In fl uence of Glutaraldehyde ’ s Molecular Transformations on Spectroscopic Investigations of Its Conjugation with Amine- Modi fi ed F

《In fl uence of Glutaraldehyde ’ s Molecular Transformations on Spectroscopic Investigations of Its Conjugation with Amine- Modi fi ed F》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

pubs.acs.org/LangmuirArticleInfluenceofGlutaraldehyde’sMolecularTransformationsonSpectroscopicInvestigationsofItsConjugationwithAmine-ModifiedFe3O4MicroparticlesintheReactionMediumRobinsonKarunanithy,TorreyHolland,andPoopalasingamSivakumar*CiteThis:Langmuir2021,37,5242−5251ReadOnlineACCESSMetrics&MoreArticleRecommendationsABSTRACT:Glutaraldehyde(GA)isawidelyusedcross-linkingagentinbiologicalresearchduetoitssuperiorcharacteristics,suchashighreactivitytowardproteins,highstability,andcost-effectiveness.Inthisregard,analyzingspectralchangesinitiatedbyvariousmolecularformsandtransformationsofGAinareactionmediumanditsreactionwithsurfacefunctional-modifiedsolidspheresisvitalforasuccessfulbioconjugationprocesstargetingthebiomoleculesofinterest.Inthiswork,wepresentFouriertransform-infrared(FT-IR),Raman,andUV−visiblespectroscopicanalysesofglutaraldehyde-modifiedFe3O4microparticles(mag-neticbeads)toconfirmtheconjugationbetweenGAandmagneticbeads.Wealsostudiedthemoleculartransformationsofglutaraldehydeduringthereactionwithamine-modifiedmagneticbeadsviainvestigatingthereactionmediumoftheglutaraldehydesolution.OurFT-IRandRamanstudiesconfirmedthatglutaraldehydewassuccessfullycoupledonthemagneticbeads.Furthermore,FT-IRandUV−visstudiesontheglutaraldehydesolutionrevealedthemultiplemolecularformsofGAinanaqueousmedium,andtheyalsoconfirmedthatglutaraldehydetransformsintoothermolecularformswhilethereactionoccurswiththemagneticbeads.■INTRODUCTIONhasbeenquitecomplexandhasbeenadebatabletopicfor10−15scientists.Theorganiccompoundglutaraldehyde(GA)isafive-carbonThefollowingliteraturereviewbrieflyexplainsthecomplexhomo-bifunctional,colorless,andstrong-smellingaldehydenatureofitsstructuralanalysis.Figure1showssomeofthethatwasfirstproducedcommerciallyin1951intheUnited16−18DownloadedviaUNIVOFNEWMEXICOonMay16,2021at06:48:34(UTC).1−3moreprominentmolecularformsofGAthattypicallyStatesforvariousapplications.Sincethen,ithasbeenusedexistinanaqueousmedium,andthesemoleculartrans-inindustrial,laboratorial,medical,andhouseholdapplications,formationscanemergeintherespectivespectraleadingtohavingawiderangeofuses,particularlybecauseofitspossiblemisinterpretationsinspectralanalyses.16,19−21Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.sterilizingproperty.UsesasanantimicrobialagentinthesugarDespiteextensiveexplorationsofGA’smolecularforms,theindustry,wastewatertreatment,andhouseholdcleaningareapresenceandrelativeproportionsofeachformarestillunclear2−5fewexamplesofsuchapplications.Moreover,GAhasaaspreviouslyreportedstudiesshowsomeinconsistencies.Inwiderangeofbiologicalapplicationsduetosomeofits1968,RichardsandKnowlesreportedthatGAislargelycompatiblechemicalproperties.Itissolubleinwater,alcohol,polymeric,particularlywithgreaterlevelsofα,β-unsaturated13,19andorganicsolventsandhastworeactivegroupsthatarealdehyde(Figure1,structureD)viaaldolcondensation.Acapableofconjugatingspontaneouslywithamine,amide,andyearlater,anothergroup,Hardyetal.,foundusingnuclearthiolgroupsinantibodies(orotherproteins).2,6,7Further-magneticresonance(NMR)withUV−visthatcommerciallymore,commercialavailabilityatalowcostdrewattentionfromavailableGApossessesminoramountsofα,β-unsaturatedmanyresearcherstouseitasaneffectivecross-linkingagentin2,7−9bioconjugationandenzymeimmobilization.However,Received:January20,2021workingwithGAischallengingasitundergoesmultipleRevised:April6,2021moleculartransformations,includingpolymerizationwithPublished:April20,2021changesintemperatureandpH.Typically,GAexistsinamixtureofvariousmolecularformsinambientconditions.Duetothisreason,structuralanalysisofGAinanaqueousmedium©2021AmericanChemicalSocietyhttps://doi.org/10.1021/acs.langmuir.1c001825242Langmuir2021,37,5242−5251

1Langmuirpubs.acs.org/LangmuirArticleFigure1.(A−D)DifferentmolecularformsofGAinanaqueousmedium.30aldehyde.Theycametothisconclusionbecauseoftheweakapplicationfromlong-termdrugdelivery.So,asanabsorbanceat235nm(responsibleforα,β-unsaturatedalternativesolutiontothisproblem,couplingGAtoanaldehyde).Furthermore,inthatarticle,theyexplainedthatinsolublemicrospherecapturedtheattentionofscientists.GAexistsinmultiplemolecularforms(Figure1):structureARemarkably,thecross-linkingmechanismhasagreatadvantageexistsinequilibriumwithstructuresBandC,andasmallforthepurificationanddetectionofspecificproteinslike22amountofpolymerization(D)isalsopresent.Aconsistentcancerantigensfromhumanfluidsusinginsolublesurface-resultwasreportedbyKornetal.inthattheydidnotfindmodifiedsolidsupportssuchasironoxide,silica,ortitanium31significantamountsofα,β-unsaturatedaldehyde.Additionally,dioxidemicro-andnanoparticles.theirstudyrevealedthataqueousGAconsistsofasimpleSurface-modifiedmicro-andnanoparticlesarereadilyaldehydeform(A)andsomeotherformssuchashydratedavailablecommerciallytocouplewithcross-linkingagentscyclichemiacetal(structureB)andoligomerformsofcyclicandthentoimmobilizetheproteins.Typically,−NH2,−NHS,17hemiacetal(structureC).Also,WhippleandRutaconcludedand−COOHfunctionalgroup-modifiedmicrospheresare23thatGAmostlyexistsinthecyclichemiacetalform.In1975,widelyusedwithGAforsuchapplications.Cross-linkageisMonsanetal.confirmedtheresultsobtainedbyHardyetal.,achievedthroughacovalentbondbetweenGAandamineKornetal.,andWhippleandRutausingvariousmethods:IRgroups,forexample,bylosingawatermolecule.SinceGAspectrometry,HNMR,massspectrometry,andthin-layerexistsinmultiplemolecularformsandundergoessignificantchromatographyinacidicconditions.Theyfoundthataddingmoleculartransformationsinanaqueousmedium,potentially,sodiumhydroxideto25%aqueousGAproducesaprecip-therearevarioustypesofcross-linkedproductsthatareitationofpolyglutaraldehyde,whichistheproductfromaldolpossiblewithavarietyofintermediateconjugates,forexample,2432condensation(structureD).Schiffbases.ThereactionpH,temperature,concentration,Inadditiontotheabovemolecularforms,otherpotentialandtimearetheparametersthatcorrelatetotheproportionofmolecularstructureshavebeenreportedthatmaybelesseachmolecularformandcross-linkedproduct.Inmanyearlycommon.Forexample,thepolymerizationofGA(Figure1,studies,lowtemperaturewasmaintainedforthecross-linking33structureD)isalsopossibledependingonthereactionprocess,butittookalongtime(6to18h).However,condition,whereitmayundergohydratedformsanddimericaccordingtonewstudies,roomtemperatureisalsoafavorableringstructuresthatcouldcontaincarboxylicandhydroxylconditiontoachievethereactionwithinashortperiod,uptoa34,35groups.However,thesereactionsrarelyhappenathigherpHmaximumof4h.24−27(above8.5).In1992,areportmadebyKawaharaetal.Inthisstudy,wereportonlythecouplingofGAonaminesummarizedmanyoftheothers’results.TheyanalyzedtheGA(−NH2)-modifiedmagneticbeadsandthemoleculartrans-aqueoussolutionbyUV−visandlightscatteringandformationsofGAduringthisconjugationprocess.FT-IRandconcludedthatmostoftheGAsolution(70%)containedRamanspectroscopictechniquesareuser-friendlyandallowapolymericforms,includingcyclichemiacetal.Theirstudyalsostraightforwardinvivoanalysisoftargetbiomolecules.So,suggestedthatinadilutedsolutionandinthepHrangeof3−understandingtheeffectsofglutaraldehydetransformationson8,GAexistsinamonomericform,mainlythecyclichemiacetalspectralanalysesisvitaltodeterminethesuitableconditions18form.ThereportedpossibleandtransientformsofGAcouldforproperbioconjugation.TheseareessentialfirststepsinpotentiallyinfluencespectroscopicinvestigationsasGAsuccessfullypurifyingcancerbiomarkers,suchascancerundergoeschangesinpHorduringachemicalreaction,suchantigen125(CA125)andhumanepididymisprotein(HE4)aswhenattemptingtoconfirmconjugationtospecializedthroughbioconjugation.Analyzinghumanserumthatconsistsbeadscommonlyusedasanefficientmethodforproteinofarangeofbiomolecules,suchasproteins,lipids,aminopurificationinbiomedicalresearch.acids,sugars,andsalts,isinevitableindetectingmanytypesofAwiderangeofGAapplicationshavebeenreportedutilizingdiseases.However,thesecomponentsexistatlowconcen-thecross-linkingtechniqueinthefieldofbiochemistry,trations,andcertainproteinconcentrationsarereducedevenbiotechnology,andengineering.Inthelate1970s,GAwaslowerforparticularkindsofdiseases.Therefore,sensitive28usedfortherapeuticandimmunologicalapplications.Gelatindetectionmethodsarerequiredtoaidintheidentificationof36microspheresweremadefromproteinfordrugdeliveryandmanyformsofdiseases.Assuch,sensitivetechniquesare29fixingtissues.However,gelatinisadissolvablecompoundinneededtodetectmanytypesofcancers,particularlyforthoseanaqueousmedium,andtherefore,thatpropertylimiteditsthataretreatablebutasymptomaticattheearlystage,like5243https://doi.org/10.1021/acs.langmuir.1c00182Langmuir2021,37,5242−5251

2Langmuirpubs.acs.org/LangmuirArticle37,38smallportionsofeachofthepreparedsampleswerecollectedatovariancancer.Therearesomemethodssuchascolumnchromatography,liquid-phasechromatography,sizeexclusiondifferentperiods(3min,30min,1h,2h,and3h)duringthechromatography,andpolyacrylamidegelelectrophoresisthatconjugationprocess.Thebeadswereseparatedfromthesupernatants36,39−44byamagneticseparationprocessforspectroscopicinvestigations.areusedtopurifytheproteins.DespitetheseavailableToconfirmthepresenceofcyclichemiacetalonmagneticbeads,methods,researchersarelookingforotherpromisingmethodsweusedaRamanmicroscopesystem(HoribaiHR550imagingthatcouldbeuser-friendlywithhighersensitivityinordertospectrometerinconjunctionwithanOlympusBX41microscope).overcomethechallengesassociatedwiththeothermethods.Near-infrared(NIR)lightatawavelengthof785nm(iBeam-smart-Bioconjugationissuchatechniquethathasdrawnpeople’s785-S-WS,TOPTICAPhotonics)wasusedasanexcitationlightattentiontoproteinpurificationandidentification.Itallowsthesource.Forspectralacquisition,anoutputpowerof10mW,a50×usertotargetthespecificproteinofinterestusingpredefinedobjectivelens,anda600gr/mmgratingblazedat500nmwereused.agentstoconjugatethetargetmoleculesthroughcovalentorTwotothreemeasurementswereperformedonthreedifferentnoncovalentinteractioninacontrolledmanner.45−47Whilesamplestoensurethereproducibilityofthemeasurements.UV−VisAnalyses.ANanoDropOnec(ThermoFisherScientific)manyresearchgroupsareactivelyworkingonthisarea,UV−visspectrometerwasusedtoanalyzetheGAsupernatant.Wesandwich-typemicroparticlemethodshavebeensuccessfullyusedthreedifferentsamplesforeachtrial,andtwotothreeemployedforpurificationanddetection.Forexample,replicationswereperformedforeachtrial.BeforeeachspectralMelikechietal.usedsilicaandagarosebeadstopurifyandacquisition,2μLof0.01MPWBwasusedtogeta“blank”detecttheCA125antigenbyconjugatingtheparticlestoanbackgroundspectrum.Then,foreveryreplication,2μLofthesample48,49antibody−antigencomplex.Markushinetal.usedasimilarwaspipettedontheNanoDroppedestal.SimilartotheFT-IRtechniqueusingtitaniumandironmicroparticlestopurifyandmeasurements,theUV−visspectrawerecollectedonsamplesfromdetectCA125.50Thisstudywillpaveapathforabettertheprecouplingsolution,andthen,theywerecollectedontheunderstatingofongoinginvestigations,andhere,amine-supernatantstakenatdifferentperiodspost-coupling(3min,30min,1h,2h,and3h).Theexaminedsupernatantportionsofthepreparedmodifiedmagneticbeadswerecoupledwithavidin,andGAsamplesfortwoofthethreetrialsateachrespectivetimeintervalforwasusedasacross-linkingagentbetweentheavidinandthetheUV−visstudywerefromthesameportionsofthesamplesamine-modifiedbeads.Subsequently,theavidinwillbecollectedattherespectivetimeintervalsfortheFT-IRstudy.Thecoupledtoabiotinylatedantibody−antigencomplexinthirdtrialforeachrespectivetimeintervalusedasamplepreparedbloodserumtoisolatetheproteinofinterest.independentlyfromthesamplespreparedfortheFT-IRstudy.■MATERIALSANDMETHODS■RESULTSANDDISCUSSIONPreparationofGlutaraldehydeSolution.ForpreparingtheFT-IRAnalysisofGA-ModifiedMagneticBeads.BeforeGAsolution,0.1Mpyridinewashbuffer(PWB)purchasedfrominvestigatingthechangesinmolecularformsofGAintheBangsLaboratories,Inc.wasdilutedtenfoldbydeionizedwatertoamedium,theconjugationofGAwithmagneticbeadswaspHof6.Then560μLof0.01MPWBwasaddedto140μLof25%confirmedbystudyingtheabsorbanceofGAonthemagneticaqueousGA(pH∼5)(BangsLaboratories,Inc.)andmixedusingarotator(minituberotatorfromFisherScientific),sothefinalbeadsovertimeusingFT-IR.Further,wetookanFT-IRconcentrationofGAwas5%.Fromthissolution,100μLofGAwasmeasurementof0.01MpyridinetoinvestigatetheinterferenceusedforprecouplingFT-IRandUV−vismeasurements.StandardofpyridinewithGAsignatures.However,therewerenosafetyprecautionsandregulationswerefollowedtohandleGAasnoticeablesignaturesobserved.Thisisduetothedominationrecommendedbythematerialsafetydatasheetsuchasusingofwaterinthepyridinesolutionwheretheamountofpyridineprotectiveglovesandclothingtopreventskinexposureandusinganisbelowthelevelofdetectionorpyridineevaporatedwhileoccupationalsafetyandhealthstandard(OSHA)respiratororfumedryingthesample.51hoodtolimitinhalation.Figure2showsthespectrumofGA-modifiedbeadstakenatConjugationofMagneticBeadswithGA.Avolumeof300μL3h(red)andareferenceFT-IRspectrumoftheGAsolutionofcommerciallyavailableaminepremodifiedmagneticbeads(Fe3O4;diameter,1.5μm,fromBangsLaboratories,Inc.)waswashedthree(black)andunmodifiedbeads(blue).FromtheGA-modifiedtimeswith0.01MPWBtoremoveanylooselycoupledaminegroups.magneticbeadspectrum(red),onecannoticethecharacter-Thewashedparticlesweremagneticallyseparatedusingamagneticseparator(Polysciences,Inc.),andthesupernatantwasdiscarded.Finally,600μLof5%aqueousGAwasaddedtothebeadsandmixedusingaminituberotator.AfteraddingtheGAsolution,80μLofsampleswascollectedeachtimefromthemixtureafter3min,30min,1h,2h,and3h.Thebeadsandthesupernatantwerethenmagneticallyseparated,andthebeadswerewashedthreetimeswithdeionizedwatertoremoveanynoncoupledGAonthemagneticbeads.Finally,GA-modifiedbeadswereanalyzedbyFT-IRandRamanspectrometry.TherelevantsupernatantswereanalyzedbyFT-IRandUV−visspectrometry.FT-IRandRamanAnalyses.ToconfirmthepresenceofGAonmagneticbeadsandinvestigatethechangesinmolecularformsofGAusingFT-IR,sampleswerepreparedonZnSewindowsandanalyzedusinganFT-IRspectrometer(Nicolet,Nexus:670).Fiftyscansat4cm−1resolutionwereusedfortheacquisitionofspectra.Threedifferentsamplesweremadeforeachtrial,andfromtwotofourindependentreplicationswereperformedforeachtrialforatotalof6to12spectralacquisitions.Ineverymeasurement,2μLofthesamplewaspipettedatthecenterofthewindowandallowedtodry.First,FT-IRmeasurementswereperformedontheGAsolutionbeforeFigure2.FT-IRspectraofGA,GA-modified,andunmodifiedmixingwithmagneticbeads(precouplingsolution).Aftermixing,magneticbeadsat3h.5244https://doi.org/10.1021/acs.langmuir.1c00182Langmuir2021,37,5242−5251

3Langmuirpubs.acs.org/LangmuirArticleisticvibrationalbandsofGA.Thebandat1720cm−1(bluearrow)canbeattributedtoCOstretchingatthefreeendofGAonthemagneticbeads.Moreover,C−Hstretchingmodesofα-carbonandthealiphaticcarbonchainofGAcouldbeassignedat2890and2950cm−1.52,53AsonecanseeinFigure2(blackspectrum),5%aqueousGAshowedastrongabsorbanceat3420cm−1.SincetheGAsamplewasallowedtodrybeforetakingtheFT-IRmeasurements,wethinkthattheO−Habsorbanceprimarilyarisesfromthecyclichemiacetaleventhoughtherecouldbesomecontributionfromwater.Additionally,thestrongFT-IRsignaturesbelow1450cm−1,particularlythelowestofthefrequencysignatures,areassignedtothecyclicfeaturesofGA.Tocorroboratethis,wecomparedourspectrawiththeFT-IRspectraofothertypesofcyclichemiacetals,forexample,α-D-Figure3.SampleFT-IRspectraofGA-modifiedmagneticbeadsoverglucose,whichiscloseinmolecularstructuretothatofGAandtime.withaverysimilarspectralsignature.Thiscomparisonwasmadeduetothedifficultyinprocuringpurecyclichemiacetal63−65products.Particularly,onthereactionwithproteins,GAduetoitstransientnatureunderstandardconditions,differentreactionmechanismshavebeenhypothesized.whereα-D-glucosepossessesamorestableyetverysimilarringHowever,byanalyzingthespectralcharacteristicsofreactantsstructure.−1andproducts,itispossibletoknowtheexistingmolecularThevibrationalbandat1022cmcanbeassignedtothe19,64forms.CyclichemiacetalisonesuchprominentmolecularC−Oringstretchingofthecyclichemiacetal.Also,thebroader−1form,whichisformedthroughhydrationintheaqueousband(Figure2,blueoval)from950to1022cmisduetothemedium(Figure4a).ThecarbonyloxygenatomsatbothendsoverlappingofthisC−Oringstretching,C−Cringstretching,andC−O−Cringdeformations,whichtypicallyexistintherangeof930to1090cm−1.54−60WeobservedthesespectralfeaturesontheGA-modifiedbeads(Figure2,red).Addition-ally,thebandat3350cm−1couldbefromtheO−HstretchingintheconjugatedcyclichemiacetalandcouldalsobeduetotheN−Hstretchingofmagneticbeads.AllthesefeaturesprovideevidenceofcyclichemiacetalGAconjugationonthebeads.Moreover,onecannoticethattherewerenobandsintherangeof1500−1700cm−1fortheGAsolution(greenovalinFigure2)andwerealsonoticeablylacking(orfarlessprominentforthe−NH2scissoring)fortheunmodifiedbeads.However,prominentbandswereobservedinthesamerangeoncethebeadsaremodifiedwithGA,whichisevidenceforformingnewbondsduetotheconjugationofGAonthemagneticbeads.AsshowninFigure5,theobservedCNFigure4.Formationof(a)cyclichemiacetaland(b)α,β-unsaturated−1aldehydefromGA.bandat1650cm(Figure2)isassignedtothebondformedbetweenGAandtheaminegroupsonthemagneticbeads.Typically,CNstretchingexistsbetween1640and1690ofGAundergonucleophilicadditionwithahydrogenatomofcm−1.61Also,theremightbeasmallcontributionfromtheawatermolecule.Subsequently,thecarbonylcarbonattacks−NHscissoringortheN−Hbending(∼1590cm−1)mode2theoxygenatomofwater,formingastableringstructure.(ofcoupledandnoncoupledmagneticbeads)thatusuallyTypically,5-or6-sidedcarbonringstructuresareproducedasarisesbetween1580and1650cm−1.62theyhavehigherstability,withglucosebeinganotherexampleFigure3showstheFT-IRspectra(plottedasvertically66offormingthistypeofringstructure.Furthermore,theseshiftedabsorbanceforcomparison)ofGA-coupledmagneticcyclichemiacetalscanformoligomersthroughintermolecularbeadsovertime.Thevibrationalbandfeatures(CO,C−H,reactions.andO−Hstretchingmodes)relevanttoGA(indicatedbyInadditiontothat,withtheriseinpH,α-carbonoftheGAdashedblackrectangles)confirmedthepresenceofGAonthemoleculecanhaveanaddition-typereactionwiththecarbonylbeads.Duringthefirst30minperiod,nodefinitivelyuniquecarbonofanotherGAmolecule(Figure4b)followedbyanGAsignatureswereobserved.However,after1h,theGAeliminationofawatermoleculetoformα,β-unsaturated65,66features(insidetherectangles)werenoticedanddevelopedaaldehyde.progressivetrendoversubsequenttimeintervals.Additionally,thereactionofanoligomerofα,β-unsaturatedHowever,themolecularformofGA,particularlyinwater,isaldehydewithmagneticbeadsisalsopossible.Thisreactionnotlimitedtoasingleform.Itcanbetransformedintoothercanbeanadditionoftheamine(−NH2)grouptothemolecularformsthroughspontaneousreactions.Duetothisethylenicdoublebondontheα,β-unsaturatedaldehyde13,67reason,itcouldexistinmultiplemolecularformswith(Michael-typeaddition).Asanothermechanism,theintermediateproducts,invaryingproportions,dependingon−NH2groupcanalsoreactwiththealdehydepartofthepHandtemperature.Therefore,thereisnosolidagreementα,β-unsaturatedaldehydeformingaSchiffbase-typeproductaboutthecross-linkingreactionmechanismandcross-linkedbyeliminatingawatermolecule.However,thesereactionsare5245https://doi.org/10.1021/acs.langmuir.1c00182Langmuir2021,37,5242−5251

4Langmuirpubs.acs.org/LangmuirArticleFigure5.ProminentreactionsofmagneticbeadswithGA(a)andcyclichemiacetal(b).unstableandthusbreakdownintomagneticparticlesandα,β-assignedtothecyclichemiacetalringC−O−Casymmetric24unsaturatedaldehydesunlessthemediumisalkaline.So,stretchingmode,whichusuallyfallsbetween1000and1100theseproductsarelesslikely,andonlythemainproductsarecm−1.54,68,69Additionally,forpureGA,thebandat1010cm−1depictedinFigure5.Intermediatereactionsandthechangesinwasofgreaterintensitythanthebandat1025cm−1(bluetheequilibriumofGAareexplainedlater;seeFigure9.double-sidedarrow).However,thisbehaviorwasinterchangedRamanAnalysisofGA-ModifiedBeads.Figure6showsafterGAmodification.AsonecanseeinFigure6,theGA-theRamanspectrumofGA-modifiedmagneticbeads(green),modifiedbeadsdemonstratedastrongersignalat1025cm−1thanat1010cm−1asopposedtoareversedratioinpureGA.Thisarisesfromthecontributionofmagneticbeadsasbaremagneticbeadssharethesamebandpositionatapproximately1025cm−1.Moreover,GAhasastrongbandat1125cm−1that54,68,70,71arisesfromC−OHstretching.However,theGA-modifiedbeadsshowedabandat1121cm−1.Thisredshiftingcouldbeexplainedbytheconjugationofcyclichemiacetalwiththebeads,possiblyrestrictingthevibrationsofoxygenandthusreducingthefrequency.Also,acontributingfactormaybetheinteractionbetweenthefreeOHgroupofcyclichemiacetalandthe−NH2groupofthebeads.Additionally,anothervibrationalbandat1170cm−1(blueoval)existsinthespectraforboththebarebeadsandGA.ThissamebandwasobservedintheGA-modifiedbeadsaswell.ItcouldbeduetoacontributionfrombothGAandthebeadsthemselves.FT-IRAnalysisoftheGASupernatant.Figure7displaysFigure6.Ramanspectraof5%aqueousGA,GA-modifiedmagneticbeads,andbaremagneticbeads.theaveragedspectraofthereplicationsofatrialatthegiventimeintervalsandshowsthevariationsinthespectraoftheGAsolution,pre-andpost-mixing(yellowspectrumhiddenwithintheothers).Thebroadbandat3420cm−1isfromtheO−Handforreference,spectraofstraightGA(red)andbarebeads(black)areincludedafterremovingthebaseline.AsonecanstretchingofcyclichemiacetalGA.Thebandsat2720,2890,and2950cm−1areduetoC−Hstretching.TheCOseeinFigure6,cyclichemiacetalfeatureswerenoticeablebetween900and1200cm−1.Thebandat1010cm−1couldbestretchingofaldehydeshowsasharpandstrongabsorbanceatFigure7.VariationofFT-IRspectraofGAinthesupernatantovertimeandobservedformationofaCCvibrationalbandat1653cm−1(inset,magnifiedfigure)duetomoleculartransformationofGAinthesupernatantovertime.5246https://doi.org/10.1021/acs.langmuir.1c00182Langmuir2021,37,5242−5251

5Langmuirpubs.acs.org/LangmuirArticle1720cm−1.52,53TheinsetandmagnifiedfiguredemonstratesavaryingamountsofresidualwaterinthesampleasitmoleculartransformationintheGAsupernatantovertimeasevaporates.After30min,absorbanceincreasessharplyasthecomparedtoprecoupledGA.conjugationtimefurtherincreases,thuscreatinga“dip”intheFigure8showstheabsorbancevariationofeachvibrationalabsorptionlines’intensities.This“dip”patternwasobservedinbandofCO,C−H,andO−H.Theaverageabsorbanceofalleverysinglereplicationofeachtrial.Thedecreasingabsorbancefrom0to30mininCOandC−Hcanbeexplainedbythealkali-inducedaldolcondensationofGA(reaction1ofFigure9).ThisaldolcondensationisinducedbythepositivelychargedsurfaceaminegroupsonthemagneticbeadsbyincreasingthelocalpH21,73value.Therefore,aldolcondensation(Figure9,reaction1)isfacilitatedbytheeliminationofwater,thusinducingadecreaseintheCOandC−Hconcentrationlevelsinthesolution.ThisisseeninFigure9,structureD;forexample,ifthreeGAmonomerscombineinachain,twoCObondswouldbelost,andforeverycentralbackbonechainofthesemoleculesaddedtogether(shownintheredsquarebracketsofstructureD),n−1moleculesofwaterareproduced,thusdecreasingC−Hconcentrations.Furthermore,theshoulderthatarisesaround1653cm−1(themagnifiedportionoftheplotinFigure7)correspondingFigure8.VariationsinabsorbanceofCO,C−H,andO−HtoCCstretchingconfirmsthisaldolcondensation,74andstretchingmodes.Thespectraldatawerenormalizedto1bydividingthisisalsoseeninFigure9,structureDinthecentralthedatabyitsmaximumvaluepriortoplottingtheabsorbancebands.backbone.ThissuggeststhatGAtransformsintoα,β-unsaturatedaldehydeuponmixingwiththemagneticbeads.Thedecreaseinabsorbanceat3420cm−1couldbeunderstoodreplicationsofeachtrialandtherelevantstandarddeviationswereestimated.Theseaverageabsorbancevalueswerebytwofacts.First,cyclichemiacetalisconvertedintoitsnormalizedbydividingbythemaximumvalue(absorbanceoligomerform(Figure9,structureC)duetothepH’sriseoftheprecouplingsolution)ofthecorrespondingbanduponmixingwiththemagneticbeads.Second,itcould,inaposition.Thepurposeofnormalizationistocomparethesmallpart,beduetotheaffinityreactionbetweenthe(−NH)+groupandtheoxygenatomofthecyclichemiacetalvariationofeachbandwiththeotherbands.Itisevidentthat3theabsorbancedrasticallydecreasedateachofthebandsafterviahydrogenbonding(Figure9,reaction6).Withthemixingcoupling.Consideringallthreetrials,thepercentagedropsinofGAandthemagneticbeads,theO−H-containingcyclicabsorbancevalueswere19−22%at1720cm−1,17−30%athemiacetalanditsoligomer(structuresBandC,respectively)2950cm−1,and16−38%at3420cm−1astheconjugationtimeareadsorbedonthesurfaceofthebeadsandproduceanofGAwithmagneticbeadsincreasesuntilthe30minmark.intermediatestructureE,asshowninFigure9,thatmayextractThevariationsbetweentrialsarisefromthecomplexnatureofsomelooselyboundmoleculartransformationsofGAoutofglutaraldehyde.Thepolymerizationrateandtheamountofthethesupernatantduringmagneticseparationduetohydrogenpolymerizedproductvarywitheachtrialandaredifficulttobonding.ThesefactorsmaylowerO−Hconcentrationinthesupernatantandthusthedecreasedabsorbanceat3420cm−1replicatewiththesameconditionsandthesamemolecularformproportionseachtimeinGA,leadingtodifferencesinfrom0to30min.72absorbancebetweentrials.Particularly,thehighervariationHowever,overtime,reversereactionsbecomefavorabletoinO−H(16to38%)mightalsobeduetothecontributionofcompensateforthedecreaseinGAandcyclichemiacetalFigure9.(A−E)DifferentmolecularformsofGAinareactionmedium:(A)GA,(B)cyclichemiacetal,(C)cyclichemiacetaloligomer,and(D)α,β-unsaturatedaldehyde,and(E)intermediatecomplexofcyclichemiacetalwithmagneticbeads.5247https://doi.org/10.1021/acs.langmuir.1c00182Langmuir2021,37,5242−5251

6Langmuirpubs.acs.org/LangmuirArticleFigure10.(a)UV−visspectraof0.1MPWBand25%aqueousGA,(b)UV−visspectraoftheGAsupernatantovertime,(c)GaussiancurvefittedspectraforasampleGAsupernatantUV−visspectrum(black),and(d)variationsinGaussianfittedpeaksat235and280nm.concentrations,andthus,thesereversereactionsmaintainAsshowninFigure10b,therewasanupwardshiftobservedequilibriumintheaqueousmedium.So,from30mintointhesmallshoulder(redcircle)at235nmovertime.Thisaround1h,thereversereactions3,5,and7inFigure9arepeakat235nmcouldbeassignedtotheπ−π*excitationin18nowexpectedtohappen.Furthermore,thesereversereactionsCCofα,β-unsaturatedaldehyde(Figure9,structureD).couldbefasterthanforwardreaction1becausetheincreasingTypically,commerciallyavailableGAhasvirtuallyzerooratconcentrationofα,β-unsaturatedaldehydecandecreasetheleastanegligibleamountofα,β-unsaturatedaldehydeinacidicspeedofreaction1overtime.So,allofthesereactionscouldconditions.However,dependingonthereactionenvironment,73itcouldundergoaldolcondensation.leadtoanincreaseintheconcentrationoftheGAmonomerFigure11showstheestimatedpeakheightat235and280andcyclichemiacetal.Wehypothesizethatthiscouldincreasenmfortheaverageofallthreetrialsovertime.Theabsorbancetheabsorbanceagainuntilabout1hlater,andthen,theabsorbanceslowlydecreases.Thesebehaviorswerenoticedinalltrials.Theslightdecreaseintheabsorbance,after1handuptoatleast3hlater,couldbeduetothecouplingofGAwiththemagneticbeadsandcouldalsobeduetothepreviouslymentionedslowedforwardreaction1thatcontinuesuntilGAreachesequilibrium.Therefore,thesepeculiarchangesinabsorbancerevealthatGAundergoesmoleculartrans-formationsuponmixingwiththemagneticbeadsasitreactsandchangestheequilibriumofGAinthesolution.Afteratleast1hagain,iteventuallycomestoequilibrium.UV−VisAnalysisoftheSupernatant.TounderstandthepeculiarchangesofFT-IRabsorbance,weexaminedtheGAsupernatantwithaNanodropOneC(ThermoScientific)UV−Figure11.Variationsinabsorbanceat235and280nm.visspectrometer.Figure10ashows25%aqueousGAabsorbanceindistilledwater(asprovidedbyBangsat235nmincreaseduntil3haftermixing,buttherateofLaboratories,Inc.)versus0.1MPWB.Acleardifferencewaschangewashigherinitiallyandthendecreased.Thisisnoticedbetweentheabsorbancespectra.evidenceofaldolcondensationofGAintoα,β-unsaturatedInFigure10b,thevariationsintheUV−visspectraofthealdehydeovertime.AsexplainedbeforeintheFT-IRanalysis,GAsupernatantovertimeareshown.WeusedaGaussianasamine-modifiedbeadsweremixedwiththeGAsolution,functionforthecurvefittingtoidentifytheindividualpeaksofreaction1wasstimulatedduetotheincreaseinthepHtheGAsupernatanttoanalyzeeachspectrum(Figure10c)level.18,21,22Furthermore,itcanalsobeconcludedthatthemorethoroughly.Thecurvefittedpeaksat235and280nmcondensationratewashigherattheearlystagesandthenovertime(Figure10d)wereanalyzedtounderstandthesloweddownwithtime.Theseresultsshowconsistencywithmoleculartransformationsovertime.theresultsrevealedbytheFT-IRstudy.5248https://doi.org/10.1021/acs.langmuir.1c00182Langmuir2021,37,5242−5251

7Langmuirpubs.acs.org/LangmuirArticleAsseeninFigure10a,25%aqueousGAhastwodistinctNotesabsorbancebandsat198nmandabroadabsorbancecenteredTheauthorsdeclarenocompetingfinancialinterest.at280nm.Theabsorbanceat198nmmightbeduetotheconjugatedoraromaticformationofGA,wherethesetypesof■75ACKNOWLEDGMENTSstructurestypicallyabsorbinthe200−380nmrange.ThisThisworkwassupportedbySIUC’snewfacultystart-upfund.peakisfoundtobeshiftedto193nm(darkblueGaussianpeakinFigure10c)inthesupernatant.ThiscouldbeduetothepHchangesonGAasitwasinthereactionmedium.75■REFERENCESTherefore,weanalyzedthevariationofthepeakat280nm(1)McGinley,H.R.ASIAdhesive&SealantsIndustry,thatarisesfromthefreealdehyde(CO)n−π*carbonylGlutaraldehydeusesandcounterfeits.https://www.adhesivesmag.18,76com/articles/91766-glutaraldehyde-uses-and-counterfeits(accessedexcitation.AsonecannoticeinFigure11,theabsorbanceJul4,2020).keptdecreasinguntil30min,whichisattributedtothealdol(2)NationalLibraryofMedicine.Glutaraldehyde;https://pubchem.condensationofGA.AsweconfirmedbeforeintheFT-IRncbi.nlm.nih.gov/compound/Glutaraldehyde(accessedJul4,2020).study,uponmixingwiththemagneticbeads,theGAsolution(3)CentersforDiseaseControlandPrevention.https://www.cdc.gov/transformsintoanα,β-unsaturatedstructurethroughthealdolniosh/topics/glutaraldehyde/default.html(accessedNov4,2020).condensation.ThiscausesadecreaseintheGAconcen-(4)Gorman,S.P.;Scott,E.M.;Russell,A.D.AntimicrobialActivity,tration’sCOlevelinthesolution,andthus,absorbanceUsesandMechanismofActionofGlutaraldehyde.J.Appl.Bacteriol.decreased.However,after30min,asthereversereactions3,5,1980,48,161−190.and7fromFigure9werefavoredtomaintainequilibrium,the(5)Angelillo,I.F.;Bianco,A.;Nobile,C.G.A.;Pavia,M.EvaluationGAconcentrationstartedtoincrease.ThismadetheoftheEfficacyofGlutaraldehydeandPeroxygenforDisinfectionofDentalInstruments.Lett.Appl.Microbiol.1998,27,292−296.absorbanceincreaseuntil1to2hlater.After1to2h,the(6)Salehi,N.;Moghimi,A.;Shahbazi,H.PreparationofCross-absorbanceslowlydecreaseduntil3hasequilibriumwasLinkedMagneticChitosanwithMethionine-Glutaraldehydeforapproached.Again,theseresultsagreewiththeFT-IRstudy.RemovalofHeavyMetalsfromAqueousSolutions.Int.J.Environ.Anal.Chem.2020,1−17.■CONCLUSIONS(7)Wang,Y.;Mo,X.;Sun,X.S.;Wang,D.SoyProteinAdhesionThisstudyinvestigatedchangesintheFT-IR,Raman,andEnhancedbyGlutaraldehydeCrosslink.J.Appl.Polym.Sci.2007,104,UV−visspectraovertimeduetotheconjugationand130−136.moleculartransformationsofGAuponthereactionwith(8)ThermoFisherScientific.BioconjugationTechnicalHandbook;ThermoFisherScientific,p72.−NH2-modifiedmagneticbeads.OurRamanandFT-IRstudy(9)Park,S.K.;Bae,D.H.;Rhee,K.C.SoyProteinBiopolymersonthemagneticbeadsshowedthesignaturesofGAanditsCross-LinkedwithGlutaraldehyde.J.Am.OilChem.Soc.2000,77,othermolecularformsofcyclichemiacetalonthebeads.879−884.Additionally,theappearanceofnewvibrationalbandsshows(10)Ferrer,I.;Thurman,E.M.AnalysisofHydraulicFracturingtheformationofnewbondsbetweenGAandthesurfaceAdditivesbyLC/Q-TOF-MS.Anal.Bioanal.Chem.2015,407,6417−groupsonthebeads.Therefore,theseconfirmthepresenceof6428.GAonthemagneticbeads.Furthermore,itwasshownthatthe(11)Rogers,J.D.;Ferrer,I.;Tummings,S.S.;Bielefeldt,A.R.;equilibriumstateofGA,alongwithitsothermolecularforms,Ryan,J.N.InhibitionofBiodegradationofHydraulicFracturinginadditiontothepreviouslyreportedinfluenceofthepHCompoundsbyGlutaraldehyde:GroundwaterColumnandMicro-medium,canalsobedistortedbythefunctionalgroupsonthecosmExperiments.Environ.Sci.Technol.2017,51,10251−10261.(12)Okuda,K.;Urabe,I.;Yamada,Y.;Okada,H.ReactionofmagneticbeadsastheyreactwithGA.Basedonourresults,weGlutaraldehydewithAminoandThiolCompounds.J.Ferment.Bioeng.concludethatGAundergoespolymerizationsthroughthealdol1991,71,100−105.condensationofmonomericGAandalsogeneratesoligomer(13)Richards,F.M.;Knowles,J.R.GlutaraldehydeasaProteinformsofthecyclichemiacetalduringconjugationwiththeCross-LinkingReagent.J.Mol.Biol.1968,37,231−233.magneticbeads.However,thereactionsfromaldehydeto(14)Aso,C.;Aito,Y.Intramolecular-IntermolecularPolymerizationhydratedcyclichemiacetalstructuresandfromhydratedcyclicofGlutaraldehyde.Bull.Chem.Soc.Jpn.1962,35,1426−1426.hemiacetaltooligomerformsofcyclichemiacetalstructures(15)Walt,D.R.;Agayn,V.I.TheChemistryofEnzymeandProtein(reactions2and4inFigure9)canbereverseddependingonImmobilizationwithGlutaraldehyde.TrAC,TrendsAnal.Chem.1994,theconcentrationofeachmolecularformandthepHasthey13,425−430.changeovertime.(16)Gowda,S.G.B.;Nakahashi,A.;Yamane,K.;Nakahashi,S.;Murai,Y.;Siddegowda,A.K.C.;Hammam,M.A.S.;Monde,K.■FacileChemoselectiveStrategytowardCapturingSphingoidBasesbyAUTHORINFORMATIONaUniqueGlutaraldehyde-FunctionalizedResin.ACSOmega2018,3,CorrespondingAuthor753−759.PoopalasingamSivakumar−DepartmentofPhysics,Southern(17)Korn,A.H.;Feairheller,S.H.;Filachoine,E.M.IllinoisUniversity,Carbondale,Illinois62901,UnitedStates;Glutaraldehyde:NatureoftheReagent.J.Mol.Biol.1972,65,525−orcid.org/0000-0001-5250-9325;Phone:+1-618-453-529.2272;Email:psivakumar@siu.edu(18)Kawahara,J.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