Layer-by-Layer Assembly of Polycations and Polyanions for the Sensitive Detection of Endotoxin - Posha, Kuttoth, Sandhyarani - 2021 - Un

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pubs.acs.org/LangmuirArticleLayer-by-LayerAssemblyofPolycationsandPolyanionsfortheSensitiveDetectionofEndotoxinBiyasPosha,HarithaKuttoth,andN.Sandhyarani*CiteThis:Langmuir2021,37,257−265ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Bacterialendotoxindetectionisanessentialsafetyrequisiteinbiomedical,food,andpharmaceuticalindustries.Endotoxininasufficientconcentrationonenteringthehumanbloodstreamcausesdetrimentaleffectssuchassepticshock,whichcanleadtodeath.Hence,thesensitiveandselectivedetectionofendotoxinalsoknownaslipopolysaccharide(LPS)isofparamountimportance.Herein,alayer-by-layer(LBL)assemblyofgold-chitosannanocomposite(CGNC)−poly(acrylicacid)(PAA)−polymyxinB(PmB)ongold(Au)electrodeisemployedforthesensitiveandselectivedetectionofendotoxin.Thesurfaceelectricchargestudiesusingdynamiccontactmodeelectrostaticforcemicroscopy(DC-EFM)revealedthesuccessfulformationofeachlayerontheAuelectrode.ThepolycationicPmBisaspecificbioreceptorofLPS,whichbindswithhighaffinitytotheanionicgroupsofthecarbohydrateportionsofLPSmoleculesandfacilitatestheselectiveelectrochemicaldetection.Thissurfacemodificationmethodpresentedasensitiveandselectivedetectionofendotoxindowntotheattogramlevel.678■INTRODUCTIONincludingcolorimetric,fluorimetric,electrochemical,and9ThemajororganismsthatcausesepsisareGrambacteriasuchchemiluminescentmethods,inwhichelectrochemicalmeth-asEscherichiacoli,Streptococcuspyogenes,Staphylococcusaureus,odsofferrapiddetectionwithaverylowdetectionlimit.Klebsiellaspp,andPseudomonasaeruginosa.E.coliisoneoftheVariouselectrochemicalapproacheshavebeendemonstratedmostwidespreadGram-negativebacteria(GNB),whichforthesensitivedetectionofananalyte,suchasenzyme-producesfood-bornediseasesduetoitstoxicnature.Thecatalyzedrecyclingofelectrodematerial,redoxcompound-reasonforthetoxicityofGNBisendotoxin,alsocalledmediatedelectrochemicalsensing,andsignalamplificationby10−12lipopolysaccharides(LPS),acomponentoftheouterDNAhybridization.Comparedwiththesemethods,1modificationofelectrode−biointerfaceusingasimpleelectro-membraneofthebacterialcellwall.WhenbacteriadieandDownloadedviaUNIVOFCONNECTICUTonMay16,2021at11:26:04(UTC).dissolve,LPSisreleasedtothesurroundings.LPSiscomposedstaticlayer-by-layer(LBL)assemblyismoreviable.ofthreedifferentparts:nonpolarlipidA,O-specificantigen,Goldnanoparticlesareoneofthemostwidelyexploredandcorepolysaccharide,inwhichlipidAisresponsibleforthemetalnanoparticlesinthemedicalfieldforvariousapplicationsSeehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.toxicity.2EvenasmallamountofLPSreleasedintothehuman13suchasbiosensing,bioimaging,anddrugdelivery.Specifi-bloodstreaminitiatesanexcessiveinnateimmuneresponse,cally,extensiveutilizationofAunanoparticlesinbiosensingisresultingininflammatoryreactionsleadingtovascularbloodnoteworthy.Varioustypesofcappingagentshavebeenusedtoclotting,hypotension,septicshock,andmultipleorganstabilizeAunanoparticles,whicharefurtherutilizedtomodify3,4failure.Thisseriousmedicalemergencytermedsepsiscantheelectrodesurface.Dependinguponthefunctionalityofthebedefinedasasystemicinflammatoryresponsesyndromecappingagent,differenttypesofchemicalgroupscanbe(SIRS)causedbyabacterialinfection.Thesepathogenicimmobilizedontheelectrodesurface.Theaptselectionofbacteriaalsoinducecontaminationindrugs,food,andcappingagentsisvitalfortheefficientusageoftheelectrodetobiomedicalequipment.Therefore,thesensitiveandaccurateachievespecificbindingofthebiomolecule.DevelopmentofmeasurementofLPShashighimportanceinclinicaldiagnosis,environmentalmonitoring,andfoodandpharmaceuticalsafety.Received:September28,2020ThestandardtechniqueusedforLPSdetectionisLimulusRevised:December10,2020amebocytelysate(LAL)assay,whichhasgoodsensitivityandPublished:December23,20205highselectivitytoLPS.However,thisassayisrelativelyexpensiveandtime-consuming.Therefore,severalLAL-reagent-freetechniqueshavebeendevelopedforthedetection,©2021AmericanChemicalSocietyhttps://dx.doi.org/10.1021/acs.langmuir.0c02852257Langmuir2021,37,257−265

1Langmuirpubs.acs.org/LangmuirArticlebiocompatiblenanoparticlesishighlydesirableforreal-timeTetrachloroauricacidwasobtainedfromSpectrochem,India.BSA,applications.Chitosanisanaturallyoccurringbiopolymerpotassiumferricyanide,potassiumchloride,glucose,sulfuricacid(98obtainedbythedeacetylationofchitin,whichisisolatedfrom%),andaceticacidwerepurchasedfromMerck,India.Phosphate-theshellsofcrustaceansandthecellwallsofmanyfungi.14bufferedsaline(PBS,pH7.4)preparedfrom0.1MKH2PO4(100mL)and0.1MNaOH(78mL)wasusedforthedetectionofanalyte.Chitosanisthesecond-mostabundantnaturalpolymerandhas15KH2PO4andNaOHweresuppliedbyMerck.TheCentralInstituteofbeenusedforanumberofapplications.IthasuniqueFisheriesTechnology(CIFT),Cochin,India,providedbiomedical-physicochemicalpropertiessuchaschelation,polycationic,gradechitosan.Thedegreeofdeacetylationofchitosanwas85%16biodegradable,nontoxic,andfilm-formingcharacteristics.(molecularweight:270kDa).Thiswasusedasreceived.DeionizedThus,itactsasanexcellentmatrixfortheimmobilizationofdistilled(DI)water(resistivity:18.2MΩcm)collectedfrombiomolecules.MilliporeMilli-QwaterpurificationsystemwasusedforallInthiswork,anLBLassemblyofpolycation−polyanion−experiments.polycationhasbeenutilizedforthemodificationoftheSynthesisofChitosan-GoldNanocomposite(CGNC).CGNCelectrodeinterface.MostofthepreviouslyreportedLPSwassynthesizedusingthereportedprocedurefromourresearch21group.Briefly,12mLof1%chitosansolutionwaspreparedin2%electrochemicalsensorshaveusedbioreceptorssuchasaceticacid.HAuCl4solution(25mL,5mM)wasaddedtothisprotein,aptamer,andenzymetofixLPSontothesensing17,18solutionandstirredfor1h.Tothissolution,2mLof10mMice-coldelectrodesurface.Comparedtootherbioreceptors,sodiumborohydridesolutionwasadded.StirringwascontinuedforpolymyxinB(PmB)islessexpensive,morestable,highlytwomorehours.TheformationofCGNCwasclearfromthevisiblespecific,andmoreuniversal.Thehighpositivechargeonthecolorchangefrompaleyellowtorubyred.ThepHofthecolloidalPmBhelpstoneutralizethetoxicityofLPS.Thepolycationicsuspensionwas3.4,andthenthepHwasadjustedto4.5byaddingmoleculePmBhashighaffinitytowardLPS19withanNaOHsolution.Thecolloidalsuspensionwascentrifuged,washedequilibriumassociationconstantof∼2.5×106M−1.20Herein,threetimeswithdistilledwater,andstoredatroomtemperature.wechosePmBastheLPSbindingmoleculeontheelectrodeFabricationofPmB/PAA/CGNC/AuElectrode.Goldfoil(0.5cm×1cm)wasusedasthesubstratefortheconstructionoftheLBLsurface.ToimmobilizethePmBonthesurface,theLBLassembly.Beforemodification,thegoldfoilwasmechanicallyrubbedtechniqueisusedwithanionicpoly(acrylicacid)(PAA)andwithaluminaslurries(1and0.5μm)onaflatpad,washedwithDIcationicchitosangoldnanoparticles(CGNCs).Theelectrodewater,andsonicatedsuccessivelyinconc.H2SO4,ethanol,andDIinterfacemodificationisbythesimpleelectrostaticinteraction.waterfor10mineach.Afterward,electrochemicalcleaningwasCGNCsareutilizedtoovercometheinsulatingpropertyoftheperformedinHClO4bycyclingthepotentialfrom0.2and1.5Vforpolyanionsandpolycations.10min(scanrate,0.1Vs−1).FigureS1exhibitstherepeatedCVsofScheme1indicatesthedifferentstepsinvolvedintheLBLtheAuelectrodeduringelectrochemicalcyclingin0.1MHClO4(22assemblyforelectrodemodification.Initially,athinfilmofcycles).TherepeatedcyclesleadtotheformationofstableAuoxideandreductionpeaks.ThefinalinvariablereductionpeakcorrespondstothecleanAuelectrodesurface.TheCGNCwasdepositedontheScheme1.SchematicRepresentationoftheElectrodeelectrochemicallycleanedanddriedelectrodebyapplyingavoltageofSurfacePmB/PAA/CGNC/Au3VusingaDCpowersupplyfor1minatasolutionpHof4.5.ThemodifiedCGNC/AuwaswashedwithDIwater.Afterdrying,theelectrodewasimmersedinPAAsolution(50μLof35wt%PAAin2mLwater)for3h.ThePAA/CGNC/AuwaswashedwithwatertoremovetheunboundPAA.ThePAA/CGNC/AuwasimmersedinPmB(100μM,130μgmL−1)andkeptfor12h.Afterwashinganddrying,PmB/PAA/CGNC/Auwasincubatedin1mMbovineserumalbumin(BSA)for1mintoavoidanynonspecificbinding.Afterthesemodifications,theelectrodewaswashedwithDIwaterandkeptfordrying.ThisPmB/PAA/GCNC/AuisusedforLPSdetection.Techniques.UV−visabsorptionmeasurementsweredonewithaUV-1800Shimadzuspectrometer.Aquartzcuvetteofpathlength1cmwasusedforcollectingthespectrum.Fieldemissionscanningelectronmicroscopy(FE-SEM)imageswereobtainedusingafieldemissionscanningelectronmicroscope(SEM,SU6600pressure-CGNCwaselectrochemicallydepositedontheAuelectrode.variable(Hitachi,Japan)andhigh-magnificationimageswereThen,theCGNCdepositedelectrodewasimmersedinanionicobtainedusingCarlZeissGeminiSEM300).APerkinElmerfrontierPAA,followedbytheimmersionincationicPmB.TheFouriertransforminfrared(FT-IR)spectrometerwasusedtorecordelectrostaticinteractionbetweenthepolycationandpolyaniontheFT-IRspectra.Attenuatedtotalreflection-infrared(ATR-IR)withwasheldtogetherwiththepositivelychargedCGNC,adiamondtipwasusedtorecordthedata.AParkatomicforcenegativelychargedPAA,andpositivelychargedPmBonthemicroscope(AFM)XE100wasemployedfordynamiccontactmodeAuelectrodesurface.Then,PmB/PAA/CGNC/Auwaselectrostaticforcemicroscopy(DC-EFM)usinganNSC14-Cr/Autipwitharadiusof<50nm,springconstantK=1.8Nm−1,andresonantimmersedinbovineserumalbumin(BSA)for1mintoeliminatethenonspecificbinding.Thiselectrodewasusedforfrequencyof160Hz,whichprovideasemiquantitativeanalysisoftheelectrostaticpotentialasasurfacephenomenon.Thetopographicthedetectionofendotoxin.ThisLBLassemblyproducedainformationandelectrostaticpotentialoftheelectrodecanbewidelinearvoltammetricdetectionofLPSdowntothemonitoredusingDC-EFMasitworksinthedynamiccontactmode.attogrampermilliliterlevel.Alock-inamplifier(LIA)ofStanfordresearchsystemsSR830wasusedtoacquiretheelectrostaticpotentialalongwithDC-EFM.■EXPERIMENTALSECTIONElectrochemicalexperimentswereperformedonCHI760Eelectro-Materials.Endotoxinstandard(E.coli0113:K10),dopamine,chemicalworkstation.Astandardthree-electrodesystemwasused,poly(acrylicacid)(averageMw:100000),sodiumborohydride,andwhichincludeselectrochemicallycleanedAuelectrode(workinguricacidweresuppliedbySigma-Aldrich,India.PolymyxinBsulfateelectrode),Ag/AgCl(referenceelectrode),andPtwire(counter(PmB)andascorbicacidwerepurchasedfromHiMedia,India.electrode).Cyclicvoltammetry(CV)andelectrochemicalimpedance258https://dx.doi.org/10.1021/acs.langmuir.0c02852Langmuir2021,37,257−265

2Langmuirpubs.acs.org/LangmuirArticleFigure1.SEMimagesofAuelectrode(A),CGNC/Au(B),PAA/CGNC/Au(C),andPmB/PAA/CGNC/Au(D).Theinsetshowstherespectivehigh-magnificationimages.spectroscopy(EIS)wereusedfortheelectrochemicalcharacterizationSurfacemorphologyofthedifferentstagesofAuelectrodeofelectrodesurfacemodification.EISexperimentswerecarriedoutatmodificationwasstudiedbySEM.Figure1AshowstheSEManamplitudeof0.005Vandinafrequencyrangeof1−106Hz.imageofthepolycrystallineAusurface(Auelectrode).FigureZsimpWinsoftwarewithanappropriateequivalentcircuitwasusedto1BexhibitstheSEMimageofCGNC/Au.ThepresenceoffittheEISdata.Analyticalstudieswerecarriedoutin0.1MPBSusingdifferentialpulsevoltammetry(DPV)with0.2spulseperiod,10Hzchitosanfilmwithembeddedgoldnanoparticlesisevidentonfrequency,0.02ssamplingwidth,50mVamplitude,0.06spulsetheCGNC/Ausurface.Thehigh-magnificationimageshownwidth,and0−0.8Vpotentialwindow.intheinsetclearlyillustratesthehighcoveragedensityofAuPreparationofEndotoxinStockSolution.Thestandardnanoparticlesonthesurface.Thesurfacemorphologywasendotoxinsolutionswerepreparedfromthestocksolutionat1μgchangedwiththesuccessiveimmobilizationofpoly(acrylicmL−1endotoxin.Asetofnineendotoxinstandardsolutions(100,10,−1−1−1acid)(Figure1C)andpolymyxinB(Figure1D)layers.The1ngmL,100,10,1pgmL,and100fgmL)werepreparedfrom−1high-magnificationimageofthePAA/CGNC/Aushowninthethestocksolution(1μgmL)bydilution.Thestocksolutionwasvortexedfor3min,andallotherstandardsolutionswerevortexedforinsetofFigure1CillustratestheformationofthePAAlayeron1minwithmaximumspeedbeforedilution.EachLPSstocksolutionCGNC.Thegoldnanoparticlesarelessvisiblenowduetothe(10μL)wassuccessivelyaddedto10mLof0.1MPBSsolutioncoverageofthePAAlayer.UpontheformationofthePmBduringquantitativeelectrochemicalmeasurements.layer,theelectrodesurfaceshowschargingandtheclarityoftheimagedecreasesduetotheincreasedinsulatingnatureof■theelectrode.RESULTSANDDISCUSSIONTheAuelectrodemodificationateachstagewasalsoThechitosan-stabilizedAunanoparticlesarepositivelymonitoredbydynamiccontactmodeelectrostaticforcecharged,andtheLBLassemblybasedonelectrostaticmicroscopy(DC-EFM).EFMprovidesinformationaboutinteractionrequiresnegativelychargedpolyions;thus,PAAtheelectrostaticpotentialatthesurfaceinadditiontothewasimmobilizedontheCGNClayer.Thenegativelychargedtopographicalinformation.A2μm×2μmareaofthesurfacePAAactsasaperfectlinkbetweentheCGNCandthepositivelychargedLPS-specificpolymyxinB.wasscanned.Togetamoreaccurateresult,multipleareasof2CGNCwaselectrodepositedonanindiumtinoxide(ITO)μm×2μmwerescannedandsimilarvaluesofthesurfaceplate,andthenPAAandPmBlayersweresuccessivelychargepotentialareobservedinallofthemeasurements.immobilizedonCGNC/ITOtoperformUV−visiblespectro-Figure2showstheEFMamplitudesandthethree-dimensionalscopicanalysis.FigureS2showstheUV−visiblespectraofthe(3D)topographicimagesofCGNC/Au(A),PAA/CGNC/AuCGNC,PAA/CGNC,andPmB/PAA/CGNClayersonITO.(B),andPmB/PAA/CGNC/Au(C).TheinsetshowstheThecharacteristicpeakat534nmisduetothesurfacehistogramindicatingthesurfacechargepotential.TheplasmonresonanceofAunanoparticleconfirmingthepresenceelectrodesurfacechargepotentialateachlayerisgiveninofAunanoparticleinCGNCnanocomposites.Anoverallthetable(Figure2).ThechangesinthetopographyofeachdecreaseinabsorbanceisobservedwiththesuccessivemodificationaccountforthesuccessiveformationofeachlayerformationofeachlayerofPAAandPmB.ontheAuelectrode.UponadditionofaPAAlayer,thesurface259https://dx.doi.org/10.1021/acs.langmuir.0c02852Langmuir2021,37,257−265

3Langmuirpubs.acs.org/LangmuirArticleFigure2.SurfaceelectricchargestudiesatmodifiedelectrodesCGNC/Au(A),PAA/CGNC/Au(B),andPmB/PAA/CGNC/Au(C);EFMamplitude,3Dtopographicimage,andthecorrespondingelectrodesurfacechargepotentialinmV(table).TheinsetsintheEFMamplitudeimagesshowhistogramsshowingthesurfacecharge.becomessmootherandfurtheradditionofPmBresultsinanchargeinthe2μm×2μmarea,thePAAcontributionaloneincreasedroughness,probablyduetotheimmobilizationofthecannotbedetected.Thisresultsinanon-negativesurfacebulkyPmBmolecule.TheEFMamplitudeimagesrevealthecharge.AftertheassemblyofthepolycationPmBonPAA/surfaceelectricchargeateachlayer.CGNC/AuexhibitedaCGNC/Au,theelectricsurfacechargeincreasedto11.90andminimumsurfaceelectricchargeof11.29mVandamaximum20.75mVwithameansurfacechargeof+15.55mV,astheof20.44mVwithameanchargeof+14.34mV.ThepositivecationicPmBincreasesthepositivesurfaceelectricpotentialofvalueshowsthepresenceofcationicchitosanmoleculesonthetheelectrode.Thechangesinthesurfaceelectricchargeuponsurface.OnimmobilizingtheanionicPAAonthesurface,thetheadditionofeachlayerrevealthesuccessfulassemblyofchargevariesintherange8.24−14.64mVwithameanvalueofeachpolycationic−polyanionic−polycationiclayer.+11.50mV.ThedecreaseinsurfacechargeisattributedtotheThesuccessiveformationofeachlayerontheAuelectrodeanionicnatureofimmobilizedPAAmolecules.FromSEM,itiswasfurtherconfirmedusingATRFT-IRspectroscopy.ItmayclearthattherearedefectsandporesinthelayersofPAA,andbenotedherethatthepenetrationdepthinATRisbetweenhencethereareregionswhereunderlyingCGNCcontributes500nmand2μm;hence,afteradditionofeachlayer,thetothesurfacecharge.SinceEFMmeasurestheaveragesurfaceprominentpeakfromtheunderlyinglayeralsoisvisible.Some260https://dx.doi.org/10.1021/acs.langmuir.0c02852Langmuir2021,37,257−265

4Langmuirpubs.acs.org/LangmuirArticleFigure3.CV(A)andEIS(B)atAu,CGNC/Au,PAA/CGNC/Au,andPmB/PAA/CGNC/Auin0.1MKClcontaining5mM[K3Fe(CN)6].InsetmodifiedRandlescircuit;Rssolutionresistance,Cdldouble-layercapacitance,Qimperfectcapacitor,Rctchargetransferresistance,Rdldouble-layerresistance,WWarburgimpedance.oftheminorpeaksinthetoplayeraregettingmaskedbytheseelectrochemicallydepositedCGNC/Au(Ipa=1120μA).Thebroadandintensepeaksfromtheunderlyinglayer.Hence,allredoxpeakcurrentagainreducedto956.8μAforPAA/ofthevibrationalfeaturesofindividuallayersarenotclearlyCGNC/Au.AfurtherdecreaseinIpawasobtainedfortheBSA-observed.ThoughthepeaksfromdifferentlayersareblockedPmB/PAA/CGNC/Au(Ipa=695μA).Comparedoverlapping,theadditionalpeaksoriginatedbytheadditionwiththebareAu,asuccessivedecreaseinpeakcurrentwasofeachlayerisusedtoconfirmtheimmobilizationofobservedforeachmodification,resultingfromtheinsulatingsuccessivelayers.FigureS3exhibitstheFT-IRspectraofnatureoftheimmobilizedpolycationsandpolyanions,whichCGNC/Au,PAA/CGNC/Au,andPmB/PAA/CGNC/Au.confirmsthesuccessfulformationofeachlayer.TheFT-IRspectrumofCGNC/AushowsalloftheEISexperimentswerecarriedouttoinvestigatetheinterfacecharacteristicpeaksofchitosan.Thepeaksat3293and3363propertiesatdifferentlayersontheAuelectrodesurface.cm−1representN−HandO−Hstretchingvibrations,Figure3BexhibitstheNyquistplotderivedfromtheEISrespectively.The2906and2881cm−1peakscorrespondtomeasurementsateachlayer,andthemodifiedRandlescircuittheC−Hasymmetricandsymmetricstretchingvibrations.Theusedfordatafittingisalsogiveninthefigure.peaksat1642cm−1(COstretchingofamideI)and1325TableS1showstheequivalentcircuitelementvaluescm−1(C−NstretchingofamideIII)originatefromresidualN-obtainedbysimulatingtheEISdata.Rcorrespondstothesacetylgroupsofchitosan.Thepeakat1071cm−1isassignedtosolutionresistancebetweenreferenceandtheworkingtheC−Ostretchingvibrations.ThepresenceofaC−O−Celectrode.Thesemicircledomaininthehigh-frequencyrangebridgewasconfirmedfromthepeakat1054cm−1.ThepeaksoftheNyquistplotindicatestheelectrontransferresistanceat1416and1376cm−1areattributedtoCHbendingand(R)attheelectrode−electrolyteinterface.Cisthedouble-2ctdlCHsymmetricaldeformations,andthepeakat893cm−1islayercapacitanceparalleltoRformedbythecharge3ctduetotheCHout-of-planebendingofthemonosaccharideseparationoftheelectrodesurfaceandthesurrounding22ring.TheFT-IRspectrumofPAA/CGNC/Aushowstheelectrolyte.TheresistanceintheinterfacialionicchargecharacteristicspeaksofPAA.Thepeaksat1710cm−1(COtransferfromthesolutionphasetotheelectroderesultsinthestretchingvibration),1173cm−1(C−Ostretchingvibration),combinationofR(double-layerresistance)andQ(imperfectdland1405cm−1(symmetricCOO−stretchingvibration)capacitororconstantphaseelement,CPE).25ThepresenceofindicateCOOHgroupsinPAA.Thebroadbandnear3550−CPEindicatesthesurfaceroughnessoftheelectrodesurface.3000cm−1isoriginatedfromtheOHstretching.ThesmallForpurecapacitance,thenvaluewillbeequalto1,shiftinthecharacteristicpeakoftheCOOHgroupinPAAisrepresentingasmoothsurface.Anydeviationfromn=1duetotheintertwistingwiththechitosannetworkthroughanindicatessurfaceroughness.TheWarburgimpedance(W)23electrostaticforce.TheFT-IRspectrumofPmB/PAA/representsthediffusionresistanceoriginatedfromthemassCGNC/AushowsthecharacteristicpeaksofPmB.Thepeaksdiffusionoftheionsfromthebulktotheelectrode.TheAuat2934and2857cm−1representthesymmetricandelectrodeexhibitedasmallresistance,andtheRisfoundtoctasymmetricstretchingvibrationsofC−H.Thepeakat1655be9.18Ω.AfterdepositingCGNContheAuelectrode,thecm−1correspondstotheCOstretchingofamideIpresentinresistanceslightlyincreasedto12.34Ω.ThechargetransferPmB.Thepeakataround1533cm−1isduetotheN−Hresistancefurtherincreasedto19.34Ωafterincubatingin24deformationofamideII.ThebroadpeakspanningfromPAA.Theincrementinresistancecanbeassignedtothe3650cm−1isduetotheO−Hstretching.ThesmallpeakatassemblyofPAAonCGNC/Au.TheRagainincreasedtoct∼3250cm−1,whichislargelymaskedbytheO−Hstretching,25.66ΩatthePmB/PAA/CGNC/AuelectrodeafterblockedindicatestheNH2stretchinginPmB.TheseresultssuggestthewithBSA,sincebindingofPmBattheelectrodesurfacesuccessfulformationofeachlayeronAuelectrode.inducessterichindranceandtherebyslowsdowntheelectronTheelectrodemodificationwasmonitoredusingcyclictransferrate.ThesuccessiveincrementinRctconfirmsthevoltammetry(scanrate100mVs−1)andEISin5mMformationofeachlayerontheAuelectrode.TheseresultsK3[Fe(CN)6]containing0.1MKClatthemetalelectrodeconfirmthesuccessfulformationofeachlayerontheelectrodesurface.TheCVsoftheAuelectrode,CGNC/Au,PAA/surface.CGNC/Au,andBSA-blockedPmB/PAA/CGNC/AuareThesuccessfulformationoflayersandtheirelectrochemicalillustratedinFigure3A.Inthefigure,thetypicalredoxpeakbehaviorwasfurtherevaluatedbymonitoringthecyclicofK3[Fe(CN)6]isobservedontheAuelectrodewithanIpaofvoltammetryinPBSaftertheadditionofeachlayer.FigureS41240μA.AslightdecreaseinpeakcurrentwasobservedontheshowsthecyclicvoltammogramofAuelectrode,CGNC/Au,261https://dx.doi.org/10.1021/acs.langmuir.0c02852Langmuir2021,37,257−265

5Langmuirpubs.acs.org/LangmuirArticleFigure4.(A)DPVplotsofPmB/PAA/CGNC/AuincubatedinvariousLPSconcentrationsin0.1MPBS.(B)LinearplotofΔIpvsthelogarithmvalueofanalyteconcentration.Scheme2.ProposedMechanismofLPSSensingbytheLBL-AssembledPmB/PAA/CGNC/AuElectrodePAA/CGNC/Au,andPmB/PAA/CGNC/Au0.1MinPBS.foundtobe73.0agmL−1at3σbasedonthestandardThepeakat0.43VisduetothesurfaceoxidationofAu−OHdeviationoftheblanksignal(n=3).ThecurrentresponseoftoAuoxideortheadsorbedphosphatefromthePBS.UponfivesuccessivemeasurementswasevaluatedusingDPV.ThetheadditionofCGNC,thepeakcurrentenhancedduetotherelativestandarddeviation(RSD)wasfoundtobe1.91%forimprovedreactioninthepresenceofAunanoparticles.0.1ngmL−1oftheanalyte,whichconfirmstheexcellentHowever,uponsubsequentadditionofPAAandPmB,thereproducibilityofthebioprobe.TostudytheroleofPmB,LPSpeakcurrentgetsdecreasedduetotheinsulatingnatureofdetectionwasmonitoredatthecationicCGNC/Au,andthePAAandPmB.TheCVconfirmsthesuccessfulformationofresultsareshowninFigureS5.Thealmostsamecurrenteachlayer.Sincethepeakcurrentdependsonthelayersresponsewasobtainedforvariousconcentrationsofanalytepresentonthesurface,thisistakenasthesensingpeak.(100agmL−1to0.1ngmL−1)atCGNC/Au,whichindicatesTheelectrochemicalsensingperformanceofPmB/PAA/thatsensitiveandselectiveLPSdetectionispossibleonlyinCGNC/AuwasevaluatedusingDPVin0.1MPBS.Figure4AthepresenceofPmB.AslightdecreaseincurrentobservedshowstheelectrochemicalresponseofthesensorforvariouswithincreasingconcentrationofLPSisduetothebindingofconcentrationsofLPSatPmB/PAA/CGNC/Au.ThePmB/insulatingLPSonCGNC.PAA/CGNC/Aushowsapeakcurrentof16.52μAat0.31V.IntheDPV,theanodicpeakat0.4VismonitoredfortheThepeakcurrentobtainedforthesensorat0.31Vincreasedsensingpurpose.ItisobservedthatthepeakcurrentincreaseswithincreasingconcentrationofendotoxinandshowsashifttowithincreasingconcentrationofLPS.Asdiscussedearlier,the0.4V.Thelinearplotofquantitativedetection,ΔIpvspeakmaybeduetothesurfaceoxidationofAuhydroxideorlogarithmofconcentration,isshowninFigure4B.AlineartheadsorbedphosphateintheAuelectrodeandthegoldrangeofdetectionisobservedfrom100agmL−1to0.1ngnanoparticleinCGNC.ThepeakcurrentisfeeblewhenthemL−1withcorrelationcoefficientsof0.98(forlowerinsulatingPAAandPmBlayersarepresentonthesurfaceconcentrations)and0.99(forhigherconcentrations).Theblockingtheelectrontransfer.TheLBLassemblyofthelimitofdetection(LOD)oftheconstructedelectrodewasCGNC,PAA,andPmBwasachievedonthesurfacebythe262https://dx.doi.org/10.1021/acs.langmuir.0c02852Langmuir2021,37,257−265

6Langmuirpubs.acs.org/LangmuirArticleelectrostaticattraction.FromtheDC-EFMstudy,itisevidentselectivityisduetothehighspecificityofPmB/PAA/CGNC/thatthePAAlayeriseithernotveryuniformorformedasaAuelectrodetowardLPS.thinlayer,resultinginanetpositivechargeonthesurface.ThestabilityofPmB/PAA/CGNC/AuwasalsomonitoredBasedontheresultsobtained,thedetectionmechanismisbystoringCGNCatroomtemperaturefor1month.A92.91proposedasfollows.AlthoughtheimmobilizationofPmBon%signalretentionwasobserved,whichconfirmsthelong-termthesurfaceensues,theinteractionofPmBwiththesurfaceisstabilityofthesensor.weakasaresultofthenetpositivechargeonthePAAmodifiedAcomparativestudyoftheanalyticalperformanceofthesurface.DuetothestrongbindingofLPSandPmB,thereportedelectrochemicalbioprobesintheliteratureandthisadditionofLPSatalowerconcentrationinthesolutioncausesLBLassayforLPSdetectionisgiveninTable1.ItisevidentsomeofthePmBtogetdetachedfromthesurfacewhereitwasthatthesimpleandcost-effectiveLBLassemblyofCGNC-bondedthroughtherelativelyweakinteractionbetweenPmBPAA-PmBproducedabroaddetectionrangeandanexcellentandPAA.AstheconcentrationofaddedLPSinthesolutiondetectionlimitforLPScomparedtoothermethodsreported.increases,itbindstomorePmBfromthesurfaceandmoreTodemonstratethepertinenceofthesensorinpracticalinsulatingPmBgetsdetachedfromthesurface,resultinginaclinicalapplications,thePmB/PAA/CGNC/Ausensorwasbetterelectrontransferandanincreaseinanodicpeakcurrent.usedtodeterminetheLPSconcentrationinwholebloodbyTheincreaseinanodicpeakcurrentisfoundtobethestandardadditionmethod.Thewholeblood(50μL)wasproportionaltotheconcentrationofLPS.Theschematicoftakenin10mLof0.1MPBS(pH7.4)andspikedwiththeproposedmechanismofsensingisshowninScheme2.differentknownconcentrationsoftheLPS.DPVwasusedforTheselectivityoftheelectrodewasmonitoredbycheckingthebloodsampleanalysis.Linearityisobtainedfrom100agthecross-reactivityofpossiblecoexistinginterferingcom-mL−1to10pgmL−1(R2=0.949).PmB/PAA/CGNC/Aupoundswithanalyte,suchasglucose(Glu),ascorbicacidsensorproducedexcellentrecoverytowardvariousLPS(AA),bovineserumalbumin(BSA),uricacid(UA),andconcentrationssuchas100agmL−1(104.31%),1fgmL−1dopamine(DA).DPVwasusedtoconducttheselectivitytest(97.60%),10fgmL−1(97.36%),100fgmL−1(99.62%),1pgatPmB/PAA/CGNC/Au.Figure5illustratesthehistogramofmL−1(99.34%),and10pgmL−1(102.72%).TheDPVplotsandthecorrespondingcalibrationplotofΔIpvslogC(gmL−1)ofLPSaregiveninFigureS6.Here,asthedilutionusedis200×,theconcentrationofLPSinbloodvariesfrom20fgmL−1to2ngmL−1.Itwasreportedthattheserumendotoxinconcentrationisintherangeof500pgmL−1inpatientswith37endotoxemiaandsepticshock.ThedetectablerangeofLPSwellbelowthislevelindicatestheusefulnessofthepresentsensorinthedetectionofsepsis.■CONCLUSIONSInshort,alow-costLBLassemblywasusedfortheFigure5.SelectivityofthePmB/PAA/CGNC/Ausensor;detectionelectrochemicaldetectionofendotoxin.Thelayer-by-layerofLPS100pgmL−1againsttheinterferencecompounds(10ngassemblywasdevelopedusingelectrostaticinteractionbetweenmL−1):AA,DA,glucose,BSA,andUA.cationicCGNC,anionicPAA,andcationicPmB.TheelectrostaticinteractionbetweenthepolyionsledtoasensitivedetectionofanalytewithoutusinganycomplicatedsignaltheresponseofPmB/PAA/CGNC/Autowardthetarget(100amplificationstrategyandredoxmediator.TheelectrochemicalpgmL−1)anddifferentinterferentswitha100timeshigherPmB/PAA/CGNC/AuoffersanexcellentLODof73.0agconcentration(10ngmL−1).TheLPSshowedahighresponsemL−1forLPSwithawidelineardetectionrangeof100agmL−1to0.1ngmL−1LPS.ThissensoralsoexhibitedgoodsignalatPmB/PAA/CGNC/Aucomparedtotheinterferentssuggestingthehighselectivityofthebioprobe.Thisexcellentstability,selectivity,andreproducibility.ThesuccessfulTable1.OverviewofRecentlyReportedWorksfortheDetectionofLPSelectrochemicaltechniqueelectrodemodificationmethodcalibrationrangepermLlimitofdetectionpermLrefs1EISaptamer/AuAC/Au1×10−6−100pg0.79×10−3fg262EISTLR4/NTA-Ni2+/SAMsofMuA/Au1−10000ng1ng273EISaptamer/AuNPs/Au0.01−10.24ng5pg284DPVCu-AuNAs/aptamer/EDC-NHS/MPA/Au0.05−10pg33fg295EISaptamer/MPA/Au1pgto1ng1pg306DPVpeptideassay/Au0.1−50EU0.04EU317DPVPPy-NWs/AptandCu-MOF/Au1.0pgto1.0ng0.29pg328chronocoulometrydual-enzymeamplification(ExoIIIandTdT)-aptamer/Au2.5−1000pg1pg339CVPmB/MPA/EDC-NHS/Au(portabledevice)1−100μg1μg3410EISPmB/EDC-NHS/DTBA/Au0.2−0.8ng0.2ng3511DPVPmB/poroussiliconnanochannel/Au1−10000ng1.8ng3612DPVPmB/PAA/CGNC/Au100agto0.1ng73agthiswork263https://dx.doi.org/10.1021/acs.langmuir.0c02852Langmuir2021,37,257−265

7Langmuirpubs.acs.org/LangmuirArticlerecoveryofLPSwithintheminimumerrorlimitinwhole(3)Mizumura,H.;Ogura,N.;Aketagawa,J.;Aizawa,M.;Kobayashi,bloodrevealsthefeasibilityofthesensortowardpracticalY.;Kawabata,S.;Oda,T.Geneticengineeringapproachtodevelopclinicalapplications.ThisLBLassemblycanbeusedforthenext-generationreagentsforendotoxinquantification.InnateImmun.sensingofotherrelevantbiomoleculesbyplacingaspecific2017,23,136−146.cationicbioreceptorinsteadofpolymyxinB.(4)Alahi,M.E.E.;Mukhopadhyay,S.C.Detectionmethodologiesforpathogenandtoxins:AReview.Sensors2017,17,1885.■(5)Ding,J.L.;Ho,B.Anewerainpyrogentesting.TrendsASSOCIATEDCONTENTBiotechnol.2001,19,277−281.*sıSupportingInformation(6)Li,D.;Sun,T.;Zhang,W.;Shen,Z.;Zhang,J.ColorimetricTheSupportingInformationisavailablefreeofchargeatanalysisoflipopolysaccharidesbasedonitsself-assemblytoinhibitionhttps://pubs.acs.org/doi/10.1021/acs.langmuir.0c02852.transport.Anal.Chim.Acta2017,99,85−93.CyclicvoltammogramofAuelectrodecleaningby(7)Lan,M.;Wu,J.;Liu,W.;Zhang,W.;Ge,J.;Zhang,H.;Sun,J.;electrochemicalcycling;UV−visspectraofLBL-Zhao,W.;Wang,P.Copolythiophene-derivedcolorimetricandassembledsurface;FT-IRspectraofCGNC/Au,PAA/fluorometricsensorforvisuallysupersensitivedeterminationoflipopolysaccharide.J.Am.Chem.Soc.2012,134,6685−6694.CGNC/Au,andPmB/PAA/CGNC/Au;EISequivalent(8)Posha,B.;Sandhyarani,N.Highlysensitiveendotoxindetectioncircuitelementsvalues;cyclicvoltammogramofAuusingagoldnanoparticleloadedlayeredmolybdenumdisulfide-electrode,CGNC/Au,PAA/CGNC/Au,andPmB/polyacrylicacidnanocomposite.Analyst2020,145,3939−3947.PAA/CGNC/AuinPBS;DPVplotsofdifferent(9)Zhao,M.;Chen,A.-Y.;Huang,D.;Chai,Y.-Q.;Zhuo,Y.;Yuan,concentrationsofLPSatCGNC/Au;andDPVanalysisR.MoS2quantumdotsasnewelectrochemiluminescenceemittersforofwholebloodsamplebystandardadditionmethodandultrasensitivebioanalysisoflipopolysaccharide.Anal.Chem.2017,89,thecorrespondingcalibrationplot(PDF)8335−8342.(10)Shen,W.-J.;Zhuo,Y.;Chai,Y.-Q.;Yuan,R.Cu-basedmetal−■organicframeworksasacatalysttoconstructaratiometricAUTHORINFORMATIONelectrochemicalaptasensorforsensitivelipopolysaccharideDetection.CorrespondingAuthorAnal.Chem.2015,87,11345−11352.N.Sandhyarani−NanoscienceResearchLaboratory,Schoolof(11)Yuan,Y.;Li,L.;Zhao,M.;Zjou,J.;Bai,L.;Chen,Z.AnMaterialsScienceandEngineering,NationalInstituteofaptamerbasedvoltammetricbiosensorforendotoxinsusingaTechnologyCalicut,Calicut673601,Kerala,India;functionalizedgrapheneandmolybdenumdisulfidecompositeasaorcid.org/0000-0002-6623-3347;Phone:91495newnanocarrier.Analyst2019,144,1253−1259.2286537;Email:sandhya@nitc.ac.in;Fax:91495(12)Iijima,S.;Kato,D.;Yabuki,S.;Niwa,O.;Mizutani,F.2287250Enzymaticallyamplifiedelectrochemicaldetectionforlipopolysac-charideusingferrocene-attachedpolymyxinBanditsanalogue.AuthorsBiosens.Bioelectron.2011,26,2080−2084.BiyasPosha−NanoscienceResearchLaboratory,Schoolof(13)Biju,V.ChemicalmodificationsandbioconjugatereactionsofMaterialsScienceandEngineering,NationalInstituteofnanomaterialsforsensing,imaging,drugdeliveryandtherapy.Chem.TechnologyCalicut,Calicut673601,Kerala,IndiaSoc.Rev.2014,43,744−764.HarithaKuttoth−NanoscienceResearchLaboratory,School(14)dosSantos,D.S.;Goulet,P.J.G.;Pieczonka,N.P.W.;ofMaterialsScienceandEngineering,NationalInstituteofOliveira,O.N.;Arora,R.F.Goldnanoparticleembedded,self-TechnologyCalicut,Calicut673601,Kerala,Indiasustainedchitosanfilmsassubstratesforsurface-enhancedramanscattering.Langmuir2004,20,10273−10277.Completecontactinformationisavailableat:(15)Hirano,S.Chitinbiotechnologyapplications.Biotechnol.Annu.https://pubs.acs.org/10.1021/acs.langmuir.0c02852Rev.1996,2,237−258.(16)Shan,C.;Yang,H.;Han,D.;Zhang,Q.;Ivaska,A.;Niu,L.NotesGraphene/AuNPs/chitosannanocompositesfilmforglucosebiosens-Theauthorsdeclarenocompetingfinancialinterest.ing.Biosens.Bioelectron.2010,25,1070−1074.(17)Amini,K.I.;Ebralidze,I.C.;Chan,N.W.;Kraatz,H.-B.■ACKNOWLEDGMENTSCharacterizationofTLR4/MD-2-modifiedAusensorsurfacesTheauthorsthanktheNanoMissionprogramofDepartmenttowardsthedetectionofmolecularsignaturesofbacteria.Anal.ofScienceandTechnology,GovernmentofIndia,fortheMethods2016,8,7623−7631.(18)Kato,D.;Iijima,S.;Kurita,R.;Sato,Y.;Jia,J.;Yabuki,S.;financialsupportthroughtheprojectDST/NM/NB/2018/Mizutani,F.;Niwa,O.Electrochemicallyamplifieddetectionfor152.BiyasPoshaacknowledgesUniversityGrantCommissionlipopolysaccharideusingferrocenylboronicacid.Biosens.Bioelectron.fortheresearchfellowship.Theauthorsacknowledgethehelp2007,22,1527−1531.receivedfromDr.C.K.SubashfortheDC-EFMmeasure-(19)Kato,D.;Oda,A.;Tanaka,M.;Iijima,S.;Kamata,T.;ments.TheygratefullyacknowledgetheCentralInstrumenta-Todokoro,M.;Yoshimi,Y.;Niwa,O.Poly-ε-lysinemodifiedtionFacilityatIndianInstituteofTechnologyPalakkadforthenanocarbonfilmelectrodesforLPSdetection.Electroanalysis2014,high-magnificationSEMimages.26,618−624.(20)Thomas,C.J.;Surolia,A.Kineticsoftheinteractionof■REFERENCESendotoxinwithpolymyxinBanditsanalogs:asurfaceplasmon(1)Ulevitch,R.J.;Tobias,P.S.Receptor-dependentmechanismsofresonanceanal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