Sterilization of PMMA microfluidic chips by various techniques and investigation of material characteristics

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Sterilization

of

PMMA

microﬂuidic

chips

by

various

techniques

and

investigation

of

material

characteristics

Cansu

Yavuz

a

_,

_Samad

_Nadimi

_Bavil

_Oliaei

b

_,

_Barbaros

_Cetin

c

_,

_Ozlem

_{Yesil-Celiktas}

a,∗

a_Novel_Fluidic_Technologies_and_Applications_Group,_Department_of_{Bioengineering,}_Faculty_of_Engineering,_Ege_University,₃₅₁₀₀_Bornova,_Izmir,_Turkey b_Microsystem_Design_and_{Manufacturing}_Center,_Mechanical_Engineering_Department,_Ihsan_Dogramacı_Bilkent_University,₀₆₈₀₀_Ankara,_Turkey c_{Microﬂuidics}_&_{Lab-on-a-chip}_Research_Group,_Mechanical_Engineering_Department,_Ihsan_Dogramacı_Bilkent_University,₀₆₈₀₀_Ankara,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11February2015

Receivedinrevisedform28August2015 Accepted28August2015

Availableonline7September2015 Keywords: Microﬂuidicdevice Microchip PMMA Material Sterilization SupercriticalCO2

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b

s

t

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Thesterilizationofmicroﬂuidicchipsisavitalstepofthefabricationprocesspriortothecustomer useinbiomedicalapplications.Theaimofthisstudywastoanalyzetheinﬂuenceofdifferent steril-izationtechniquesandtocomparethecharacteristicsofthematerialbeforeandaftersterilizationof polymethylmethacrylate(PMMA)microchips.Forthis,supercriticalcarbondioxide(SC-CO2)alongwith

standardsterilizationmethodssuchasultraviolet(UV),heat(autoclaving),ethyleneoxide(EtO)and hydrogenperoxide(H2O2)wereapplied.ThetreatedmicrochipswereanalyzedbyScanningElectron

Microscopy,DifferentialScanningCalorimetry,FourierTransformInfraredSpectroscopyandLaser Scan-ningMicroscopyinordertoascertainanychangesinthechemicalstructureandsurfacemorphology.The optimumsterilizationparametersforSC-CO2wereelicitedas120bar,40◦Cand60minwhichprovided

completesterilityanddidnotalterthemainpropertiesofthepolymeralongwithEtOandH2O2

ster-ilizationsunlikeheatandUVtreatments.However,surfaceroughnessandmicrochannelproﬁleswere negativelyaffected.Althoughcompletesterilitywasachieved,eachprotocolhasitsownstrengthsand weaknesses.

1. Introduction

Inthepastdecade,theapplicationofmicroﬂuidictechnology hasgrownrapidlyinlifesciences[1,2].Recentdevelopmentsin microﬂuidicsrevealedsomefunctionsthatisincludedinsynthesis andanalysisofchemicalandbiologicalmaterialssuchasgenesand proteins,drugdelivery,medical diagnostics,cellculture, under-standingcell behavior and cell-handling[3,4].Microchips have severaladvantages suchas fast screening of parameters, rapid massandheattransfer,lowreagentconsumption,smalland con-trolledmicroenvironmentsandreproducibility[5].Thematerials usedforconstructionofmicroreactorsareofprimeimportanceand varybasedonthetargetedapplication.Polymethylmethacrylate

(PMMA)iscommerciallythemostimportantmemberofarange

ofacrylicmaterialsandusedprimarilyinplasticapplicationsdue toexcellentmechanicalandchemicalproperties[6].AsPMMAis disposable,biocompatible,transparent,impermeabletoair, effec-tivetofabricateandsuitableformassfabrication,itispreferredas

∗ Correspondingauthor.

E-mailaddresses:[email protected],[email protected]

(O.Yesil-Celiktas).

apolymericmaterialinthefabricationofmicroﬂuidicdevices[2,4]

andmedicalapplications[6].Applicationsofmicrofluidicsystems basedoncellandtissueculturearenowemergingas physiolog-ically relevant microenvironmentscan befabricatedby invitro cellculture.Microfluidic cellcultureposes elicitationofspecific sterilizationrequirements.Currentmethodsofsterilizationforthe

removalofmicroorganismsfrommedicaldevicesincludesteam

autoclaving,ethyleneoxidetreatment,UVandhydrogenperoxide treatment[7–9].However,thetechniqueshaveobviousdrawbacks andcannotbeappliedforsterilizationofsomematerialssuchas polymericmedicaldevicesandheat-sensitivebiomaterials[8–10]. Sterilization with supercritical carbon dioxide (SC-CO2) is

reportedasanalternativetechniqueforsterilizationofbiological pathogens[7].Carbondioxidehasmanyfeaturessuchaslow crit-icaltemperatureandpressure,isinexpensive,non-toxic,inertand

non-ﬂammable.Moreover,itcanbecompletelyremovedfromthe

materialthatisprocessedwithSC-CO2[11–13].Steamsterilization

isthemostcommontechniquebecauseofitslowcost, effective-ness,easinesstoprocessandnotproducingtoxiccompounds[14]. However,steamsterilizationoperatesat121◦Csoheat-sensitive materialswillbedamagedordestroyed.Therefore,UVand ethyl-eneoxidesterilizationcanbeappliedtoheat-labilematerials[10]. Ethyleneoxideisﬂammableandtoxic,ethyleneoxideresidueson

http://dx.doi.org/10.1016/j.supﬂu.2015.08.019

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Fig.1.InjectedpartsofPMMA(A)andthesinglechannelmicroﬂuidicchip(B).

thesurfacescancausehemolysisandbecomecarcinogen[7,10].In addition,thesterilizationperiodisverylong[8].Hydrogen per-oxideisnottoxic andit hasahighlymicrobicidaleffectiveness againstabroadspectrumofmicroorganisms[15].Hydrogen per-oxideplasmaproduceslargeamountsoffreeradicalsinorderto achievesterilization.Thesefreeradicalsmayadverselyaffectthe chemistryofthesterilizedmaterial[9].Thistechniqueissafefor thehumanbodyandharmlesstotheenvironment[8].UVradiation hastheDNAasmaintargetformicroorganismselimination[14].

Inthisstudy,supercriticalCO2sterilizationofPMMAmicrochip

wasinvestigatedandcomparedwiththoseofstandard steriliza-tiontechniques both in terms ofsterility andpossible changes in thecharacteristics of thepolymericmaterial. Scanning Elec-tron Microscopy(SEM), DifferentialScanning Calorimetry(DSC) andFourierTransformInfraredSpectroscopy(FTIR)wereusedto determinephysicalandchemicalpropertiesofmaterialafter ster-ilization.Although,various studies werereportedin regardsto terminalsterilizationofbacterialendospores[9,16]andmicrobial inactivationusinghighpressurecarbondioxide[17–19], steriliza-tionofamicroﬂuidicchipusingsupercriticalcarbondioxidehas notbeenpresentedbeforetothebestofourknowledge.

2. Materialsandmethods

2.1. Materials

TrypticSoyBroth(TSB)and ThioglycollateBroth(TGB)were

obtained from Sigma. Sterilization paper was purchased from

Tyvek rolland chemical indicator waspurchased formSterrad

for hydrogen peroxidetreatment. Ethanolwas purchased from

Merck.Nanopurewaterusedintheanalysiswaspreparedbyusing in-housenanopurewatersystem(SartoriusArium611,

Sartorius-Stedim, Gottingen, Germany). Sterilecabin and incubator were

usedforincubationundersterilconditions(Jauan-MSC12/ClassII, FormaseriesII-Hepaclass100respectively).Ethyleneoxide ster-ilizatorwasused(AxisAX-60&AX135series)andUVsterilizator

wasused(GOLDTERM,UV1679model).

2.2. Fabricationofmicroﬂuidicchips

ThePMMAmicrochipwasfabricatedusinginjectionmolding

process,wherethemoldwasfabricatedoutofblank(unmachined)

moldbyusingthehighprecisionCNCsystem(DeckhelMahoDMU

50)atBilkentUniversityMicroSystemDesignandManufacturing Center.Themoldmaterialwaschosenasthestainlessmoldsteel CK–50AISI1.1050.Four-toothcoatedcarbidetoolswerepreferred inthemachiningprocessanda5◦draftanglewasintroducedat theside-wallsofthemicrochannelsandthemoldcavityforthe easeofdemolding.Duringthemachiningofthemold,ﬁrstly,the

runner,gateandmoldcavitieswereroughlymachinedandthen

thebaseandside-wallsofthecavityweremachinedwith0.5mm tolerance.Afterthemachining,asurfaceﬁnishoperationwas per-formedbyusingagrindingmachineandagrindingpasteinorder toincreasethesurfacequality.Lengthsofthemicrochannelswere 20mmandeachhasawidthandadepthof240␮m.Evonik plex-iglas6N(PMMA-Acrylics)wasusedasthematerial.Theinjection wasperformedusingaplasticinjectionmoldingmachinewitha maximuminjectionpressureof90MPa[20].Theinjectedpartscan beseeninFig.1.

3. Sterilization

3.1. SupercriticalCO2sterilization

Supercritical CO2 sterilizationwascarriedout usingSFE-100

System(TharInstruments,Inc.,UK,2006).Microchipswerepacked insterilizedcartridgesandplacedintothereactionchamber. Oper-atingparametersforsterilizationweretemperature(25,40,50and 60◦C),pressure(70,100,120,150and250bar)andtime(30,45,

60and 90min).Sterilizationswereperformedunderautomated

controloftemperatureandpressure.Theprocesswasstartedby reachingthesettemperatureandpressurevalues.Flowratewas 10g/minforallsterilizationprocesses.Attheendofsterilization process,thevalveofCO2tubewassealedofffordepressurization

andreleasingCO2fromthesystemwasapproximately45min.

3.2. Heatsterilization

Heatsterilizationorautoclavingisarelativelysimpleprocess thatexposesthedevicetosaturatedsteamat121◦Cfor20minata pressureof115kPa.Theprocesskillsmicroorganismsbydestroying metabolicandstructuralcomponentsessentialtotheirreplication.

3.3. UVsterilization

For UV radiation, the microchipswere exposed to UV light

(254nm)for45minoneachside,totaling uptoatreatmentof 90min.Microreactorswereplacedintopetridishes.

3.4. Ethyleneoxidesterilization

Theethyleneoxide(EtO)sterilizationprocessutilizesEtOwhich hasbactericidal,sporicidaland virucidaleffects innucleicacids causing cell injury or death. In this study, microreactors were exposedtheEtOconcentrationof5mg/mlfor3hat55◦Cand aer-ationfor12h.

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Fig.2.Surfaceroughnessmeasurementsonthechannelsurface(A)andonthepartsurface(B)alongwithchannelproﬁlemeasurements(C).

3.5. Hydrogenperoxidesterilization

Hydrogenperoxidehasbactericidal,virucidal,sporicidal,and fungicidalproperties.Althoughmostmicrobialformsarekilledin lessthan1h,ittakeshourstoeliminatespores.Itshouldbestored inacoolplaceandprotectedfromthelight.Themicrochipswere treatedwithhydrogenperoxide(HP)for1handstoredatacool placeprotectedfromlightforabout48h.

3.6. Determinationofsterility

The sterility was determined by incubating the sterilized

microchipsintrypticsoybroth(TSB)andthioglycollatebroth(TGB) toassessthemicrobiologicalloadsattwodifferentincubation tem-peratures(27◦Cand37◦C)foradurationof7days.Sterilitytests wererepeatedthreetimes.

3.7. Characterization

ThesterilizedmicrochipswereanalyzedusingScanning Elec-tronMicroscopy(SEM),FourierTransformInfraredSpectroscopy

(FTIR) and Differential Scanning Calorimetry (DSC) in order to assessmicrochannelsterilityandpossibledeformationsinPMMA. 3.7.1. ScanningElectronMicroscopy(SEM)

Themicrochipswereviewedbyscanningelectronmicroscope (FEI,Quanta200F).Thesamplesweredriedundervacuum(1bar)at roomtemperatureandthensputter-coatedwitha10nmthickgold layerbeforeimaging(Gatan682,precisionetchingcoatingsystem). 3.7.2. DifferentialScanningCalorimetry(DSC)

Thermalbehavior ofthemicrochipswasmeasuredby differ-entialscanningcalorimetry(TAInstruments,Q2000),wherethe sampleswerepackedintoaluminumandDSCsamplepanswiththe lidtightlycrimped.Nitrogenwaspurgedataﬂowrateof50ml/min

andmeasurementsweremadeintemperaturerangebetween30

and250◦Cataheatingrateof10◦C/min.

3.7.3. FourierTransformInfraredSpectroscopy(FTIR)

Fourier transform infrared spectra of the microchips were

recordedfrom8000cm−1 to200cm−1 (Bruker VERTEX70 with

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3.7.4. Surfaceroughnessandproﬁlecharacteristics

Inordertoinvestigatetheeffectofdifferentsterilization

tech-niques onsurface quality and proﬁleof themicrochannels, 3D

topographyofthechannelswasobtainedusingKeyenceVK-X100

laserscanningmicroscope.Arithmeticroughnessaverage(Ra)was

usedasa measureofsurfaceroughnesswhich isdeﬁnedasthe

arithmeticaverageheightofsurfaceirregularities(peaksand val-leys)fromthemeanlinewithinthescanninglength.Averaging

wasperformedonthemeasuredsurfacetopographybothonthe

microchannelsurfaceand part(chip)surface. For the measure-mentsonthechannelsurface,20sampleswithascanninglength of1.25mmatthreedifferentlocationsweremeasuredasdepicted inFig.2A.Forthemeasurementsonthechipsurface,20samples withascanninglengthof1.25mmatfourdifferentlocationswere measuredasdepictedinFig.2B.Inordertoremovetheeffectof wavinessonmeasuredroughnessvalues,acut-offfilterof0.25mm wasappliedtothemeasuredroughnessprofile.Theeffectof pri-marysurfaceprofileontheroughnesswaseliminatedbyapplying suitablemathematical(tiltandcurvature)corrections.

Theaverageproﬁleofthemicrochannelswasobtainedusing

proﬁlemeasurements at100 differentlocationsequally spaced

withintheareaofinterestand theillustration oftheprocessis giveninFig.2C.

4. Resultsanddiscussion

4.1. Sterilizationtechniquesappliedtomicrochips

Optimizationofsupercritical CO2 sterilizationprocedurewas

thecoreobjective ofthis study.Initially, variouspressures (70, 100,120,150and250bar)fromsubcriticaltosupercriticalwere appliedataﬂowrateof10g/minwhilekeepingthetemperature andtimeconstantat40◦Cand60min,respectively.Althoughrapid pressurization/depressurization cycles of SC-CO2 were reported

tocausemembranedisruptionandcelllysis[21,22],therelease ofCO2fromthesystemwaskeptapproximately45mintoavoid

swellingofthepolymer.SubsequenttoSC-CO2sterilization,treated

microchipswereincubatedintrypticsoybrothandthioglycollate brothattwo differenttemperatures(27◦Cand 37◦C)for about 7 days.Trypticsoybrothand thioglycollatebrothwasusedfor determination microbiologicalloadsfor aerobicmicroorganisms andanaerobicmicroorganisms,respectively[23].Underthese con-ditions,theexperimentat 120barrevealedthebestsolutionin termsofsterility.

Asa second step,various temperaturesweretested (25,40, 50and 60◦C),whereas thepressurewassetto120barand the

ﬂowrateto10g/minwhileandtimewas60min.Temperatures

Fig.3.SEMmicrographsregardingsurfaceofuntreatedPMMAmicrochip(A),microchipsafterSC-CO2treatmentin70bar(B),100bar(C),120bar(D),150bar(E)and250bar

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0.5 0.4 0.3 0.2 0.1 0.0 Heat Flow (W/g) 0 50 100 150 200 250 Temperature (°C) 0.4 0.3 0.2 0.1 0.0 Heat Flow (W/g) 0 50 100 150 200 250 Temperature (°C) 0.5 0.4 0.3 0.2 0.1 0.0 Heat Flow (W/g) 0 50 100 150 200 250 Temperature (°C) 0.6 0.4 0.2 0.0 Heat Flow (W/g) 0 50 100 150 200 250 Temperature (°C) (E) (F) (G) (H) (I) (J) 0.6 0.4 0.2 0.0 Heat Flow (W/g) 0 50 100 150 200 250 Temperature (°C) 0.4 0.3 0.2 0.1 0.0 0.1 Heat Flow (W/g) 0 50 100 150 200 250 Temperature (°C)

Fig.4.DSCthermogramsofuntreatedPMMAmicrochip(A),microchipstreatedwithSC-CO2at70bar(B),100bar(C),120bar(D),150bar(E),250bar(F)andalsomicrochips

treatedwithstandardtechniquesnamely,UV(G),hydrogenperoxide(H),ethyleneoxide(I)andheat(J).

above40◦Calongwiththepressurechangedthetransparencyof thepolymerentirelytoopaque.Therefore,theoptimum temper-aturewaselicitedas40◦Cwhichsustainedthetransparencyand thesterilityofthemicrochip.Finally,sterilizationtimewas con-sideredbytreatingthemicrochipsfor30min,45minand60min whiletheotherparameterswerekeptconstant.Consequently,the sterilizationprotocolwasascertainedtobeoperatedatapressure of120bar,atemperatureof40◦Candatimeof60min.

Withinthisstudy,themicrochipswerealsosubjectedto satu-ratedsteamat121◦Cfor20minbyautoclaving,whereastheywere exposedtoUVlightat254nmfor45minoneachside,totalingup toatreatmentof90min.Asforchemicalsterilizations,thechips weretreatedwithhydrogenperoxideandethyleneoxide. Subse-quenttosterilization,thesamesterilityprocedurewasappliedto thetreatedmicrochips.

4.2. Effectsofsterilizationtechniquesonmaterialcharacteristics Theeffectsofdifferentsterilizationprocedures were investi-gatedintermsofmaterialcharacteristics.Thesurfacemorphologies

wereevaluatedusingSEMmicrographsofuntreatedandtreated

PMMA microchips (Fig. 3). Increase in the pressure adversely affectedthesurfacemorphologywhichwasparticularlyobserved inPMMAmicrochipstreatedat 150and 250bar(Fig.3EandF) appearingas cavitiesand fracturesonthesurface, whereasthe surfacesofthemicrochipstreatedat70and120bar(Fig.3Band

D) seem as smooth as the untreated. However, sterility could

notbeachievedwiththetreatmentperformedat70bar.Asfor thestandard procedures,the surfacesofautoclaved microchips werenegativelyaltered rightafterthetreatmentvisiblyleading

toopaquemicrochipsandthatwassupportedbytheSEM

micro-graphs(Fig. 3G). Surprisingly, irregularities were alsoobserved inSEMmicrographsofUVsterilizedmicrochips(Fig.3J).Onthe otherhand,bothhydrogenperoxideandethyleneoxidetreatments revealedsmoothsurfaceswithoutcausinganyadverseeffectson thematerial(Fig.3HandI).

Additionally,DSCthermogramswereusedtointerprettheeffect ofpressureonglasstransitiontemperature(Tg)ofuntreatedand treatedmicrochips(Fig.4).

Theglasstransitiontemperature,thatis,onsetofchangeinthe heatcapacitywasusedtodescribethemotionofthepolymerchain segment.PMMAisreportedtohaveaglasstransitiontemperature ofabout100–105◦C[24].SimilarTgvalueswereobtainedforthe microchipstreatedwithheat,UV,hydrogenperoxideand ethyl-eneoxidebeinglittlehigherthanTgofuntreatedmicrochipwhich was91.56◦C. Considering supercritical CO2 treated microchips,

increaseinpressurefrom70to250barhasnotresultedina sig-niﬁcantdifferenceinTg values(Fig.4B–F).In a study,atlower pressuresuptoabout400bar,Tgdecreasedlinearlywithpressure

[25]asawiderangeofpressurewasapplied.Basically,fourtypes ofglasstransitionbehaviorofpolymersincompressedgaseswere reportedusingalatticetheoryandtheGibbs–DiMarziocriterion whichwereinterpretedasafunctionofthreefactors:thesolubility ofgasesinpolymer,theflexibilityofthepolymerandthecritical temperatureofthefluid.Ithasbeenstatedthattheglass transi-tionbehaviorofPMMAinCO2showedaretrogradevitrification

whichcorrespondedtoTypeIV[24].CO2inducespolymer

plasti-cizationcausinganincreaseoftheinter-chaindistancesaswellasof thedegreeofchains-segmentalmobilityandincreasethediffusion ratesinthepolymermatrix[26].AppearanceofTypeIVbehaviorin

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Fig.5. FTIRspectraofuntreatedPMMAmicrochipandmicrochipsafterSC-CO2andstandardsterilizationtreatments.

PMMAwithCO2stronglysuggeststhatsorptionofCO2intoPMMA

ishigh.Indeed,thetreatmentsat150and250barvisuallyledto

opaquePMMAmicrochips.

The FTIR spectra of PMMA conﬁrmed the presence of

dif-ferent bondsin thestructure (Fig.5).Polymethacrylatesgive a seriesofcharacteristicinfraredbandsat2950,1722,1435,1386, 1238,1190,1142,986,840,810and751cm−1[6,27,28].Untreated

PMMAshowedabandat2950cm−1 assignedtoC Hstretching.

ThepresenceofC Obendingbondswasseenbetween750cm−1

and 810cm−1. C C stretching bonds were observed between

900cm−1 and 1000cm−1, whereas C O stretching mode was

seenat1238cm−1.Additionally,CH3 deformationwasobserved

between1300cm−1and1500cm−1.Thepeakat1730cm−1

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Fig.6. Averagesurfaceroughness.

4000cm−1 was related to the O H vibrations. The ﬁngerprint

regionofPMMAisbetween600and1400cm−1range,which

rep-resents theregionwhere mostof thebendingvibrationsoccur

andprovideinformationaboutmoleculecharacteristic.Although allspectrarevealedsimilarpeaksinregardstodifferent steriliza-tiontechniques,thetransmittancevaluesforeachtreatmentvaried inregardstoabsorptionoftheIRradiation.Butformationofnew

bandswasnot observedindicating that nosigniﬁcantchemical

changesoccurredinstructureofthematerials.

ApartfromcharacterizationtechniquessuchasSEM,DSCand FTIR,surfaceroughnessandpossiblechangesinchannelproﬁles

wereinvestigatedaswell.Themeasuredroughnessvaluesfor dif-ferentsterilizedsampleswerecomparedtothatoftheuntreated case.Asseenfromthedepiction(Fig.6),eachsterilizationtechnique reducedthesurfaceroughnessbothonthechannelandsurfaceof thechipswhichwillbereferredaspart.However,thereduction islessthan20%exceptfortheSC-CO2treatment.Overall,HPand

EtOtreatmentsexhibitedmoreprominenteffectsonpartsurface thanonchannelsurface.UVtreatmenthadthesameeffectboth onthepartandchannelsurface.Ontheotherhand,SC-CO2

treat-mentsigniﬁcantlyinﬂuencedthesurfaceroughnessandaffected thepartsurfacemoredominantly.Asfortheautoclavedmicrochips,

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nomeasurementscouldbetakenduetothefactthatthesurface andmicrochannelswerebadlydistortedwhichwasalsosupported bytheSEMmicrographs.

Followingthesurfaceroughnessmeasurements,theeffectof

the sterilization techniques on the microchannel proﬁles was

investigated.Theaveragechannelproﬁlesforuntreated,UV-, HP-,EtO-treatedand SC-CO2-treated microchannelscanbeseen in Fig.7.TheeffectsoftheHP-andEtO-treatmentsinthechannel pro-ﬁleswereveryminor.However,thechannelsweretotallydistorted inautoclavedmicrochips. AsfortheSC-CO2 treatedmicrochips,

thetreatmentexhibitedadverseeffectsonthemicrochannel pro-filewhichisundesiredforthemicrofluidicapplication,sincethe distortionof themicrochannelwouldaffecttheflow insidethe microchannel.RegardingUV-treatment,althoughthereisnota sig-nificantdistortionontheleftside-wall,asignificantdeviationfrom theuntreatedprofilewasobservedattherightside-wall.

5. Conclusion

Inthispaper,thefocuswasontheoptimizationof supercriti-calCO2sterilizationandstandardsterilizationtechniquesofPMMA

microchipswhilepossiblechangesinmaterialcharacteristicswere determinedaswell.Optimumparametersofthenewsterilization

protocol were 120bar,40◦C and 60min which provided

com-pletesterility and didnot causean adverseeffect onthemain

propertiesofthepolymerbasedonSEM,DSCandFTIRanalyses

butsomehowexhibitedadverseeffectsonthemeasuredsurface

roughnessandmicrochannelproﬁle.Heatsterilizationresultedin opaquestructures,whereaschemicalsterilizationtechniqueshave not affected material characteristicsof PMMA but slight

varia-tionswereobservedin termsofsurfaceroughness andchannel

proﬁle.However thateffectwasmoreprominentin UVtreated

samplesespeciallyregarding channelsurface andproﬁle.Taken together,sterilizationisofprimeimportanceinbiomedical applica-tionsalongwithoccupationalhealthandsafetyissues,thecurrent studyhighlightsthestrengthsandweaknessesofheat,chemical, irradiationandsupercriticalCO2treatments.

Acknowledgments

ThisworkwasﬁnanciallysupportedbytheResearchFundof EgeUniversity(14MUH045).TheauthorsarethankfultoARELat

EgeUniversityandNationalNanotechnologyResearchCenterat

BilkentUniversityforaccesstothefacilities.Theauthorsalsowould liketothankDr.SelimHanayfor hisassistancetoacquireSEM images.

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