10⁹ [64].

Forthecaseofadiamagneticparticleseparation,thebio-particles need to be suspended in a paramagnetic fluid or ferro fluid.

Theselectivityofthismethodcanbeextendedbyswitchingthe magnetic field using an electro-magnet [65]. Common choices for paramagnetic fluids are MnCl₂ (Managnese (II) Chloride), GdCl3 (Gadolonium (III)Chloride)andGd-DTPA(gadolinium(III) diethylene-triaminepentaaceticacid) whichis anFDA approved MRIcontrastagent.Inferrofluids,themostcommonchoiceisthe useofEMG408fromFerrrotecwhichisawaterbasedproductwith nano-sizedironparticles.Attheendofthemanipulationprocess, theparamagneticfluidorferrofluidmayneedtobewashedaway fromthebio-particlesifinfusionintoaliveorganismistobefol- lowed.Duetothesample preparationand postprocessing,MG methodsrequirerelativelycomplexprocedures.Therearesome examplesofmicroscalesystemswhichperformtheincubationstep in-line.Onesuchsystemusessix rotatingmagnets[66] tomix micro-particlessothatincubationwiththebio-particlescanoccur onthemicrofluidicdevice.Aftertaggingthebio-particles,theincu- batedbio-particlesaresenttothemicrofluidicdeviceandcanbe positivelyselectedbymeansofamagneticfield.Theprocessof immunomagneticseparation is generallyused forseparation of rarebio-particlesratherthanfocusinglargenumberofbio-particles sincetheimmunomagnetictaggingofbio-particlesisnoteconom- icallyfeasibleandistimeconsuminginlargenumbers.

In the case of OP applications, it is important to know the physicalcharacteristicsofthefluidandthetrappedobjectduring opticaltrappingexperiments.Refractiveindexchangesaccording tomediumwhichthebio-particleisinandthebio-particletype.

Forinstance,therefractiveindexofthecellmediamaychangedur- ingcellculturegrowth,althoughtherefractiveindexofindividual cellstypicallystaysthesame(∼1.38).Itisbeneficialtomeasure themediumsrefractiveindex(usuallyaround∼1.33–1.35)with a refractometer if suspended bio-particles are used. Moreover, refractiveindexofanybio-chemical stimuliagentintroducedto microfluidic channels shouldbe knownbefore hand.The main advantageofOPmethodsistheabilitytosensedifferentcolorsof light emittedfromdifferent fluorophores. Broad-spectrumlight detectorscanbeadjustedwithopticalfilterstodetectonlycertain

portionsofspectral window.Thisability isusefulin activeand passivefeedbacksystemswheretheamountofspectralsensitivity is the identifier for the state of a bio-particle. Fluorescence or immunofluorescence staining (labeling) of bio-particles is per- formedusingin-vitromethodsdependingonthesizeofthedye molecule.Besidesdyestainingthecells,thepathogensintroduced togrowthmediaduringcellpreparationcanalsobefluorescently labeledandusedasthediagnosticmarkerduringopticalmanip- ulation[67].InOPapplications,thechannelsneedtobeprimed withcertainchemicals priortoflow of bio-particles toprevent adhesionespeciallyduringanextendedstopoftheflow.Inthecase ofbiologicalcells,thecellmediacontainingproteins,glucose,salt, lipidsandothernutrientsaregoodprimingagents,andneedto beappliedpriortotheoperation.Bovineserumalbuminisalsoan effectiveagentthatpreventsadhesionofenzymestoglasssurfaces.

3.2. Flow-rateandthroughput

Forfilteringapplications,theparticleswithalargersizecanbe immobilizedand/orparticleswithsmallersizecanflowthroughthe desiredoutlet.Cloggingistypicalforfilterapplications.Moreover, theshearstressinducedonthebio-particlesmaycauselysis.As moreandmorebio-particlesaretrappedwithinthemicrochannels, highershearforcesareinduced,andhigherpressuresarerequired to maintain the desired flow rate. For other HD applications, bio-particlesflowinamicrochannelinaforce-freemanner.How- ever,forDLD,PFFandhydrophoresisapplications,theflowneeds to controlledprecisely, and regarding both thefabrication (for hydrophoresisthegapsizeofthecontractionsiscriticalandneeds tobecomparablewiththebio-particlesize)andtheoperational concerns,theyneedtobeoperatedwithrelativelysmallflow-rates (intheorderof␮L/h).Thisleadstolowthroughputformanyclin- icalapplications.Asanexample,ithasbeenshownthatinertial effectsdecreasethesortingefficiencyforhydrophoresisapplica- tions[26].Atthispoint,inertialmicrofluidicsoffersapromising perspectively.InertialmicrofluidicsutilizesthehighRecharacteris- ticoftheflow;therefore,inherentlyflow-ratesarehighintheorder ofml/hwhichleadstohighthroughput(>∼10⁶particles/min),and thismakesinertialmicrofluidicssuitableformanyclinicalappli- cations.However,thepredictionoftheflowfieldandtheparticle motioninthisregimeisalsochallenging.Thethroughputofsev- eralHDapplicationcanbeseeninFig.6.Thehighestthroughput forHDmanipulationisaround∼10¹¹particles/min,andbelongsto anapplicationinwhichtheplasmaisseparatedfromwholeblood ratherthanadifferentiationofbio-particles.

Thesuccessfulmanipulationofbio-particlesinEKapplications dependsonthebalancebetweenthedragforceandtheEKforces.

AnincreaseindragforcerequiresanincreaseinEKforceandthis indicatestheuseofhigherelectricalfieldstrength.Sincehigher electricfieldsarenotdesirableduetoJouleheatingphenomena [42],thedragforceandtheflow-ratearelimitedfortheseapplica- tions.InthecaseofeDEPapplications,theelectricfieldisanissue onlywithinthevicinityoftheelectrodes;therefore,higherelectric fieldscanbetolerated.However,onemajordisadvantageofthe eDEPdeviceswithplanarelectrodesisthattheelectricfieldgradi- entdecreasesrapidlyastheparticlesflowawayfromtheelectrodes.

Therefore,aneffectivemanipulationregionisgeneratedwithinthe vicinityoftheelectrodeswhichreducestheeffectivenessofthe manipulation.Iftheelectrodesaredepositedonthebottomwall ofthemicrofluidicchannel,thentheheightofthechannelneeds tobelimited.Thislimitationcanbesolvedbyintroducing3Delec- trodeswithinthedevice.Manyresearchershaverecentlyrealized thisaspectandproposeddifferentfabricationtechniquestofabri- cateembedded3Delectrodes[68].Byintroducing3Delectrodes, theflow-ratesandthethroughputoftheDEP-baseddevicescanbe enhancedalthoughthroughputofEKmethodsisrelativelylow,and

atthispointnotsuitableforclinicalapplications.Thethroughput ofseveralEKapplicationscanbeseeninFig.6.

In terms of throughput, ACT devices have relatively higher throughputcompared tothat of othermethods. Theflow rates generally range from1␮l/minto 1L/h.The throughputof ACT devicesforsomestudiescanbeseeninFig.6.Thefigureshows eightordersofmagnitudedifferenceinthebio-particlethrough- puts.Thestudieswithhighthroughputsaregenerallystudieswhich processwholebloodsamples.Thenormalbloodcountofamaleis approximately5×10⁹RBCsinamilliliterofblood.Fortheappli- cationswhichdonotincludefractionationofdifferentcellgroups fromeachothersuchasseparationofplasmafromwholeblood, thethroughputreachesashighas10¹¹particles/min[69].

ThroughputofMGmanipulationmethodsisalsopromising.The throughputofseveralMGapplicationscanbeseeninFig.6.The largestofthetwothroughputvalues[70,71]belongtoasystem wheretheborediameterofthechannelisroughly8mm.Itmay bearguedthatthisdiameteristoolargeforamicrofluidicsystem.

Mostofthehigherflowratesbelongtosystemswithimmunomag- neticbio-particlemanipulationsystems,anddiamagneticparticle manipulationsystemshavelowerthroughputs.However,itshould benotedthatthethroughputsarepurelybasedonthetimethat cellsspendinthemicrofluidiccircuit.Intheimmunomagneticpar- ticlemanipulationcases,asignificantportionofthetimeisspent on thepre-processing of bio-particles (considering the incuba- tiontimefortaggingofbio-particleswiththemagneticparticles), hencethethroughputsmaynotberepresentativeofthetotaltime requiredforthebio-particlestobeprocessed.Generally,flowveloc- itiesinthemicrochannelsareintheorderofmm/sorless.

ForOPapplications,activemicrofluidiccellsorterscompared toitspassivecounterpartsuseinformationsuchasfluorescence, sizeandshapebymeansofphotodetectorsorimageprocessing to dynamically steer thebio-particles to desired outlets.These systems canoperatequitefastbasedonthetype offorceused formanipulation. Forinstance,activesorterseitherutilizeopti- cal forces for grabbing or deflection of bio-particles. Grabbing anddeflectionoccurwhenthegradientorscatteringforcedom- inates each other.Active sortersbased ondeflection are much faster since there is no need for steering the focused optical beam.6354cells/min[72] and 1320cells/min[67] hasbeenfor deflection-based sorters. Grabbing-based sorters are almost an orderofmagnitudeslowerwithreportedvaluesof300cells/min [73]and84cells/min[74](thethroughputofseveralOPapplica- tioncanbeseeninFig.6).Foractivemicrofluidicsystemsinvolving OPmanipulation,theflow-rateinthechannelandthedragforce onthebio-particlecompetesagainsttheopticalforce.Considering theopticalforceonaparticleintherangeofafewtotensofpN,the fluidvelocityneedstobeadjustedtoachievedesiredmanipulation forthebio-particle.

3.3. Channelgeometry

The filteringapplicationsin HD methods typically include a microfluidicchannelwithpoststructures.Thespacingofthepost structures is critical to trap the bio-particles with target size.

Dependingonthesizeofthetargetparticles,thespacingcanvary between5and15␮m.Tointroduceasmany poststructuresas possibleforagivenvolume,thediameter(orwidth)ofthepost structuresaretypicallyaroundcoupleoftensofmicrometers.For DLDapplications,againsomepost(orpillar)structuresarealso needed.Thesepoststructuresmayhavedifferentshapes(circular, square,etc.).Sincesomeamountoflateraldisplacementisdesired tomanipulatethebio-particles,thesectionwiththepoststructures needstohavesomethresholdlengthwhichistypicallycoupleof centimeters.ForthePFFapplications,rectangularmicrochannels withsharpcornersarerequired.Althoughtheinletsectionofthe

Fig.6. (a)Throughputdataforseveralmicrofluidicdevices.(b)Throughputrangefordifferentmethods.

deviceiscomposedofamicrochannelwith∼100␮m,toachieve fractionationwithagoodresolution,theexitsectionofthedevice needstobewide,andthegeometryoftheexitsectionneedsto becriticallydesignedtoobtainthedesiredoutput.Fortheinertial applications,sincetheflow-ratesarehightoachievehighReflows, certainlengthisrequiredtoachievetherequiredlateraldisplace- mentofbio-particles.Toutilizeanycirculationwithintheflowfield, itistypicaltointroduceanexpansion/contractionsectionsand/or poststructureswithintheflow field.Morerecently,theexpan- sion/contractionintheheightdirectionhasalsobeenutilizedfor bothsortingandfocusing[38].ToutilizetheDeanflowforparticle manipulation,spiralandserpentinechannelsarerequired,andthe radiusofcurvatureofthechannelsiscritical[32].Forhydrophore- sisapplications,expansions/contractionsintheheightdirections arenecessary.Thesizeofthesecontractionsarecritical,andcer- taindesignproceduresneedtobefolloweddependingonthesizeof thebio-particleofinterest.Typicallytheminimumchannelheight inthecontractedpartneedsbelargerthanthebio-particlesizeto avoidclogging,andlessthantwotimestheparticlesizeforsuc- cessfulmanipulation.Onecommonpoint aboutthemicrofluidic channelnetworkwithpoststructuresisthattheheightofthechan- nelislimitedconsideringthelimitoftheaspectratioduetothe fabricationconstraints.

ForEKapplicationswithoutinternalelectrodes,atypicalchan- nelwidthisintheorderof100␮mtogetherwithsomespecially designedstructures.Theheightofthemicrochannelisnotcritical

aslongasthefabricationisnotproblematic.ForeDEPapplications withinternalelectrodes,theheightiscriticalif3Delectrodesare notused.Typically,channelheightvariesbetween20and50␮m.

ForeDEPapplicationswith3Delectrodes,microchannelstructures withlargerheightispossible(typically20–100␮m).Intheseappli- cations,thelimitationsfortheheightcomesfromthefabrication stepoftheelectrodes.

Channelgeometryisquiteimportantforsuccessfulimplemen- tationofACTmethods.Thechannelsgenerallyhave rectangular crosssections. It issignificantly easiertocouplea piezoelectric patchto a rectangularsurface compared toa circular one.The widthofthechannel(widthistheprimarydirectionalongwhich acousticradiationforceactsonbio-particles)needstobecarefully selectedasanintegermultipleofhalfwavelengthoftheacoustic waveswithinthefluidfortheselectedfrequency.Ifthetransversal configurationisselectedforthepiezoelectricmaterialplacement (piezoelectricmaterialssurfacenotalignedwiththeacousticradia- tionforcedirection),thechannelwidthalsoneedstobeselectedas thehalfwavelengthoftheacousticwavesinthesuspensionfluid.

However,ifthelayeredconfigurationispreferred,thenthechannel widthcanbeseveralwavelengthsoftheacousticwavesinthesus- pensionmedium.Notonlythechannelwidth,butalsothewidthof thedeviceneedstobeselectedasanintegermultipleoftheacoustic wavelength.Thedepthofthechannelisgenerallyselectedsignifi- cantlylessthantheacousticwavelengthinthefluidtoavoidfitting astandingwavealongitsdirection.Thelengthofthechannelis

generallyintheorderofseveralcentimeterswhichcanfitseveral wavelengths.Sincetheacousticwavesarerequiredtofitbothinto thechannelandthedevice,thesensitivityoftheperformanceto thechannelanddevicegeometryissignificantlyhigh.Therefore, thedimensionsoftheACTbio-particlemanipulatordevicesneed tobecontrolledprecisely.

MGmanipulationhasveryfewconstraintsonthegeometryof thechannel.UnliketheACTmethod,theperformanceofthesystem isnothighlysensitivetochannelanddevicegeometry.Typically, thewidthofthemicrochannelsisintheorderof100–700␮m,the depthisintheorderof10–100␮m,andthelengthofthechannel isseveralcentimeters.Althoughpreferredgeometryisarectan- gularcross-sectioned microchannel,thecapillarytubescanalso beutilized[75,76].Ifpermanentmagnetsareused,generallythe polesofeachmagnetisbroughttothevicinityofthechannelwalls.

Thecloserthepolesaretoeachother,thestrongerthegradientof themagneticfieldis,the(B·∇^{)BterminEq.(15),whichresultsin} strongermagneticforceontheparticles.

InOPapplications,thechannelgeometryneedstobeadjusted dependingontheopticalbeamcharacteristics.ThefocusedTEM₀₀ modebeamsize,dependingonthemicroscopeobjectiveused,is smallerthanmostbio-particles,anddoesnotbringanychannelsize limitations. However,rectangularchannel geometriesis prefer- abletoavoidopticalbeamshapechange(e.g.circulartoelliptical), andtoobtainbetterimaging.Opticalclarityisstronglyaffected bycurvedchannelwalls.FordivergingsinglemodeGaussian-like beams, thebeamdiameter and opticalpower both have direct impactontheopticalforceandstressprofile.Fortheapplications withhighnumericalaperture(NA)objectives,theworkingdistance oftheobjectivemaybelessthanamillimeter,andanindexmatch- inggelmaybeneededtotakeadvantageofhighNA.Thedistance betweenthebeamoriginandthelight-particleinteractionarea;

hence,thechannelgeometryshouldbecarefullydesigned both toaccommodatetheopticalsetupandtoachievedesiredphysical effects.

3.4. Materialandfabrication

Whenthefabricationofthemicrofluidicdevicesisconcerned, there are basically two common approaches: direct substrate manufacturing(photolithography,etching,laserablationetc.)and mold-based techniques (hot embossing, injection molding or soft-lithography)[77].Photolithographyhasgoodabilitytomanu- factureverysmallandcomplicatedmicrochannelstructures,butit usuallyinvolvesmulti-stepprocesseswhichtakeconsiderabletime and specificchemical requirementsespecially for etchingsteps inhightechfacilitiessuchasaclean-roomenvironment.Mold- basedtechniquesrequireamold(sometimesmoldisreferredas themask)tobefabricated.Althoughthefabricationofthemold may need lithography-based, relatively complicated fabrication process;oncethemoldisfabricated,themoldmaywellbeusedfor severaltimes.Afterthecompletionofthemold,therestofthefab- ricationprocedureissimpleandhighlyreproducible(i.e.low-cost replication),whichmakesmold-basedtechniquesverysuitablefor massproduction.Acommonmaterialusedinthefabricationof themicrochannelsisthePolydimethylsiloxane(PDMS)duetoits lowcost,lowtoxityandtransparency.BondingPDMSwithglass canbeachievedusingastraightforwardsurfacetreatmentprocess withoxygenplasma(asealedPDMSmicrochannelcanwithstand pressuresuptofivebars[1]).Soft-lithographyusingPDMSisavery commontechniqueusedinthefabricationofmanymicrofluidic devicesfordifferentaforementionedapplications.

Fordirectsubstratemanufacturing,acommonapproachisto etchthewafer and seal it withPDMS.Especially, for microflu- idicchannels withhighaspect ratiopost structures(AR∼1–5), itis preferredtouseetchingof thesilicon waferinsteadofthe

fabricationofamold.Forlowaspectratiopoststructures,molding isabetterchoice.Fordirectsubstratemanufacturing,analternative techniqueislaserablation,whichislocalized,non-contactremoval ofthematerialfromthesurfacebyexposingthesurfacetoalaser beam.Unlikephotolithography,laserablationdoesnotrequirea maskandmaybeappliedtoawidevarietyofsubstratematerials [1].Althoughthecostoftheprocessisrelativelylow,theinvest- mentcostoftheequipmentisrelativelyhigh.Moreover,generally thesurfaceroughnessofthelaserablatedchannelsarenotsuperior thanthatofmold-basedtechniques[78].

For thefabrication of thedevices for HD applications, soft- lithography and photolithography is a common approach, and fusedsilica,PDMS,PMMAandsiliconarethecommonmaterials.

Insomeinertial[38]andhydrophoretic[27–31]applications,3D geometriesareneeded.3Dgeometriescanbeachievedbyutilizing two-steplithography.If3Dstructuresarebothonthebottomand thetopwall,lithographicaligningprocedureisrequired[27,28].

For the fabrication of devices for EK applications, soft- lithography with PDMS is a common method. For many EK applications, embedded electrode structures are also required whicharedesignedandlocatedstrategicallywithinthemicroflu- idicstructure.Asacommonpractice,theplanarelectrodeswithin themicrofluidicdevicesarefabricatedusingmetaldepositionona substrate.Additionalfabricationstepsneedstobeintroducedfor thedeviceswith3Delectrodes.Differentfabricationstrategiesfor DEP-basedmicrofluidicdeviceshavebeenreviewedrecentlybyLi etal.[68].Titanium,gold,silverandcarbonarecommonmaterials fortheelectrodes[68].Copperhasalsobeenusedintheliterature, butsomeadverseeffectswerereported[79,80].

ForthefabricationofdevicesforACTapplications,thereisawide varietyofmaterialsusedforthemicrofluidicdevicesuchassilicon [61,81,69],steel[82–84],glass[85,86]andPDMS[87–91]aswell asotherpolymers[92,93].Thechoiceofmaterialforthetransver- salconfiguration shouldhave highacoustic impedancesuchas silicon,whereasthelayeredconfigurationmaybemanufactured fromawiderangeofmaterialsrangingfromdifferentpolymers tosteel.Thematerialofchoiceforacoustofluidicdevices,whichis excitedwithsurfaceacousticwaves,ispolymerswithlowacous- ticimpedanceandwithgoodshearwavecarryingcapacitysuchas PDMS[87–89].Forthesilicon-baseddevices,fabricationthrough standard photolithographyand wetor dryetchingarecommon approaches[61,94,69,95,47,96].Inthecaseofpolymersandpartic- ularlyPDMSdevices,standardsoftlithographymethods[87,89,93]

andrapidprototypingarecommonlyemployed[97].Typically,a transparentmaterialwithhigheracousticimpedancesuchasglass orfusedsilicaisusedasalidtoformthechannel.Thislidisalso utilizedasanacousticreflectorintransversalconfigurations.

ForthefabricationofdevicesforMGapplications,thereisno significantconstraintotherthanmaterialofthedevicenotbeing amagneticmaterial.Duetotheeaseofprocessandpossibilityof embeddingthemagnetsintothedevice,mostwidelyusedmaterial isPDMS[48,64,98,99,65,100–102].Othermaterialsarealsousedin thismethodsuchasPMMA[66]andsilica[76].TheuseofNDFeB magnetic powdertogetherwithPDMScan formself-assembled magneticconfigurationandachievebetterdeviceperformancefor capturingapplications[99].

ForOPapplications,microfluidicchannelswithopticallytrans- parentwallsarerequired.Mosttransparentmaterialssuchasglass, fusedsilica,PDMS,PMMAaresuitableowingtotheirtransparency tovisibleandnear-infraredspectrum.Siliconisreflectivetolight aboveitsbandgap(below∼1100nm),andthetrappinglasershould emitatopticalwavelengthsabovethislimitinthecaseofsilicon asthetrappingsurface. Asnotedinthenextsection,biological viabilityisreducedathigherwavelengthsduetoincreasedwater absorption.Thus,materialstransparenttovisiblelightarepreferred over siliconfor bio-particlemanipulation. Forthefabricationof

Fig.7.(a)PhotographofthemoldfabricatedwithconventionalCNC-machine.(b)Photographofthemicro-machiningcenter,themoldandthechannels.

devicesforOPapplications,standardaforementionedmicrofabri- cationtechniquescanbeapplied.Well-knownprocessessuchas softlithographytofabricatePDMSandwetordryetchingtofabri- categlasschannelsarepopularamongothertechniquesduetoease ofuse.Alternatively,3Dlasermicromachiningandhotembossing arealsogoodcandidatestoformrigidpolymericchannels.

One alternative method tofabricate the microfluidic device istousemechanicalmicromachining(i.e.CNC-machining)either for direct substratemanufacturing or forthe fabricationof the mold.For directsubstrate manufacturing,thelimitsofthepro- cessisconstrainedbythesizeofthemillingtoolwhichmaylead tounsatisfactoryend-productformicrofluidicapplications.How- ever,for the fabrication ofthe mold,the process is limited by thexyz-accuracy ofthe tool-positionerof a CNC-machine since the negative of the microfluidic structure is fabricated as the mold.Withtoday’s technology, by usingmagnetic bearings for theirpositioningsystems,thexyz-accuracyofaconventionalCNC- machinesarearound5␮m.Therefore, amoldcanbefabricated usingmechanicalmachiningwithincoupleofhourswithoutany need for clean-room equipment within the desirable accuracy limitsformicrofluidicdevices.Moreover,CNC-machiningcangen- erate3Dstructureswithoutanydifficulty.Commonmoldmaterials formold-basedtechniquesaresilicon(quartz/glass),SU-8photo- resist,polymerbasedmaterials(e.g.plexiglas)oranymetalbased materials(titanium,stainlesssteel,etc.).Polymer-andmetal-based mold materials are superior over silicon or photo-resist based moldmaterialsintermsofdurabilityandrobustness.Inthecase of mechanical micro-machining, any of thesematerials can be selected.Machinability,costandtheexpectedlife-spanofthemold aretheimportantparameterswhichneedtobeconsideredduring theselectionofthemoldmaterial.Anotherimportantparameter istheexpectedlifeofthemold.Consideringtheuseofthemold toproducemorethan7,000–10,0000parts,metal-basedmaterials arethebestchoice.However,usingmetal-basedmaterialscomes withaprice.Machiningofmetal-basedmaterialsiscostlydueto thereducedtoollifeandincreasedmachiningtime.Ontheother hand,machiningofpolymerbasedmaterialsislessproblematicin termsoftoollifeandmachiningtime,yetthemoldstillcanbeused formanytimes.

TheaccuracyoftheCNC-machiningcanbefurtherimproved bytheintroductionofspeciallydesigned micro-machiningcen- ters.Thesemicro-machiningcenterscanoperatewithanimproved spindlespeed which leadstoa superior surface finish withan

improvedxyz-accuracy. WithinBilkentUniversityMicro System DesignandManufacturingCenter,oneconventionalCNCmachine withmagneticbearingsandonecustom-mademicro-machining centerisavailableandusedforthefabricationofthemoldsofthe variousmicrofluidicdevices[103,104].Anexamplemoldfabricated withconventionalCNC-machine,andamoldfabricatedwiththe micro-machiningcentercanbeseeninFig.7.Thefabricationof metalelectrodeswithCNCmachiningisalsopossiblewhichleadsto 3Dmetalelectrodes.Aninitialattempthasbeenmadeforthefabri- cationofaneDEPdevicewith3Delectrodes.Amicrochannelwitha widthof100␮mandaheightof100␮mwasfabricated.Thedevice andthemoldcanbeseeninFig.8.Totalmachiningtimeforthe moldandtheelectrodestookapproximately180min.Theembed- dedelectrodeswerealsoremovedfromthedeviceandreusedfor thefabricationofaduplicatedevicewithoutanyproblems.

Fig.8.TheDEP-basedmicrofluidicdevicewith3Delectrodesfabricatedbymachin- ing:(a)themoldtogetherwiththeelectrodes,(b)theassembleddevice.