Advanced Technique for In Situ Raman Spectroscopy Monitoring of the Freezing-Induced Loading Process - German et al. - 2021 - Unknown

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pubs.acs.org/LangmuirArticleAdvancedTechniqueforInSituRamanSpectroscopyMonitoringoftheFreezing-InducedLoadingProcessSergeiV.German,*GlebS.Budylin,EvgenyA.Shirshin,andDmitryA.GorinCiteThis:Langmuir2021,37,1365−1371ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Thefreezing-inducedloading(FIL)methodisapromisingtechniqueforencapsulationofbioactivesubstancesaswellasforpreparationofnanocompositematerials.Acriticallyimportantaspectforthismethodistheremotecontrolofthefreezingprocess.Theknowledgeofthemomentoffreezingprocessendingcanallowustoincreasethequalityofloadingandreducetheprocessduration,thusmakingthisapproachmorecontrollable.Herein,wepresentaphotonictechniquebasedonRamanspectroscopyasoneoftheoptimalsolutionsforremotecontrolofFIL.Asaresultofourstudy,thesetupforobtainingRamanspectraduringtheprocessofliquidvehiclecrystallizationinsuspensionshasbeendeveloped,whichallowedustoanalyzethesorptionofnanoparticlesontomicro-andsubmicronparticlesbytheFILmethodinsitu.ThemainfocusofthepresentworkistheinsituRamanspectroscopymonitoringofthecrystallizationprocess,includingtechnologicallyimportantparameterssuchastheice/waterinterfacevelocityinwatercolloids/suspensionsandthemomentofthefinaladsorptionofthenanoparticlesonthemicroparticles.Incontrasttootherapproaches,Ramanspectroscopyallowstodirectlyobservethehydrogenbondformationduringcrystallization.Additionally,aschematicandadetaileddescriptionofthesetuparepresentedhere.Thus,thedevelopedtechniquehasagoodperspectiveforscalinguptheFILapproachandincreasingtheareaofapplicationofthistechnology.■INTRODUCTION1b).TheFILmethodincludesseveralconsecutivesteps:Duringthepasttwodecades,activeworkhasbeendedicatedaddingasolutionofadsorbent(forexample,vateritesubmicro-tothedevelopmentofvariouscompositeparticles,forexample,ormicroparticles)andasolutionofadsorbate(forexample,forthediagnosticsorthedeliveryofactivesubstancesinmagnetitenanoparticlesorpeptides)inapolymercentrifuge1,2vivo.However,theloadingcapacityofsuchcarrierswithtube(Figure1a(1));samplefreezingwithgentlestirringDownloadedviaBUTLERUNIVonMay16,2021at17:32:11(UTC).activematerialsremainsextremelylowanddoesnotexceeda(Figure1a(2));andthawingthesample,centrifugingthe3certainpercentagebyweightofthecarrierparticle.Thesuspension,andwashingwithcleanwater(Figure1a(3)).Ifincreaseoftheloadingcapacitywillallowustoimprovethethementionedprocesses(adsorbateadding/freezing/thawing)efficiencyandpropertiesofcarriersforthetargeteddeliveryofarerepeatedseveraltimes,itallowsobtainingahighloadingbioactivesubstances.Also,itwillhelpindevelopingmoreSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.capacityofparticles.effectivefunctionalcompositeswithsuperparamagnetic,InthecurrentlyusedprotocoloftheFILmethod,astandardplasmonicproperties,etc.Previously,amethodwasdeveloped,whichmadeitpossibletosignificantlyincreasethemassfreezerisused,whichallowsthesampletocooldownto−20fractionofasubstanceadsorbedonmicro-andsubmicron°C.AspeciallydesignedrotatorTetraQuantR-1(TetraQuant,4Russia)withasampleisplacedintothefreezerfor1.5−2htoparticles,namely,thefreezing-inducedloading(FIL).Ithasbeenshownthatincomparisonwiththetraditionalmethods,achievethecrystallizationofthewholedispersantvolume.The56suchassorptionfromsolutionandcoprecipitation,theFILrotationspeedis7−14rpm.UsingastandardfreezerallowstotechniqueprovidesmoreefficientandcontrolledsorptionofsignificantlyreducethecostoftheFILprocess,asonlyaactivesubstancesandnanoparticlesonthesurfaceofporousfreezerandtheTetraQuantR-1rotatorarerequired.micronandsubmicronparticles.TheFILmethodfinds4applicationinobtainingcompositeparticles,drugdeliverysystems,7,8carbonnanodots(CNDs),9fluorescentmicro-Received:September1,2020capsules,7,8optoacousticcontrastagents,10contrastagentsforRevised:January10,2021MRI,11photocatalyticparticles,12etc.Published:January20,2021TheFILmethodisbasedontheeffectofpushingoutanyinclusions,includingmicro-andnanoparticles,bythecrystallizationfrontofadispersant(liquidvehicle)(Figure©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c025931365Langmuir2021,37,1365−1371

1Langmuirpubs.acs.org/LangmuirArticleFigure1.(a)SchemeoftheFILprocess;(b)pressingofnanoparticlesintomicroparticlesduringtheFILprocess;and(c)thesampleafterthawing.(1)Additionoftheadsorbentparticlesandadsorbateparticles,(2)freezingthesamplewithgentlestirring,and(3)thawingandcentrifugationofthesample;Ibasalplaneoficecrystal,IIc-axis,andIIIa-axis.However,despitetheexcellentresults,sufficientuniversality,providemoreinsightsintothefreezing-inducedloadingandreproducibilityoftheFILmethod,itsstagesandprocessandoptimizeit.encapsulationmechanismshavenotbeenstudiedindetail.Atpresent,thereareseveralmethodsforthephase-transition17Anin-depthstudyoftheFILprocesswillallowustooptimizedetermination,suchasopticalmicroscopy,calorimetric18theminimumtemperature,coolingrate,andprocessduration.method,high-resolutionX-rayabsorptionradiographyand14,1920,21Itwillalsoallowustoexpandthelistofsubstancesandtomography,dielectricspectroscopy,optoacoustic22−24nanoparticlesthatcanbeencapsulatedbytheFILmethod.spectroscopy(photoacousticspectroscopy),andRaman25−28Duringwaterfreezing,thecrystalsgrowapproximatelyaspectroscopy.Microscopymakesitpossibletostudyinhundredtimesfasterinthea-direction(Figure1b(III))ofthedetailtheshapeofthecrystallizationfrontandthezoneofhexagonalbase(Figure1b(I))ofthegrowingcrystalsthaninconcentrationovercoolinganddeterminethethicknessofthe1317thec-direction(Figure1b(II)),perpendiculartoit.Duringformedicelamellas.Youetal.presentedamethodbasedonthisprocess,theadsorbentandadsorbateparticlesarepushedmicroscopy,makingitpossibletodeterminewithreasonableoutbythegrowingcrystals,whichformalamellarstructureofaccuracythecrystallizationfrontvelocityandtheconstitutional4,14theadsorbentparticlessurroundedbyadsorbateparticles.undercoolingzone.However,asthismethodisbasedonvisualThislamellarstructureisparalleltothea-directionoftheobservationofthecrystallizationfront,itdoesnotallowcrystal’shexagonalbase.Itwasfoundthattheperiodoftheevaluatingthecrystallizationofwaterbetweenicecrystalslamellarstructuredecreaseswiththeincreasingcrystallizationdirectlynearthesurfaceoftheadsorbentparticles.Assessing15,16frontvelocity.Also,thefreezingfrontvelocityandparticlethecrystallizationofwaterneartheadsorbentparticles’surface14sizeinfluencethestructureoftheformedicecrystals.ThisisveryimportantfortheFILmethodbecauseadsorptionindicatesthatthecrystallizationfrontvelocityinfluencesthereachesitsmaximumefficiencyonlywhenthesampleisvolumeoftheareainthesamplethatwillcompress,completelycrystallized.concentratingtheadsorbateparticlesaroundtheadsorbentHigh-resolutionX-rayabsorptionradiographyandtomog-particles.Ontheonehand,itaffectsthesorptionefficiency,butraphyareefficacioustechniquesandallowamoredetailedontheotherhand,itinfluencestheadsorbentparticleinvestigationofthefrozensamples’structures.X-rayaggregation.Moreover,thecrystallizationfrontvelocityshouldabsorptionradiographyallowsustodeterminethezonesbelowerthanthecriticalvelocityofthefreezingfront,atwhichwithdifferentmorphologiesofthecrystalswithinafrozen13particlesaretrappedbythesolidifiedphase.sample.Radiographyisusedtoprovideadynamicvisualization14Thecrystallizationfrontvelocitymainlydependsontwoofthesamplesolidification.However,microscopy,X-rayquantities:thermalpropertiesofthesolidphasethathasabsorptionradiography,andtomographyareusedtostudythealreadybeenformedinthesampleandthetemperaturecrystallizationofcolloidsandsuspensionswithveryhighgradientbetweenthesolidifiedareainthesampleandtheparticleconcentrationduringdirectionalcrystallization.Suchcooledsideofthecell.conditionsarenecessaryfortheproductionofporousAheadofthesolid/liquidinterface,whichpushesoutnano-ceramics,whicharecurrentlybeingactivelystudied.Intheandmicroparticles,aregionisformedwithahighFILmethod,thetaskistopreventtheaggregationofadsorbentconcentrationoftheseparticlesand,asaresult,withaparticlesandensurethesuspension’shomogeneityimmedi-decreasedcrystallizationtemperature(concentrationsuper-atelybeforecrystallizationandduringthegrowthofice13cooling).So,theliquidvehicle’scrystallizationnearthecrystals.Therefore,itisnecessarytostirthesuspension.Asitissurfaceofthemicroparticles(adsorbent)shouldoccurataimpossibletostirthesamplewithanimmersiblemixerduringlowertemperaturebecauseofahighconcentrationofthecrystallizationofthesample,itisnecessarytorotateorshakeadsorbate.Also,thewatercrystallizationtemperatureinporesthecellwiththesample.Microscopy,X-rayabsorptionwithadiameteroflessthan100nmislowerthanthatintheradiography,andtomographyrequireasignificantincreasein13bulkwateranddecreaseswithreducingporediameter.Thethecomplexityofdevicestosynchronizethemovementoftheinvestigationoftheliquidvehicle’sphasetransitionwillsamplewiththemomentofmeasuringitsstructure.1366https://dx.doi.org/10.1021/acs.langmuir.0c02593Langmuir2021,37,1365−1371

2Langmuirpubs.acs.org/LangmuirArticleDielectricspectroscopy,optoacousticspectroscopy,andandadetaileddescriptionofsuchadevice.ThemainemphasisofthisRamanspectroscopydonotvisualizethecrystallizationfrontworkiscenteredontheRamanspectroscopyofthesamplesduringandthestructureofafrozensample.However,thesemethodstheFILprocess.Arotarystirrerwiththefunctionofcoolingdownthesampletoarerelativelyeasilyimplementedbyrotatingthecellwitha−20°Cwasdeveloped(Figure2).Forthecoolingofthecuvette,asuspension.Dielectricspectroscopyandoptoacousticspectros-copycanmakeitpossibletodeterminethephasetransitionandthespeedofthecrystallizationfront.However,thesemethodsdonothaveaveryhighspatialresolution.Also,theoptoacousticspectroscopyandRamanspectroscopyarecomplementarymethods,sincewhenstudyingsuspensions,theoptoacousticspectroscopyissensitivetochangesintheparametersofparticles,theviscosityofthemedium,andthespeedofsoundinthemedium,andRamanspectroscopyallowstodirectlydiscoverthehydrogenbondformationFigure2.RotatorwithaPeltiercooler:1motor,2coolingduringcrystallization.module,3cuvetteholder,4rubberclamp,5heatexchangerwithThus,thegoalofthepresentworkistoinvestigatethethepump,6doublebearing,7console,8crankingmechanism,crystallizationfrontvelocityanddetectthebeginningofthe9base,and10radiatorhoses.pushingoutofparticlesbythecrystallizationfrontandthemomentofthefinaladsorptionofthenanoparticlesonthethermoelectricmodule(DRIFT-2.0,Kryotherm,Russia)isused.ThemicroparticlesusingRamanspectroscopy.hotsideofthethermoelectricmoduleiscooledbytheliquidcoolingsystemwithaheatexchangerjoinedwithacoolantpump.Thecuvette■holdercontainsacopperheatexchangercoolingthethreewallsoftheMATERIALSANDMETHODScuvetteandacompactrubberclamp.TheheatexchangerhasaMaterials.Iron(III)chloridehexahydrate(99.8%),iron(II)temperaturesensor.Thecuvetteholder,thePeltierelement,andthechloridetetrahydrate(99.8%),citricacid(99.8%),calciumchloridehotsideoftheheatexchangerwiththepumpformasinglecoolingdehydrate,andanhydroussodiumcarbonatewerepurchasedfrommodule,whichrotatesby180°.ThemodulerevolvesontwobearingsSigma-Aldrich.Sodiumhydroxide(99.8%)waspurchasedfromFluka.mountedonconsolesupport.Acrankingmechanism,areductionAllchemicalswereusedwithoutfurtherpurification.Deionized(DI)gear,andamotoraremountedonthebacksideoftheconsole.Awater(specificresistivitynotlowerthan18MΩ·cm)fromaMilliporegearedmotorisusedtorotatethecoolingmodule.TheschemewithaMilli-Qsystemwasusedtomakeallsolutions.APET-Gfilamentsteppermotorcouldbemoresimpleandflexibleinoperation,but(Bestfilament,Russia)andRECrubber(REC,Russia)wereusedforsteppermotorsgeneratestrongvibrations,whichareundesirableinan3Dprinting.opticalsetup.UsingageareddrivewithabrushedDCmotorallowsMagnetiteNanoparticlePreparation.Magnetitenanoparticlesustoavoidthesevibrations.Afterthemotor,thereductiongearwitha(MNPs)arepreparedbychemicalprecipitationfromasolutionofgearratioof5/1isinstalled.Then,thecrankingmechanismconverts29,30iron(II)chlorideandiron(III)chlorideinbasicmedia.Theirontherotationalmotionofthesecondgearwheeltotheoscillationofsaltsolutionsareinjectedintothesodiumhydroxidesolutionwiththethirdgearwheel.Thethirdgearwheeloscillatesina1/4ofafullactivemixing.Aftertheformationofmagnetitenanoparticles,citricrevolution(90°),andthenthecoolingmodulerevolvesbackandacidisaddedtothesuspensionunderconstantmixingandnitrogenforthinahalfofafullrevolutionthroughthereductiongearwithapressure.Theobtainednanoparticlesolutionisdialyzedforthreedaysgearratioof1/2.Suchaschemeallowsreducingthestrainontheunderslowmixing.Weusedanautomaticchemicalreactortopreparecrankingmechanismatthedeadpointsoftheconnectingrod.nanoparticlesandtofurtherstabilizethem.ChemicalreactionandWeuseddisposablesquarepolystyrenecuvettesinthepresentedwashingstepswerecarriedoutundertheatmosphereofnitrogen.Thesetupbecauseglassorquartzcuvettescanbedestroyedwhenthenanoparticles’averagesizeandzetapotentialmeasuredbythewatercrystallizes.ThewatercondensationbetweenthecuvettewallsdynamiclightscatteringwithaZetasizerNanoZS(Malvern,UK)andtheholderduringsamplecoolingaffectstheholder-cuvettewere11±3nmand−25±10mV,respectively.Theconcentrationcontact’sthermalconductivityand,consequently,thesamplecoolingofthemagnetitecolloidmeasuredbythecolorimetrictitrationwasrate.Toavoidthis,wecoverthecuvettewalls(theopencuvettewall0.64mg/mL.remainsclean)withaspecialgelconsistingofamedicalultrasoundVateriteMicroparticlePreparation.Wesynthesizedvateritegelandglycerininaratioof3/1.Thisgeldoesnotcrystallizeat31microparticlesusingthemethoddescribedbyVolodkinetal.temperaturesdownto−20°CsoitprovidessufficientthermalSolutionsofCaCl2andNa2CO3areinjectedintodeionized(DI)contactbetweenthecuvettewallsandtheholder.waterunderactivemixing.Afterthereaction,westoppedstirringforAsimplifiedschemeofthesetupforRamanmeasurementsduring30sandthentransferredthesuspensiontothesuctionfilter.UsingtheFILprocessisshowninFigure3.Thesetupincludesalaser,athesuctionfilter,wewashedthemicroparticlestwotimeswithDIlongpassdichroicmirrorwithacut-onwavelengthof550nm,awaterandoncewithethanol.Finally,wedriedthevateritepowderinlongpassfilterwithacut-onwavelengthof532nm,objectives,afiberanelectricovenwithforcedairconvection.spectrometer,andasamplefreezercombinedwitharotator.TheInallexperiments,wefilledthecuvettewith3.8mLofthesample.setupcontainsa532nmlaserwithapowerof130mW.AnOceanInthiswork,weinvestigatedthefreezingprocessinthesamplesofOpticsMaya2000Prospectrometerwasusedforobtainingthedeionizedwater,magnetitenanoparticlescolloidinaconcentrationofopticalspectra.Thelaserisreflectedbyalongpassdichroicmirror0.64mg/mL,andCaCO3suspensioninaconcentrationof10.5mg/(Figure3(6))andisfocusedthroughthe10Xobjective(Figure3(7))mL.onthesample.TheRamanspectrumwasobtainedintheModifiedApproachforRealizationoftheFILMethodwithbackscatteringconfigurationthroughtheobjectiveandthelongpassInSituControlofFreezingbyRamanSpectroscopy.Tofilter(Figure3(5)).ThespectrometercollectsthebackscatteredlightinvestigateandfurtheroptimizetheFILprocessbyRamanandthrougha0.2mmopticalfiber.Thesetupisshownintheattachedphotoacousticspectroscopies,itisnecessarytodevelopandconstructvideos(SupportingInformations1and2).adevicethatallowscreatingconditionsascloseaspossibletotheTopreventtheformationoffrostontheopenwallofthecuvette,previouslydevelopedprotocolsoftheFIL.Indoingso,itisnecessarytheairflowisappliedbetweenthewallandtheobjective(Figuretoleaveopenatleastonewallofthecuvettewiththesampleforthe3(9)).Forpreventinglaserbeamreflectionfromthecuvettewall,theinputandoutputofalaserbeam.Inthiswork,wepresentaschematicrotatorismountedatanangleof3−6°tothelaserbeam.1367https://dx.doi.org/10.1021/acs.langmuir.0c02593Langmuir2021,37,1365−1371

3Langmuirpubs.acs.org/LangmuirArticlecuvette,whichstronglyscattersthelaserbeam(Figure5a).So,theRamansignalfromthisareaisweakerthanthatfromwaterFigure3.Opticalschematicofthemeasurementsetup.1spectrometer,2laser,3samplefreezercombinedwitharotator,4lenswithanadapterforopticalfiber,5longpassfilter,6longpassdichroicmirror,7objective,8cuvettewithasample,9airnozzle.■RESULTSANDDISCUSSIONFigure5.ApproachesforRamanspectrummeasurementfromaInthiswork,weaimedatstudyingthepropertiesanddynamicsrotatingcuvette.(a)MeasuringRamanspectrafromthespliceofopposingcrystallizationfronts;(b)shiftingthespliceofopposingoftheice/waterinterfaceduringFILusingtheRamancrystallizationfronts;(c)shiftingtherotatorrelativetothelaserbeam.spectroscopytechnique.Therelativeliquid/solidfraction1cuvette,2laserbeam,3spliceofopposingcrystallizationcouldbecalculatedastheratiobetweentheintegratedfronts,4cuvetteholderandheatexchanger,5thermalinsulator,intensitiesofspectralbandsaroundwavenumbersthat6directionofthecuvetteoscillating,7pathofthelaserbeamcorrespondtosymmetricandasymmetricO−Hstretches.duringthecuvetterotation,and8objective.Ds:rotatorshifting25SuchanapproachwasalreadypresentedbyDurickovič́etal.distance.AlthoughthisinterpretationofthewatervalenceRamanband32,33isdebatable,theratiometricapproachisconvenientfororiceinothersampleregions.Therefore,itismoreconvenientassessingthechangesintheRamanspectrumbandshapeandtodetectthespectrumfromaregionwithlowerscattering.wasusedinourworktofollowthefreezingprocessduringFIL.ThethinlayerofathermalinsulatorisplacedbetweenthewallAccordingtothisapproach,awater-phasetransitioncanbeofthecuvetteandthecuvetteholderforshiftingthesplicetodeterminedusinganondimensionalratiometricindicator,Sd,thecorrespondingsideofthecuvette(Figure5b).Inthiscase,whichcanbecalculatedbythefollowingequationtheinvestigatedvolumeislocatedatapointlocatedontheΔI2cuvetterotationaxis.Sd=AnotherwayisshiftingtherotatorinaplaneperpendicularΔI1(1)tothelaserbeamondistanceDs(Figure5c).Inthiscase,whereΔI1andΔI2areintegratedintensities(areasunderduringthecuvetterotation,thelaserbeampassesthroughacurves)correspondingtothesymmetricandasymmetricOHsemicirclethathasaradiusequaltotherotatorshifting25stretchingbands.ForcalculationofSd,weusedthefollowingdistance.Theadvantageofthisschemeisthepossibilitytospectralbands:3123−3245cm−1forΔIand3365−3483cm−11collecttheinformationalongwiththewholedistanceoftheforΔI2(Figure4).Forexample,Sd=0.59forspectraoficecrystallizationfrontmovement.SuchaschemeallowsandSd=1.86forspectraofwateratatemperatureof23°Cobtaininginformationonthecompletecrystallizationofthe(Figure4a).liquidvehicleinthesample.Moreover,inthiscase,thelaserDuringthecuvettecooling,thecrystallizationfrontmovesheatingofthesampleintheinvestigatedvolumeislowered,fromoppositewallsandformsaspliceinthemiddleofthewhichreducestheinfluenceofthelaseronthemeasurementresults.TheresultsobtainedusingthisapproacharepresentedinFigure6.Figure6ashowsthedependenceofSdontimeduringthecrystallizationoftheliquidvehicleinthesample.Thegraphshowsthreeareasindicatedwithreddots:1waterat23°C,2waterat0°C,and3iceat−2°C.Usingthegraph,wecandistinguishtheprocessintotwostages:coolingofwater(betweenpoints1and2)andwatercrystallization(betweenpoints2and3).Duringfurthercoolingofice,theSdvaluedoesnotchange.Sdoscillationontheslopeofthecurvebetweenpoints2and3isconnectedwiththefactthatduringcrystallizationthelaserscansthesampleandacquiresasignalconsequentlyfromareaswithliquidandsolidifiedsamples(Figure5c).RamanspectraofthesampleinthecorrespondingpointsinFigure6aarepresentedinFigure6b.TheRamansignalintensityfromiceismuchlowerthanthatfromthewaterbecause,duringthecuvetterotation,thelaserbeampassesthroughthecrystallizationfront’ssplice,whichexhibitsFigure4.MeasuredRamanspectraoficeandwaterinthecuvette.strongscattering.1368https://dx.doi.org/10.1021/acs.langmuir.0c02593Langmuir2021,37,1365−1371

4Langmuirpubs.acs.org/LangmuirArticleFigure6.(a)TimedependenceoftheratiometricindicatorSdforice/waterfractions;(b)Ramanspectraofthesysteminthetimepointsindicatedinpanel(a).TheobtainedtimedependenceoftheSdcanbeusedforcrystallizationfrontvelocity.Itisconnectedtothefactthatthecalculatingthecrystallizationfrontvelocity.Sincethelaserdifferenceintheoverallcrystallizationratewouldberelatedbeamscansthesampleintheplane,whichisparalleltothenotonlytothespeedofthecrystallizationfrontbutalsotothedirectionofthecrystallizationfrontmovement,thespeedofformationofaconstitutionalundercoolingzonethatsolidifiesthefreezingfrontcanbecalculatedusingthefollowingatalowertemperaturethanbulkwater.Duringthesampleequationfreezing,thecrystallizationfrontexpulsesparticlesandformsaregionwithanincreasedconcentrationofparticlesandaDsv=decreasedsolidificationtemperature,calledtheconstitutionaltundercoolingzone.13,14,17Thus,whentheFILmethodispt(2)carriedout,regionswithanincreasedconcentrationofwhereDsistherotatorshiftingdistanceandtptisthetimeimpurities(adsorbentandadsorbate)and,therefore,withaintervalofphasetransition,calculatedfromthetimelowercrystallizationtemperatureareformedbetweentheicedependenceofSd.lamellas.Whenwatersolidificationoccurs,thereisatemperatureAtthemoment,therearesupposedtobetwotypesofdifferencebetweenthesampleandthecuvetteholder(ΔT),processesinvolvedinconstitutionalundercooling:solutewhichincludesthetemperaturegradientinthesample,cuvetteconstitutionalsupercoolingcausedbyadditivesinthesolventwall,holder,andholder-cuvettecontact.Asmentionedintheandparticulateconstitutionalsupercoolingcausedbyparticles.introduction,thetemperaturegradientbetweenthesolidified34However,JiaxueYouetal.showedthatparticulateareainthesampleandthecooledcuvettewallsevaluatestheconstitutionalsupercoolingcontributeslesssignificantlytofreezingfrontvelocity.Inourexperiments,ΔTdependsonthetheformationoftheconstitutionalundercoolingzonethancoolingpowerofthePeltierelementandevaluatesthefreezingsoluteconstitutionalsupercooling.Atthesametime,thefrontvelocity.Usingthepresentedmethod,wemeasuredtheauthorsoftheworkassumethattheexperimentallyobserveddependenceoffreezingfrontvelocityofpurewaterontheconstitutionalundercoolingzonesduringcrystallizationoftemperaturedifferencebetweenthesampleandthecuvettecolloidsandsuspensionsareassociatedmainlywithionsandholder(ΔT)(Figure7),whichdemonstratesadirectadditivesinthem.Unfortunately,accordingtothemethodrelationshipbetweenthesetwovalues.describedinthepresentwork,thecuvettewiththesamplerotatesconsequentiallyclockwiseandcounterclockwise,sowecannotsimultaneouslyobservethereferenceandthesample.Thisdoesnotallowustodeterminethepresenceofaconstitutionalundercoolingzone;wecanonlydeterminetheoverallcrystallizationrateofthesample.However,thismethodmakesitpossibletostudycolloidsandsuspensionswithlowsedimentationstability,whichsignificantlyexpandsthenomenclatureofsubstances.AlloftheseallowustosaythatcombiningopticalmicroscopywithRamanspectroscopywouldgivethepossibilitytostudytheFILprocessinmoredetail.Figure7.DependenceoffreezingfrontvelocityofpurewaterontheTakingintoaccounttheparameterSd,themomentoftemperaturedifferencebetweenthesampleandthecuvetteholdercompletecrystallizationoftheentirevolumeofwaterinthe(ΔT).Theerrorbarscorrespondtothestandarddeviationcalculatedcuvettewasdetermined.ThisparameterisamarkeroftheFILforthreereplicatesoftheexperiment.processendingandoneoftheprocessoptimizationcriteria.NotethattheFILprocessduration,usingthepresenteddeviceInFigure8,theoverallcrystallizationrateinsuspensionsofinourexperiments(10−17min),ismuchlessthanthemagnetitenanoparticlesandcalciumcarbonatemicroparticlesdurationoftheFILprocessusingtherotatorTetraQuantR-1ispresented.Additionally,wemeasuredtheoverallcrystal-andafreezer(>1h).lizationrateofwaterwhilethemagnetitenanoparticlesadsorbontothevateritemicroparticlesbyFIL.Whenstudying■CONCLUSIONSsuspensionsandsolutionsbythepresentedapproach,itisWehavedevelopedanewsetupforobtainingRamanspectrabettertoconsidertheoverallcrystallizationrateinsteadoftheduringthefreezing-inducedloadingofbioactivesubstanceson1369https://dx.doi.org/10.1021/acs.langmuir.0c02593Langmuir2021,37,1365−1371

5Langmuirpubs.acs.org/LangmuirArticleFigure8.(a)TimedependenceofSdforpurewaterandsuspensionsofMNPsandCaCO3;(b)freezingfrontvelocityofpurewaterandsuspensionsofMNPsandCaCO3.Theerrorbarscorrespondtothestandarddeviationcalculatedforthreereplicatesoftheexperiment.ThetemperaturedifferencebetweensamplesandthecuvetteholderΔTis14°C.asurfaceofmicro-andnanoparticlesthatcanbeusedasSechenovFirstMoscowStateMedicalUniversity(Sechenovcarriersfordrugdelivery.ThismethodhasbeendevelopedforUniversity),119991Moscow,Russiameasuringthecrystallizationfrontvelocityinwatersuspen-DmitryA.Gorin−SkolkovoInstituteofScienceandsionsusingRamanspectroscopy.ThedependenceoftheTechnology,121205Moscow,RussiacrystallizationfrontvelocityofwateronthecoolingpowerisCompletecontactinformationisavailableat:obtainedusingthepresentedtechnique.When3.8mLofwaterhttps://pubs.acs.org/10.1021/acs.langmuir.0c02593isfrozeninapolystyrenecuvette,thefreezingfrontratevariesfrom7.6±1to11.6±0.7μm/satΔTof10−14°C.ItwasAuthorContributionsshownthatthepresenceofmagnetitenanoparticlesandThemanuscriptwaswrittenthroughcontributionsofallvateritemicroparticlesaffectsthecrystallizationfrontvelocityauthors.Allauthorshavegivenapprovaltothefinalversionofoftheliquidvehicle.Moreover,thepresenteddevicecanbethemanuscript.usedforstudyingsuspensionsandcolloidswithlowsedimentationstabilitybyRamanspectroscopy.ThecurrentFundingprotocolsoftheFILtechniquecouldbeoptimizedusingtheThereportedstudywasfundedbyRFBR,projectnumber19-presentedapproachtogetvariousstructures.Optimized33-60089.TheworkofEvgenyA.ShirshinwassupportedbyprotocolsfortheproductionofnanocompositeparticleswiththeRFPresident’sgrant(GrantNo.MK-2999.2019.2).Themuch-increasedmassfractionsofactivesubstancescanbestudywaspartlysupportedbytheInterdisciplinaryScientificwidelyusedinotherareas,forexample,toobtainphoto-andEducationalSchoolofMoscowUniversity“Photonicandcatalyticstructures,photoluminescentsubmicronobjects,orQuantumTechnologies.DigitalMedicine”.carbonnanodots(CNDs).Notes■Theauthorsdeclarenocompetingfinancialinterest.ASSOCIATEDCONTENT*sıSupportingInformation■ABBREVIATIONSTheSupportingInformationisavailablefreeofchargeatFIL,freezing-inducedloading;MNPs,magnetitenanoparticleshttps://pubs.acs.org/doi/10.1021/acs.langmuir.0c02593.Supportinginformation1:Ashortvideopresentsthe■REFERENCESmeasuringsetupinoperation(avi)(1)Bah,M.G.;Bilal,H.M.;Wang,J.FabricationandApplicationofSupportinginformation2:AshortvideopresentstheComplexMicrocapsules:AReview.SoftMatter2020,16,570−590.rotatingofcuvetteduringmeasuringprocess(avi)(2)Liu,D.;Yang,F.;Xiong,F.;Gu,N.TheSmartDrugDeliverySystemandItsClinicalPotential.Theranostics2016,6,1306−1323.■(3)Dvorak,H.F.;Tavares,A.J.;Ohta,S.;Audet,J.;Chan,W.C.AUTHORINFORMATIONW.;Wilhelm,S.;Dai,Q.AnalysisofNanoparticleDeliverytoCorrespondingAuthorTumours.Nat.Rev.Mater.2016,1,No.16014.SergeiV.German−InstituteofSpectroscopyoftheRussian(4)German,S.V.;Novoselova,M.V.;Bratashov,D.N.;Demina,P.AcademyofSciences,108840Moscow,Russia;SkolkovoA.;Atkin,V.S.;Voronin,D.V.;Khlebtsov,B.N.;Parakhonskiy,B.V.;InstituteofScienceandTechnology,121205Moscow,Russia;Sukhorukov,G.B.;Gorin,D.A.High-EfficiencyFreezing-Inducedorcid.org/0000-0002-4239-3741;Email:s.german@LoadingofInorganicNanoparticlesandProteinsintoMicron-andskoltech.ruSubmicron-SizedPorousParticles.Sci.Rep.2018,8,No.17763.(5)DeCock,L.J.;DeKoker,S.;DeGeest,B.G.;Grooten,J.;AuthorsVervaet,C.;Remon,J.P.;Sukhorukov,G.B.;Antipina,M.N.GlebS.Budylin−InstituteofSpectroscopyoftheRussianPolymericMultilayerCapsulesinDrugDelivery.Angew.Chem.,Int.AcademyofSciences,108840Moscow,Russia;MedicalEd.2010,49,6954−6973.(6)Svenskaya,Y.I.;Pavlov,A.M.;Gorin,D.A.;Gould,D.J.;ScientificandEducationalCenterofM.V.LomonosovParakhonskiy,B.V.;Sukhorukov,G.B.PhotodynamicTherapyMoscowStateUniversity,119991Moscow,RussiaPlatformBasedonLocalizedDeliveryofPhotosensitizerbyVateriteEvgenyA.Shirshin−InstituteofSpectroscopyoftheRussianSubmicronParticles.ColloidsSurf.,B2016,146,171−179.AcademyofSciences,108840Moscow,Russia;Facultyof(7)Novoselova,M.V.;German,S.V.;Sindeeva,O.A.;Kulikov,O.Physics,M.V.LomonosovMoscowStateUniversity,119991A.;Minaeva,O.V.;Brodovskaya,E.P.;Ageev,V.P.;Zharkov,M.N.;Moscow,Russia;InstituteforRegenerativeMedicine,Pyataev,N.A.;Sukhorukov,G.B.;Gorin,D.A.Submicron-Sized1370https://dx.doi.org/10.1021/acs.langmuir.0c02593Langmuir2021,37,1365−1371

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