Microfluidic bio-particle manipulation for biotechnology

ContentslistsavailableatScienceDirect

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

_,

_Mehmet

_Bülent

_Özer

_,

_Mehmet

_Ertu˘grul

_Solmaz

a_{MechanicalEngineeringDepartment,Microfluidics&Lab-on-a-ChipResearchGroup, ˙IhsanDo˜gramacıBilkentUniversity,Ankara06800,Turkey} b_{DepartmentofMechanicalEngineering,TOBBUniversityofEconomicsandTechnology,Ankara06560,Turkey}

c_{DepartmentofElectricalandElectronicsEngineering, ˙IzmirKatipC¸elebiUniversity, ˙Izmir35620,Turkey} d_{NationalNanotechnologyResearchCenter,Ankara06800,Turkey}

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r

t

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c

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Articlehistory: Received23March2014

Receivedinrevisedform8July2014 Accepted14July2014

Availableonline21July2014 Keywords: Biomedical Bioprocessdesign Bioseparations Fluidmechanics Microfluidics Bio-particlemanipulation

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Microfluidicsandlab-on-a-chiptechnologyoffersuniqueadvantagesforthenextgenerationdevices fordiagnostictherapeuticapplications.Forchemical,biologicalandbiomedicalanalysisinmicrofluidic systems,therearesomefundamentaloperationssuchasseparation,focusing,filtering,concentration, trapping,detection,sorting,counting,washing,lysisofbio-particles,andPCR-likereactions.The combi-nationoftheseoperationsledtothecompleteanalysissystemsforspecificapplications.Manipulationof thebio-particlesisthekeyingredientfortheseapplications.Therefore,microfluidicbio-particle manip-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]since1980sledtotheusageofdevices hav-ingthedimensionsofmicrometersandnanometersinmanyfields. Thistrendhashelpedmicrofluidics,whichistheflowphysicsat micro scale,becomean activeresearchareaat theintersection ofchemistry,physics,biologyandengineering.Thisintersection eliminatedtheboundariesbetweenthesedisciplines.The elimi-nationoftheseboundarieshasposedmanychallengesandnew directionsfororganizationsofeducationandresearch.Oneofthe importantchallengesistherapiddevelopmentofbiochips, minia-turizedanalysissystemsorlab-on-a-chip(LOC)deviceswhichare microfluidicplatformsonwhichonecanhandlechemicaland bio-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,hundredsofdifferent reac-tionsand/oranalysescanbeperformedatthesametimethrough hundredsofparallelmicrochannels.Originallyitwasthoughtthat themostsignificantbenefitoftheseLOCdeviceswouldhavebeen theanalyticalimprovementsassociatedwiththescalingdownof thesize.Furtherdevelopmentsrevealedothersignificant advan-tagessuchas:(i)smallamountofsample(inthenanotopicoliter range,openingthedoortothepossibilityofanalyzingcomponents fromsinglecells),(ii)smallamountofreagents,(iii)veryshort reac-tionandanalysistimecomparedtobench-topcounterparts,(iv) reducedmanufacturingcosts,(v)increasedautomation,(vi)high portability,and(vii)opportunityformassivelyparallelchemical analyseseitheronthesameormultiplesamples[3].

Forchemical,biologicalandbiomedicalanalysesin microflu-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 ledtothe com-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 geometryortopologychangestomanipulatethemotionof par-ticles.Ontheotherhand,activesystemsutilizeanexternalforce fieldsuchaselectric,acoustic,magneticandoptictomanipulatethe motionofparticles.Thesemethodscanalsobecategorizedas label-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) require cell-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(whichwillbediscussedinthefollowing sub-section)otherthandragforcegeneratedontheparticlecomeinto

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

Re= UmaxDh  , Rep= Umaxd2 D_h =Re



2

whereUmax isthemaximumvelocity inthemicrochannel,is

thefluiddensity,isthedynamicfluidviscosity,distheparticle diameter,andDhisthehydraulicdiameterofthechannel.

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,asizedependentlateral displace-ment of bio-particles canalso beachieved, which is knownas deterministiclateraldisplacement(DLD)(seeFig1a)[16–20].DLD canbeutilizedforbio-particleseparation,sortingandfocusing.The presenceofslantedoranisotropicobstacleswithinthe microchan-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,sortingand focus-ing[27–31].

TheStoke’sflowregimeisvaliduptoRe∼1.WhenRereaches unityandbeyond,theinertialeffectsbecomesignificantand mod-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 secondary rota-tionalflowbeginstobeobserveddue totheinertiaofthefluid whichisknownasDeanflow.Thedimensionlessnumberswhich characterizesthissecondaryflowaretheDeannumber(De)and thecurvatureratio(ı)[15,32]:

De=Re



1/2

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,sincethetrajectoryoftheparticleswithina microflu-idicchannelmayalsopossessshapedependencedependingonthe channelgeometryandcharacteristicsofflow.Morespecifically,the flowofasphericalparticleandnon-sphericalparticlemaydiffer. Separationandsortingofsphericalandnon-sphericalparticlesis importantforclinicalapplicationssuchasseparationofyeastcells atdifferentcellstageandseparationofparasitesformblood.More recently,someresearcheffortshavebeenfocusedonthe imple-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-conducting parti-clesandcanbegeneratedeitherbyusingDCoralternatingcurrent (AC)field.

TheDEPforceonasphericalparticlesubjectedtoaDCfieldofE

canbewrittenas[8]:

FDEP=2εmfCMR3

∇

∇





, (4)

whereEistheelectricfieldvector,εmistheabsolutepermittivity

ofthesuspendingmedium,andRistheparticleradius.fCMisthe

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 (whichisinthedirectionofminimaofthegradientofthe elec-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

∇

whereErmsistheroot-mean-squareoftheAC-field,Re[fCM]isthe

realpartoftheClausius-Mossottifactorwhichisdefinedas fCM(˜εp, ˜εm)=

˜εp− ˜εm

˜εp+2˜εm

, (7)

where ˜ε isthecomplexpermittivityanddefinedas ˜ε=ε−j



ω



. (8)

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

FDEP(t)=2εmRe[fCM]R3

∇



E2_rms,i

∇



, (9)

whereϕisthephaseoftheAC-field.Subscriptireferstoeach com-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.Thefrequencyatwhichthis tran-sitionoccurs(i.e.thefrequencyatwhichDEPforcebecomeszero) iscalledthecross-overfrequency.Actually,theremayexist multi-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.Sincethebiologicalbasisofthe dielec-tricsignatureofthebio-particlesisnotwell-known,theprediction ofthedielectrophoreticmotionofthebio-particlesinanelectric fieldis notstraightforward.For EKmanipulation,both negative andpositiveforcescanbegeneratedwithdifferentconfiguration oftheelectrodesand/orthemicrofluidicchannelstructures,and switchingthepolarityand/orthefrequencyoftheelectricfield.In additiontothat,DEPhasafavorablescalingeffectwhichmakes itperfectcandidateforthemanipulationofmicro/nano-sized par-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-DEP appli-cations.However,specialcareisneededsincethehighelectrical voltagemayleadtoaseriousJouleheatingeffectinsidethe chan-nel.Thisseveretemperatureincreaseinside thechanneldueto Jouleheatingmayleadtoabubbleformationwhichcanseverely interferewiththeoperationofthedevice[42].Ontheotherhand, duetotheabsenceoftheelectrodesinsidethedevice,iDEPdevices arerobust,chemicallyinertanddonotrequireany photolithogra-phy,thin-filmdepositionandlift-offand/oretchingforelectrode fabrication.

2.2.2. Electrode-basedDEP(eDEP)

Thenon-uniformelectricfieldcanbegeneratedbymeansofthe speciallydesignedmicroelectrodearrays(i.e.interiorelectrodes) patternedwithinthemicrochannels.Toavoidtheadverseeffects ofDCfieldsuchasmigrationofchargedparticlestowardsthe elec-trodes,ACfieldisappliedforeDEPapplications.Mostofthetime, embeddedinternal electrodesareplanar(2D) (i.e.heightofthe electrodesareintheorderofhundrednanometers),andare fabri-catedwithinthedevicebymeansofcomplex,timeconsumingand relativelyexpensivemanufacturingtechniquessuchasthin-film deposition,sputtering,chemicalvapordeposition,etc.Moreover, foulingoftheelectrodesmaydistorttheoperationofthedevice whenworkingwithbio-particles[43].However,withan appropri-atedesign(i.e.closelyspacedelectrodes),operatingvoltagecanbe lowered(whichpreventsanysufferingfromJouleheating)foreDEP applications.Moreover,lowvoltagessimplifytheequipmentand circuitry.ForeDEPapplications,theinterfacialeffectsmayoccur attheinterfacebetweenthefluidmediumandtheelectrode sur-face,andmayleadtoadverseeffectssuchaselectrodepolarization, localheatingaroundtheelectrodes(whichmayresultinAC elec-troconvection),bubbleformationanddissolutionoftheelectrodes

[8].Therefore,someconstraintsneedtobeconsideredinthedesign ofsuchsystems.

2.3. Acoustic-based(ACT)

Theuseofultrasonicstandingwavesforbio-particle manipu-lationreliesonthecreationofultrasonicstandingwaveswithina channel.Thegenerationofacousticradiationforcehasbeen stud-iedforalongtime.Theformulationforacousticradiationforceon inelastic[44]andelasticsphereswasderived[45]andlater general-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:

Frad=−

∇

whereUradis theradiation potential.Under theassumptions of

particlesbeingsmallwithrespecttothewavelengthofthe acous-ticwavesandnotconsideringacousticwavescatteringfromother particles(i.e.smallparticleswithlowconcentration),theacoustic radiationforcecanbeobtainedas:

Urad= 4 3 R 3





Eq.(12)containsthevariablesrelatedtotheacousticproperties ofthefluidmediumaswellastheparticlestobemanipulated. pinandvinaretheincidentacousticpressureandacousticparticle

velocity,andfandcfindicatesthedensityandthespeedofsound

ofthesuspensionfluid,respectively.pandcpindicatesthedensity

andthespeedofsoundofparticles,Ristheradiusoftheparticle. Theaboveexpressionsaretrueforanyacousticfield.Forthecase ofanacousticstandingwavewhereacousticpressureismaximum, theparticlevelocityisminimumatthechannelwalls.Thefollowing

Fig.2.Arepresentationofacousticbasedbio-particle(a)washingand(b)separation. equationshowstheacousticradiationforceduetoa1Dstanding

waveasfollows:

Frad=4R2(kR)Eac sin(2ky)ˆj, (13)

wherekisthewavenumber,yisthelocationdirectionalongwhich theacousticpressurewavechanges,andistheacoustophoretic contrastfactorwhichcreatesdifferentamountofforcingon parti-clesduetotheiracousticproperties

=p+2/3(p−f) 2p−f − fc2_f 3pc2p , Eac= 1 4fu 2 y· (14)

ThesignofEq.(14)alsodeterminesthedirectionofthe parti-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.Thedifferenceinthe mag-nitudeof theacousticradiationforcecausestheparticles tobe manipulatedintothedifferentlocationswithinthemicrochannel basedontheirproperties.Propertydependentparticle manipula-tioncanbeexploitedfordifferentapplicationsinbiotechnology. Bio-particlewashingisoneoftheseapplicationsinwhichdeflection ofbio-particlesisusedtomovethebio-particlesfromonecarrying mediumtoanother.Forthistooccur,thereneedtobetwodifferent typesof carrying medium(buffer) flowing inthe microchannel andtheReynoldsnumberoftheflowneedstobelowtosatisfy

minimalmixingofthetwobuffersolutions.Theideabehindthe bio-particlewashingistomovetheparticlesfromonebufferto anotherthroughtheuseofacousticradiationforceinducedbythe ultrasonicstandingwavesinsidethemicrofluidicchannel.The pro-cesscanbeseeninFig.2(a).Ultrasonicwavescanalsobeutilized totrapbio-particles(acoustictrapping)tocertainlocations(nodal planes)ofamicrochannel.Theacousticradiationforcemaybeused toovercomethedragforceonthebio-particlesandholdthem sta-tionary(i.e.trappedparticles).Thesameprinciplecanbeusedto cleanthemediumfrombio-particlesorincreasethenumber con-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 particleswillreachthecenterchannelfirstwheretheycanbe chan-neledoutfromthecenter,whereasthesmallerdiameterparticles canbechanneledoutfromlocationsthatareclosertothechannel sidewalls.ThisprocessisschematicallyshowninFig.2(b). 2.4. Magnetic-based(MG)

Magneticfieldcanbeusedasanexternalforcetomanipulate bio-particles.Theforceappliedbythemagneticfieldonaparticle canbewrittenas[48]:

FMG=V

p−m

0

Fig.3.Theprinciplestepsofimmunomagneticbio-particleseparation.

whereprepresentsmagneticsusceptibilityoftheparticle,mis

themagneticsusceptibilityofthemedium,0isthemagnetic

per-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-particlesaresuspendedina param-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]and biol-ogy[51]haveshownitstruepotential.Thescientificcommunity hassincebeenusingthetermopticaltweezerstoidentifytheuseof opticalforcesasameanstomanipulatenanometerto micrometer-sizedobjects.Someofthesignificantachievementsusingoptical trapsarestudyingmolecularmotorswithsubnanometerresolution

[52],fundamentalpropertiesofcolloidsandinterfacescience[53], mechanicalpropertiesoflivingcells[54],andpolymerelasticity

[55].Theabilitytopreciselycontrolsmallobjectswithno mechan-icalcontactisdefinitelyadvantageousformicrofluidicapplications. Recentadvancesonmicrofluidicdevicefabricationbroughta dif-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,receivesthe inci-dentphotons,whicharescatteredand/orabsorbed.Scatteringof incidentphotonscreatesamomentumtransfer,hencetheoptical forcecomponent.Thephysicalprinciplebehindopticaltrapscan beexplainedbythebalanceoftwoforces:(i)scatteringforcesin

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

thedirectionofpropagationand(ii)gradientforcesinthe direc-tionofopticalfieldgradient.Scatteringforcegeneratesradiation pressurethat pushesthedielectricparticleawayfromthelight source.Gradientforce,ontheotherhand,actstheoppositeway andattractstheparticletowardsthepeakspatiallightintensity. Inthecaseoftightlyfocusedbeam,stabletrappingoccursifthe gradientforceexceedsthescatteringforce.Trappingdependson factorslikeparticlesize,refractiveindex,laserpowerandspatial characteristics.Theoreticaltreatmentofopticaltrapping catego-rizestheforcesonparticlesdependingontheirsizes.Thecondition wherethesizeoftheparticleismuchlargerthanthewavelength oflight(d)iscalledtheMieregime.Inthisregime,theforces actingonaparticlecanbecomputedbyRayopticsprinciples,i.e. reflectionand refraction. Reflection and refraction onthe front andbacksurfacescreatesperpendicularmomentumchangeona sphereasseeinFig.5(a).Inthefigure,adashedcirclerepresents crosssectionofthesphereandwaschosenfordemonstration pur-poses.

AssumingaGaussianintensityprofile,therayclosertobeam axiscreatesmorepointforcethantheraysituatedfurtheraway. Thenetforcepullstheobjecttowardsthebeamaxis,andpushesthe objectduetoscattering.Gradientforceonlydominatesandpulls theobjectwhenthereisasteepfieldgradientachievedbytightly focusingthebeam.InMieregime,theforceactingonasphereat anincidenceangleof andrefractionangleofˇisduetoasingle rayofpowerPis(n1QP/c).Therefractiveindexofthemedium

sur-roundingtheparticleisn1andtheparticlerefractiveindexisn2.Q

describestheamountofmomentumtransferatthefrontandback surfacesformultiplebounces,andisafunctionofFresnel reflec-tion(R)andtransmission(T)coefficients.Qvalueforalltherays actingonacircleweregivenbyAshkin[56],andthetotal scatter-ingandgradientforcesactingonadielectricparticleweregivenby Roosenandcoworkers[57].ThecircleonFig.5(a)canbetreatedas theintersectionofaplaneandasphereonFig.5(b),andthetotal scatteringandgradientforcescanbeintegratedovereverypoint onthesphereas:

Fs=−

Rcos2 +1−T2cos2( −ˇ)+Rcos2 1+R2_+2Rcos2ˇ

sin 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= Ion1 c 1285_r6 34

2

∇

wheremistheindexcontrastratio,n2/n1,andIoistheintensity

oflight.Thescatteringforceisdependentonthelightintensity, theparticlesize,thewavelengthofthelight,andrefractiveindex contrast.Thegradientforceisdependentonthespatialvariation ofelectricfield,theparticlesizeandtherefractiveindex.Unlike scattering,gradientforcescaleswiththethirdpowerofparticle size.Hence,itiseasiertoachievetrappingforasmallerparticle thanalargeparticle.

Sincetheopticaltrappingtheoryissizeandgeometry depend-ent, researchers depend on empirical determination of optical forces.Theprocedureiscalledopticalforcecalibration(standard methodswiththeiradvantagesanddisadvantagescanbefound elsewhere[13]).Themainobjective oftheopticalforce calibra-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, accuratemeasurementsofdisplacementarecrucialforthe calcu-lationofappliedforceandeitheraquadrantphotodetectororvideo trackingisnecessary.

3. Assessmentofthemethods

3.1. Samplepreparationandselectivity

TheselectivityoftheHDmethodsisbasedonbio-particlesize anddeformability.Ifthereisanappreciablesizedifferenceamong bio-particles,theycanbemanipulatedtodifferentlocationswithin themicrofluidicnetwork.Onetypicalaspectofbio-particlesisthat theydonotpossessspecificsize,insteadtheypossesasize dis-tribution.Ifthere isasizeoverlapfortwo differentbio-particle populations,theselectivityoftheHD methodsdiminishes.One majoradvantageofHDmethodsisthattheydonotneedany label-ing.Althoughlabelingisnotnecessary,samplepreparationmay stillbeneeded,sincetheconcentrationoftheparticlesmayaffect theflowfield,andresultinaundesiredflowpatterns.For exam-pleinthecaseofwholeblood,dilutionofthewholebloodmaybe usefuldependingontheapplication.

TheselectivityoftheEKapplicationsrelyonthedielectric prop-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 responsealsodependsonpermittivityofthemediumandthe bio-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.Therearesomeproposedmethodsinthe lit-eratureforthemeasurementofDEPresponse[8,59,60],however theprocessisnotstraightforward.Astand-alonemicrofluidic plat-formwithsomespecialcircuitryand/oropticaldetectionsystemis neededtodeterminetheDEPresponseofabio-particlepopulation. Ifthischallengeishandled,theselectivityofDEPcanbeutilizedto manipulatedifferentbio-particles.

Thepresence ofthe electricfield in EK applicationsinduces Jouleheatingwithinthefluid.Dependingontheconductivityof thebuffersolutionandthestrengthoftheelectricfield,theJoule heatingeffectcanbesevere,andcausedeteriorationofthesystem performance[8,42].Moreover,Jouleheatingmayalsocause unde-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)andlabelingisnecessaryforacoustic manip-ulation,labelingofparticleswereutilizedtohavebetterselectivity in a very limited number of studies where bio-particles were taggedwithbeadswhichhavenegativeacousticcontrastfactors

[62].Thiswaywhentheacousticfieldwasemployed,thelabeled bio-particlescanmoveintheoppositedirectionoftheremaining particles.Moreover,thebio-particlemaybelabeledwith fluores-centdyetoimprovethevisibilityoftheparticlesmovement[63].

Considering the MG applications, both the diamagnetic and immunomagneticmanipulationmethodsrequiresample prepara-tionpriortothebio-particlemanipulation.Inimmunomagnetic bio-particlemanipulation, theparticles need toincubated with thebio-particlesamplesothat thetargetcells arelabeled. The labelingoccurs dueto biochemicalreactionsbetween the anti-gensandantibodiesonthebio-particlesandthemagneticparticles. The duration of the incubation is in between 10 and 30min dependingonthemixing,temperatureandbead-cell concentra-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-particlesifinfusionintoaliveorganismistobe fol-lowed.Duetothesample preparationand postprocessing,MG methodsrequirerelativelycomplexprocedures.Therearesome examplesofmicroscalesystemswhichperformtheincubationstep in-line.Onesuchsystemusessix rotatingmagnets[66] tomix micro-particlessothatincubationwiththebio-particlescanoccur onthemicrofluidicdevice.Aftertaggingthebio-particles,the incu-batedbio-particlesaresenttothemicrofluidicdeviceandcanbe positivelyselectedbymeansofamagneticfield.Theprocessof immunomagneticseparation is generallyused forseparation of rarebio-particlesratherthanfocusinglargenumberofbio-particles sincetheimmunomagnetictaggingofbio-particlesisnot econom-icallyfeasibleandistimeconsuminginlargenumbers.

In the case of OP applications, it is important to know the physicalcharacteristicsofthefluidandthetrappedobjectduring opticaltrappingexperiments.Refractiveindexchangesaccording tomediumwhichthebio-particleisinandthebio-particletype. Forinstance,therefractiveindexofthecellmediamaychange dur-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 labeledandusedasthediagnosticmarkerduringoptical manip-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).Thisleadstolowthroughputformany clin-icalapplications.Asanexample,ithasbeenshownthatinertial effectsdecreasethesortingefficiencyforhydrophoresis applica-tions[26].Atthispoint,inertialmicrofluidicsoffersapromising perspectively.InertialmicrofluidicsutilizesthehighRe characteris-ticoftheflow;therefore,inherentlyflow-ratesarehighintheorder ofml/hwhichleadstohighthroughput(>∼106_{particles/min),and}

thismakesinertialmicrofluidicssuitableformanyclinical appli-cations.However,thepredictionoftheflowfieldandtheparticle motioninthisregimeisalsochallenging.Thethroughputof sev-eralHDapplicationcanbeseeninFig.6.Thehighestthroughput forHDmanipulationisaround∼1011_{particles/min,andbelongsto}

anapplicationinwhichtheplasmaisseparatedfromwholeblood ratherthanadifferentiationofbio-particles.

Thesuccessfulmanipulationofbio-particlesinEKapplications dependsonthebalancebetweenthedragforceandtheEKforces. AnincreaseindragforcerequiresanincreaseinEKforceandthis indicatestheuseofhigherelectricalfieldstrength.Sincehigher electricfieldsarenotdesirableduetoJouleheatingphenomena

[42],thedragforceandtheflow-ratearelimitedforthese applica-tions.InthecaseofeDEPapplications,theelectricfieldisanissue onlywithinthevicinityoftheelectrodes;therefore,higherelectric fieldscanbetolerated.However,onemajordisadvantageofthe eDEPdeviceswithplanarelectrodesisthattheelectricfield gradi-entdecreasesrapidlyastheparticlesflowawayfromtheelectrodes. Therefore,aneffectivemanipulationregionisgeneratedwithinthe vicinityoftheelectrodeswhichreducestheeffectivenessofthe manipulation.Iftheelectrodesaredepositedonthebottomwall ofthemicrofluidicchannel,thentheheightofthechannelneeds tobelimited.Thislimitationcanbesolvedbyintroducing3D elec-trodeswithinthedevice.Manyresearchershaverecentlyrealized thisaspectandproposeddifferentfabricationtechniquesto fabri-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-particle through-puts.Thestudieswithhighthroughputsaregenerallystudieswhich processwholebloodsamples.Thenormalbloodcountofamaleis approximately5×109_{RBCsinamilliliterofblood.Forthe}

appli-cationswhichdonotincludefractionationofdifferentcellgroups fromeachothersuchasseparationofplasmafromwholeblood, thethroughputreachesashighas1011_{particles/min[69].}

ThroughputofMGmanipulationmethodsisalsopromising.The throughputofseveralMGapplicationscanbeseeninFig.6.The largestofthetwothroughputvalues[70,71]belongtoasystem wheretheborediameterofthechannelisroughly8mm.Itmay bearguedthatthisdiameteristoolargeforamicrofluidicsystem. Mostofthehigherflowratesbelongtosystemswith immunomag-neticbio-particlemanipulationsystems,anddiamagneticparticle manipulationsystemshavelowerthroughputs.However,itshould benotedthatthethroughputsarepurelybasedonthetimethat cellsspendinthemicrofluidiccircuit.Intheimmunomagnetic par-ticlemanipulationcases,asignificantportionofthetimeisspent on thepre-processing of bio-particles (considering the incuba-tiontimefortaggingofbio-particleswiththemagneticparticles), hencethethroughputsmaynotberepresentativeofthetotaltime requiredforthebio-particlestobeprocessed.Generally,flow veloc-itiesinthemicrochannelsareintheorderofmm/sorless.

ForOPapplications,activemicrofluidiccellsorterscompared toitspassivecounterpartsuseinformationsuchasfluorescence, sizeandshapebymeansofphotodetectorsorimageprocessing to dynamically steer thebio-particles to desired outlets.These systems canoperatequitefastbasedonthetype offorceused formanipulation. Forinstance,activesorterseitherutilize opti-cal forces for grabbing or deflection of bio-particles. Grabbing anddeflectionoccurwhenthegradientorscatteringforce dom-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](thethroughputofseveralOP applica-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, certainlengthisrequiredtoachievetherequiredlateral displace-mentofbio-particles.Toutilizeanycirculationwithintheflowfield, itistypicaltointroduceanexpansion/contractionsectionsand/or poststructureswithintheflow field.Morerecently,the expan-sion/contractionintheheightdirectionhasalsobeenutilizedfor bothsortingandfocusing[38].ToutilizetheDeanflowforparticle manipulation,spiralandserpentinechannelsarerequired,andthe radiusofcurvatureofthechannelsiscritical[32].For hydrophore-sisapplications,expansions/contractionsintheheightdirections arenecessary.Thesizeofthesecontractionsarecritical,and cer-taindesignproceduresneedtobefolloweddependingonthesizeof thebio-particleofinterest.Typicallytheminimumchannelheight inthecontractedpartneedsbelargerthanthebio-particlesizeto avoidclogging,andlessthantwotimestheparticlesizefor suc-cessfulmanipulation.Onecommonpoint aboutthemicrofluidic channelnetworkwithpoststructuresisthattheheightofthe chan-nelislimitedconsideringthelimitoftheaspectratioduetothe fabricationconstraints.

ForEKapplicationswithoutinternalelectrodes,atypical chan-nelwidthisintheorderof100␮mtogetherwithsomespecially designedstructures.Theheightofthemicrochannelisnotcritical

aslongasthefabricationisnotproblematic.ForeDEPapplications withinternalelectrodes,theheightiscriticalif3Delectrodesare notused.Typically,channelheightvariesbetween20and50␮m. ForeDEPapplicationswith3Delectrodes,microchannelstructures withlargerheightispossible(typically20–100_␮m).Inthese appli-cations,thelimitationsfortheheightcomesfromthefabrication stepoftheelectrodes.

Channelgeometryisquiteimportantforsuccessful implemen-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 (piezoelectricmaterialssurfacenotalignedwiththeacoustic radia-tionforcedirection),thechannelwidthalsoneedstobeselectedas thehalfwavelengthoftheacousticwavesinthesuspensionfluid. However,ifthelayeredconfigurationispreferred,thenthechannel widthcanbeseveralwavelengthsoftheacousticwavesinthe sus-pensionmedium.Notonlythechannelwidth,butalsothewidthof thedeviceneedstobeselectedasanintegermultipleoftheacoustic wavelength.Thedepthofthechannelisgenerallyselected signifi-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.Althoughpreferredgeometryisa rectan-gularcross-sectioned microchannel,thecapillarytubescanalso beutilized[75,76].Ifpermanentmagnetsareused,generallythe polesofeachmagnetisbroughttothevicinityofthechannelwalls. Thecloserthepolesaretoeachother,thestrongerthegradientof themagneticfieldis,the(B·

∇

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,andanindex match-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].Photolithographyhasgoodabilityto manu-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,therestofthe fab-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,the invest-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 whicharedesignedandlocatedstrategicallywithinthe microflu-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].Thechoiceofmaterialforthe transver-salconfiguration shouldhave highacoustic impedancesuchas silicon,whereasthelayeredconfigurationmaybemanufactured fromawiderangeofmaterialsrangingfromdifferentpolymers tosteel.Thematerialofchoiceforacoustofluidicdevices,whichis excitedwithsurfaceacousticwaves,ispolymerswithlow acous-ticimpedanceandwithgoodshearwavecarryingcapacitysuchas PDMS[87–89].Forthesilicon-baseddevices,fabricationthrough standard photolithographyand wetor dryetchingarecommon approaches[61,94,69,95,47,96].Inthecaseofpolymersand partic-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,microfluidicchannelswithoptically trans-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,standardaforementioned microfabri-cationtechniquescanbeapplied.Well-knownprocessessuchas softlithographytofabricatePDMSandwetordryetchingto fabri-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,thelimitsofthe pro-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-accuracyofaconventional CNC-machinesarearound5␮m.Therefore, amoldcanbefabricated usingmechanicalmachiningwithincoupleofhourswithoutany need for clean-room equipment within the desirable accuracy limitsformicrofluidicdevices.Moreover,CNC-machiningcan gen-erate3Dstructureswithoutanydifficulty.Commonmoldmaterials formold-basedtechniquesaresilicon(quartz/glass),SU-8 photo-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-machining cen-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.Aninitialattempthasbeenmadeforthe fabri-cationofaneDEPdevicewith3Delectrodes.Amicrochannelwitha widthof100␮mandaheightof100␮mwasfabricated.Thedevice andthemoldcanbeseeninFig.8.Totalmachiningtimeforthe moldandtheelectrodestookapproximately180min.The embed-dedelectrodeswerealsoremovedfromthedeviceandreusedfor thefabricationofaduplicatedevicewithoutanyproblems.

Fig.8.TheDEP-basedmicrofluidicdevicewith3Delectrodesfabricatedby machin-ing:(a)themoldtogetherwiththeelectrodes,(b)theassembleddevice.

CNC-machining is rather an alternative method not a com-petitor for thelithography-based techniques. Especiallyfor the mold-basedfabrication,itoffersuniqueadvantages:(i)post struc-tureswithaspectratioaround1.0canbeintroducedwithoutany difficulty,(ii)the3Dgeometrieswithvaryingchannellengthcan befabricatedeasily,(iii)theangleofthemicrochannelsidewallon themoldandtheheightofthemicrochannelcanbecontrolled pre-ciselywithintheaccuracylimitofthemachiningcenter,anddeep channelscaneasilybefabricated.Ontheotherhand,structures withsharpcornersoranobstaclewithcornersisproblematicsince certainfilletsalwaysexistdependingontheradiusofthetool. 3.5. Hardware

ForHDapplications,theimportantparameteristheflowfield. Flowfieldneedstobecontrolledthroughprecisepumpsandvalves. Formicrofluidicapplications,theuseofsyringeand/orperistaltic pumpsare common.Duetothesuperior flowcontrol andease ofusesyringepumpsaremore popularin microfluidic applica-tionsespeciallyforlowflowrates(∼1–100ml/min).Morerecently, LabSmithInc.(www.labsmith.com)hasintroducedmicrosyringe pumps(flow-ratesrangingbetween50nl/minand5ml/min), auto-matedvalvesandmicrofluidiccomponentsspeciallydesignedfor microfluidicapplications.

ForEPandiDEPapplications,DCelectricfieldistypicallyapplied throughoutthechannel,andtheflowisinducedbyelectro-osmosis. Dependingontheelectrodegeometryandflowrate,ahighvoltage sourcemaybenecessary.ForeDEPapplications,ACelectricfieldis preferredandtheappliedfrequencyrangesfromfewkHztotens ofMHzwithamplitudesfrom5to100Vpeak-to-peakdepending ontheelectrodegeometrywithinthemicrochannels.Application ofDCvoltagesrequireslessauxiliaryequipment(e.g.signal ampli-ficationequipmentoranRFamplifier)withrespecttoACalthough conventionalfunctiongeneratorsmaybeenoughformost appli-cations.Typically,usingACfrequencycomplicatestheDEPsetup. Moreover,thefrequencyofACfieldneedstobetunableowingto frequencydependentCMfactor.Eventhoughitisdesirabletohave DEPoperationwithasinglesetfrequency,especiallyfor commer-cialpoint-of-careproducts,frequencytuningmayberequiredin practice.Conventionalfunctiongeneratorstypicallyareequipped withsuchfeature.TheEKsystemsand applicationsinclude fre-quencydependentcomponentswithtwoormoreelectricalports, andtheelectricalpropertiesofcomponentsandmaterialsare char-acterizedeither usingLCR meters orimpedance analyzers. LCR metersareusuallypresetandusedatcertainfrequenciesofkHz range. Impedance analyzers are preferred due to theability to sweepinfrequencydomainfromfewkHztoRFrange.Impedance measurementscanalsobeappliedtoextractdielectricproperties ofbio-particleswhentheyarenearbytheinducedelectricpotential

[105].Accurate determinationof bio-particlesdielectric proper-tiesisanothercriticalrequirementforcommercializationofDEP devicessincebio-particlesofsimilarsizesmayexhibitpositiveor negativeDEPforcesatthesameoperationfrequency.Similarto impedanceanalysis,DEPaffinityseparationorcrossoverfrequency determinationallowsmeasurementoffrequencydependentDEP forceandidentificationoftwodielectricallydissimilarbio-particles (e.g.bloodcellsandcancercells)[106].Crossoverfrequency deter-minationmethodinvolveselectro-rotationandrequiresaswept frequencygeneratorandelectronicstoenableaccuratephase shif-tingofthegeneratedsignal.

For ACTapplications,a voltagesourceis necessarytocreate theacousticwaveandacousticradiationforce.Typically,a piezo-electric patchis used asan acousticwave source.Piezoelectric patchesareidealduetotheirlowcostandefficientradiationat ultrasonic frequencies. The piezoelectric patch attachedexcites thematerialwhich carriesthewavestothemediumwherethe

particlesaresuspendedin.Theuseofpiezoelectricceramic mate-rial(suchasPZ26)iscommonduetotheirhighvoltageabilities. The position of the piezoelectric material with respect to the devicedetermines which type ofconfiguration theresonatoris in. Thereare threemain configurationswherethepiezoelectric material can be placed against the chip which are transversal

[61,92,107,94],layered[82,108,85,109]andsurfaceacousticwave (SAW)configurations[87–89].Inthetransversalconfiguration,the piezoelectricmaterialisplacedonthedevicesuchthatthe stand-ingwaveispositionedalonganaxisperpendiculartothenormalof thepiezoelectricpatchsurface.Thelayeredconfigurationrequires thesurfacenormalofthepiezoelectricpatchtobealignedwith thedirectionalongwhichstandingwavesarepositioned.Surface acousticwaveconfigurationgeneratestheacousticradiationforce throughpropagationofsurfacewavesonthedevice.Toactuatethe piezoelectricmaterial,apoweramplifierthatcanoperateatradio frequenciesandultrasonicfrequencyrangesisrequired.Typically, thepowerrequiredforACTapplicationsisintheorderofcouple ofWatts.However,insomehighthroughputstudieswithPDMS materials,itcangoupto10W[97]whichrequiresanadditional externalcoolingofthepiezoelectricmaterialandthechannel.The useofaluminumblockasaheatsinkmaybeapplied[85,97,110].

ForMGapplications,theonlyhardwarerequiredisamechanism tocreatemagneticfieldgradientinsidethemicrochannel. Gener-ally,permanentmagnetsareusedtocreatetherequiredmagnetic field,andthechoiceforthepermanentmagnetmaterialisalmost alwaysNdFeB(Neodymiumironboron)magnets.Theuseof per-manentmagnetsratherthanelectro-magnetssimplifiesthedesign andeliminatestheneedforpower.Onthedownside,theuseof permanentmagnetsmakesitdifficulttocontrolthemagneticfield strength,anditdoesnotallowtheuseofswitchingfieldwhichmay addadditionalselectivitytotheprocess[65].Thepole configura-tionofthepermanentmagnetdependsonthetypeofmanipulation beingperformed(focusing,concentrationorseparation)aswellas thechoiceofmethodformanipulation(diamagneticor immuno-magnetic).Thestrengthofthemagneticfieldfluxisgenerallyinthe orderof0.1–0.2Tesla,butthetotalmagneticfluxcanbeashighas 1Tesla.Thedistancebetweenthemagnetpairscanrangefromtens of␮m[111]toseveralmm[75].Bio-particlescanalsobe manipu-latedusingthemagneticfieldinducedbythepermanentmagnet onaferromagneticwireplacedinsidethemicrochannel[112,113]. Additionally,electrodescanalsobeintroducedwithinthedevice toswitchthemagneticfield[65],andtoprovidehighresolution controloverpositioningofbio-particles[100].

Anopticaltweezersetupisusuallybuiltaroundamicroscope, andalltheelectronicandopticalelementsareintegrated. Com-mercialopticaltweezersetupsareavailableforpurchase(Thorlabs Inc.,www.thorlabs.de),yetmostsetupsarebuiltfromscratch,and customizedaccordingtotheneed.Handlingbiologicalspecimen requiresextraattentionduringselectionofthelaserdueto opti-caldamageand heatingeffects.Sincebiological specimenshow lowindexcontrastwithrespecttoitssurroundings,theamountof laserpowertotrapandmovesuchanobjectishigherthanthat ofapolystyrenebead.Thetypicallaserpowersrangefromafew milliWattstoaWatt,andabsorptionincreaseslinearlywithpower. Thechoiceoflaserwavelengthisevenmorecriticalforbiological objectsduetolowwaterandproteinabsorptionrequirement. Visi-blespectrum(400–700nm)hasthelowerwater,buthigherprotein absorption.Onthecontrary,wavelengthspectrumabove1250nm introducesmany ordersof magnitudehigher waterabsorption; therefore,aheatingproblemthatmaydamagethebiological spec-imencanoccur.Themostpreferredwavelengthsfortrappingare around800and1064nm.Laserdiodesatthesewavelengthswith opticalfiberends,notonlyprovideshighopticalpower,butalso hasaverygoodqualitysingle-modebeamprofilesuitablefor opti-caltrapping.TraditionalopticaltweezersrequireacleanGaussian