Cascading chirality from molecule to twisted microstructures with amplified circularly polarized luminescence

Domain: chemistry
Published: 2026-01-16
Editorial outcome: minor_revision

Abstract
Controlofchiralassemblyacrosslengthscalesisdesirablebutchallenging.Herein,wedemonstratecontrollableconstructionofmicroscopicchiraltwiststhroughhierarchicalassemblyofenantiomericstyrylpyridinium-derivedglutamideamphiphiles(L/D-SPG).Bycombiningvariable-temperaturespectroscopywithin-situreal-timeopticalmicroscopicobservation,thedynamicchiralitytransferprocessacrossspatialandlengthscalesduringassemblyiselucidated.ItisrevealedthathydrophobicinteractionsbetweenalkylchainsandintermolecularhydrogenbondsdominatetheinitialassemblyofSPGoligomers,whichsubsequentlyelongateintomicroscopictwiststhroughsynergisticactionofmultiplenon-covalentbondsunderstereochemicalcontrol.Thecoherentcross-length-scalechiralitytransferdrivesadramaticamplificationofchiropticalsignals,evolvingfromundetectablecircularlypolarizedluminescence(CPL)inthemonomericstateandweakCPLwithasmallluminescencedissymmetryfactor(|g |=2.8×10−3)inthenon-hierarchicalnanostructurestoa|g |valueof0.11amongsingle-componentsupramoleculargelsystems.Thisworknotonlyprovidesadirectspatio-temporalobservationofchiralitytransferfrommoleculartomicronscales,butalsoestablishesasupramoleculardesignstrategyforhigh-performanceCPLmaterialsthroughoptimizedhierarchicalorganization.

Introduction
Biologicalsystemsaremasterfulatconstructingfunctionalmaterialsacrossmultiplelengthscalesinnature.Fromtheprecisefoldingofpolypeptidechainsintofunctionalproteinstothecross-scaleintegrationoforganellesandtissues,livingorganismsachieveextraordinaryfunctionalcomplexitythroughthespatiotemporalorchestrationofnon-covalentinteractions(NCIs)1–4.Thissophisticatedbiologicalarchitecturehasinspiredsupramolecularchemistrytodevelophierarchicalassemblystrategiesthatreproducesuchstructuralsophistication.ThroughthestepwiseandprogrammedincorporationofNCIs,artificialsystemsachievefunctionalorganizationacrosssingle-moleculetomacroscopicscales5–13.Contemporaryresearchhasamassedsubstantialdesignprinciplesforconstructingintricatearchitecturesviacontrolledmultisteppathways.Exampleslikerosettesandultralongnanoladdersdemonstratehowhierarchicaldrivingforces,includinghydrogenbonding,ionicinteractions,microphaseseparation,yieldorderedstructureswithemergentproperties14–23.Crucially,thishierarchicalapproachfacilitatesmacroscopicsupramolecularassemblywhileachievingprecisedimensionalcontrolacrossmultiplelengthscalesandconcurrentfunctionalenhancement,particularlyintermsofefficientchiralexpressionandamplification24.

Thetransmissionofchiralityacrossspatialandlengthscalesposesbothfundamentalscientificchallengesandtransformativeopportunitiesinmaterialfields25–34.Biologicalsystemspervasivelyutilizechiralasymmetry:fromthestereochemicalprecisionofL-aminoacidsatamolecularleveltothebiconcavediscoidmorphologyandefficientoxygen-bindingcapacityoferythrocytesatthemicronscale,demonstratinghowmolecular-levelchiralitycanbetransferredacrossscalestopreciselyorchestratemacroscopicfunctionaloutcomes35,36.

Inartificialhierarchicalassemblies,systematicinvestigationofchiralitypropagationthroughstepwiseformationpathwayshasuncoveredaoperativeenhancementofchiropticalpropertiesarisingfrommolecularhandedness,witheachassemblystagecontributingtothecumulativeamplificationofasymmetry37–42.Forinstance,hierarchicalassemblystrategiesallowenantiomericdopantstomanifestmolecularchiralityinsingle-handedmacroribbons43.Anotherstudydemonstratesthathierarchicalhelicoidsderivedfromchiralbuildingblocksexhibitpronouncedcircularlypolarizedluminescence(CPL),withscrewdislocation-drivenassemblyandintermolecularchargetransferplayingkeyrolesinchiropticalenhancement44.Real-timeobservationofthemacroscopichomochiralassembliesallowsfordynamicmonitoringofchiralexpression,reducingrelianceonhighspatialresolutionwhileenhancingtemporalprecision.Thereby,thisapproachprovidesinsightsintotheevolutionofartificialchiralarchitecturesandenhancesbottom-updesignstrategiesofbiomimeticmaterials45–49.However,despiteextensiveeffortstocharacterizechiralhierarchicalmorphologies,thedynamicprocessesunderlyingchiralexpressioninhierarchicalassembliesremainpoorlyunderstood,andquantitativespatiotemporaltrackingofchiralitytransferacrossstructuralhierarchieshasyettobeestablished.

Herein,wedemonstrateahierarchicalassemblystrategyforconstructingthermodynamicallystableandchirality-controllablemicroscopictwistsusingenantiomericcationicamphiphilesL/D-SPG(Fig.1a).Thesetwistsaremacroscopicallyobservablewiththelengthover100μm,exhibitingstrongcyanfluorescence(FL)(Fig.1b).Themicron-scaledimensionsenablethereal-timeobservationoftheinsituformationprocessinasingle-handedtwistedsystemsassembledthroughenantiopuremoleculesusingopticalmicroscopy(OM)(Fig.1c).Moviesanddataanalysesshowthatthesemicroscopictwistsgrowsimultaneouslyinboththelateralandaxialdimensionsataconstantratewhilemaintaininganinvariantmicron-scalepitch.Thisobservationprovidesdirectevidenceforboththespatiotemporalevolutionofchiralityexpressionacrosshierarchicalassemblystagesandaninterface-controlledgrowthmechanism(Fig.1d,e).Furthermore,comparedtomolecularstateSPGmono(CPL-silent)andnon-hierarchicalnanostructuresSPGsol(withaluminescencedissymmetryfactor|g |=2.8×10−3)assembledviaanantisolventprotocol,thecoherentcross-length-scalechiraltransferintheproposedhierarchicalassemblystrategypresentedhereimpartstheupscaledmicrotwistswitha40-foldCPLamplification,culminatingina|g |of0.11thatsurpassesmostCPL-activesystemsandrepresentsasignificantadvancewithinsingle-componentgelsystems(SupplementaryFig.50andSupplementaryTable1).Thisstudynotonlyunderscoresthecriticalroleofhierarchicalassemblyineffectivelypropagatingchiralinformationfromthemoleculartothemacroscopiclevelbutalsoprovidesguidancefortransformingstochasticmolecularprocessesintopredictableanddesignablehigh-performancematerials.

Results
Hierarchicallyself-assembledmicroscopicchiraltwistsandasymmetryamplification
Helicalstructuresnotonlyexemplifytheeleganceofnaturalhierarchybutalsoserveaspowerfulmodelsystemsforprobingtheemergenceandpropagationofchiralityacrossscales.InspiredbythesubunitstructureofDNAdoublehelicesinbiologicalsystems,apairofchiralcationicamphiphilicmoleculesL/D-SPGwasdesignedandsynthesizedviaathree-stepreactionroute(Fig.1aandSupplementaryFigs.1–3)50,51.Inthisdesign,theglutamicacidmoietyprovidedthechiralcenter,enforcingchiralpackingviadirectionalhydrogenbondinginvolvingthreeamidegroups.ThecationicconjugatedheadgroupfacilitatedelectrostaticinteractionsthatwerefunctionallyanalogoustothoseofDNAphosphategroups,andparticipatedinadynamicinterplaywithπ–πstacking,whichcontributedtostabilizingthehydrogen-bondingorientationestablishedbythestereocenter.Afterasystematicsolventscreening,aDMF/HOmixedsolventwaschosenowingtoitssuitablesolubilityandpolarityformodulatingtheNCIequilibriaamongbuildingblocks,therebyofferingtheprospecttodirectassemblytowardlong-rangeorderedmorphologies(SupplementaryFigs.4–7and18).Throughatypicalheating-coolingprotocol,aseriesofsupramolecularassemblieswithsolvent-regulatedmorphologicalevolutionwasprepared(SupplementaryFig.8).Notably,scanningelectronmicroscope(SEM)imagesshowedthathighlyuniformsupramoleculartwistedassemblieswithclearlyvisiblemicroscalechiralityemergedatawaterfraction(f )valueof30%(Fig.2a,b).Thesemicroscopictwistsinterpenetratedtoformathree-dimensionalnetworkstructurethatimmobilizedthesolvent,affordingastablesupramoleculargel(SupplementaryFig.8).Asshownbystatisticalanalysis,theaveragewidthandhalf-pitchofthetwistswerefittedas2.11±0.91and5.82±2.79μm(mean±s.d.),respectively,demonstratingexcellentstatisticaluniformity(Fig.2c;SupplementaryFigs.9;10andSupplementaryTables2–3).Cryo-transmissionelectronmicroscope(cryo-TEM)imagesfurtherconfirmedthetwistedmorphologyanddimensionalcharacteristics,revealingcompleteconsistencywithSEMobservationsandstatisticalsizedistributions(insertedimagesinFig.2a,b).Additionally,atomicforcemicroscope(AFM)imagesprovidedinformationonheightandtwisteddirection(Fig.2dandSupplementaryFig.12a,b).ThecorrespondencebetweentheSEM-andAFM-identifiedtwisteddirectionvalidatedthefidelityofchiralmorphologycharacterization,supportingtheconclusionthatthehandednessofthesupramolecularassemblieswasgovernedbythemolecularchiralityofSPGenantiomers.Thecentralheightofthetwistedsegment(redline)wasmeasuredtobe813.34±41.60nm(mean±s.d.,SupplementaryFig.11andSupplementaryTable4),whiletheverticalrepeatingunits(blueline)displayedaperiodicityaround5μm,consistentwithhalfthewidthandthe1/2pitchdiscussedabove,respectively.Themicron-scaletwistedmorphologyinthreedimensionsrendereditpossibleforthedirectobservationunderanOM.Well-definedtwistedstructureswerereadilycapturedinthebrightfieldmicroscopicimages,withlengthsextendingtoseveralhundredmicrometers(Fig.2eandSupplementaryFig.12d).Moreover,thepresenceofπ-conjugatedheadgroupderivedfromstyrylpyridiniumcationicdyesendowedtheassembledsystemwithstrongcyanFLthroughaggregation-inducedemission(AIE)activation.Whenexcitedbyultraviolet(UV)radiation,themicroscopictwistsdisplayedperiodicalternatingbrightanddarkcyanemissionpatterns,manifestingthepresenceofmicroscalechirality(Fig.2fandSupplementaryFig.12c).TheopticalpropertiesofSPGgelwerefurthercharacterizedusingspectroscopictechniques.Ultraviolet–visible(UV–vis)andFLspectraexhibitedtheabsorptionandemissionmaximaofthemicroscopictwistsat394and493nm,respectively(Fig.2g).Thepronouncedbathochromicshiftcomparedwithstyrenepyridinewasattributedtoanenhancedintramolecularchargetransfer(ICT)effectduetothequaternizationofpyridylnitrogen,andthesubsequentefficientπ–πstackinginassemblies(Fig.4gandSupplementaryFig.13).DFTcalculationsconfirmedthatthischemicalmodificationsignificantlystrengthenedtheelectron-acceptingability,leadingtoasubstantiallyloweredlowestunoccupiedmolecularorbitalenergylevelandamoredistinctspatialseparationoforbitals,therebypromotingtheICTcharacter(SupplementaryFig.14).Consequently,theabsorptionmaximumat394nmwasattributedtothisICT-assistedπ→π*transition.Theemissionmaximumat493nmwasthenassignedtotheradiativedecayfromtheS state(π*→πtransition),whichwasenhancedduetotherestrictedintramolecularmotionthatsuppressednon-radiativepathwaysandstabilizedtheemissivestate.ItwasclearthatthepresenceofchiralcenterinSPGmoleculesintroducedchiropticalabsorptionandemissionproperties.Asconfirmedbythemorphologicalcharacterization,themicroscalechiralityofmicroscopictwistsarosefrommolecularchirality-mediatedasymmetricalassembly,whereachiralmicroenvironmentfacilitatingchiropticalexpressioninthechromophoricmoietieswascreatedbylong-rangeorderacrossmultiplespatialscales.Accordingly,steady-statechiropticalspectrawererecordedtoevaluatethesupramolecularchiralityofthemicroscopictwists.Theobviousnegativebisignatecottoneffectwithacrossoverat394nmwasfoundinthecirculardichroism(CD)spectrumofL-SPGgel,indicatingstrongchiralexcitoncouplingduetotheintermolecularstyrylpyridiniuminteractions(Fig.2h).ThecorrespondingD-SPGgelexhibitedperfectmirror-imagedCDsignals,furtherdemonstratingefficientchiralitytransferfrommoleculartosupramolecularlevel.Crucially,strongmirror-symmetricCPLsignalswereobservedfromL-SPGgel(right-handed)andD-SPGgel(left-handed),withhigh|g |valuesreaching0.11(Fig.2iandSupplementaryFig.17).Notably,comparativestructuralanalysisrevealedapositivecorrelationbetweenmicroscalechiralorderandchiropticalresponseinassembliespreparedundervaryingf .Thestrongestchiropticalsignalswereobservedinstructuresexhibitingthehighestdegreeofmicroscalechirality(SupplementaryFigs.15and16),promptingafocusedinvestigationintotheunderlyingstructure-propertyrelationships.Higher-magnificationSEMimagesunveiledthatthemicroscopictwistswerecomposedoflaterallyalignedtwistedribbonarchitectures,reflectingclearhierarchicalassemblycharacteristics(Fig.1b,cyandottedlines).Itwasknownthatthedynamicprocessoftheassemblysystem,particularlythereal-timeobservationoftheinsituformationprocess,wasvitalforunderstandingthehierarchicalbehaviorandthespatiotemporalsequencesofchiralexpression.However,duetothehighsol-geltransitiontemperatureandrelativelylowsolubilityofSPGmoleculesinDMF-HOco-solventsatroomtemperature,capturingintermediatestatesusingelectronmicroscopy(EM)orhigh-speedAFMprovedchallenging.Fortunately,themicron-scaledimensionsenabledreal-timeobservationbyOM,whichnotonlysignificantlyimprovedtemporalresolutionbutalsoavoidedthestringentvacuumlimitationsofEM.ByintegratingOMwithaheatingstage,thereal-timeimagingwasemployedtocharacterizethedynamicgrowthprocessofthemicroscopictwists.

Real-timeobservationofthein-situdynamicevolutionprocessbyOM
GuidedbythephysicochemicalpropertiesofSPGmoleculesandtheco-solventsystem,areal-timeobservationdevicewasbuiltseekingtocapturetheinsituevolutionprocessofmicroscopictwists(Fig.3a).Aseptum-cappedquartzcuvette,filledwithSPGmoleculesuniformlydispersedin-aDMF-H Oco-solvent,wasplacedontheheatingmicroscopestageandgraduallyheateduntilaclearandhomogeneoushotsolutionwasobtained(Fig.3b,leftandupper).Duetothehermeticenvironment,thisdevicewascapableofoperatingatarelativelyhightemperaturewithoutsolventevaporation.Afterwards,asthesystemtemperaturedroppedintheairatmosphere,nascentfilamentsemergedandunderwentmorphologicalevolutionbyprogressivelyelongatingintoone-dimensionalmicroscopictwistedarchitectures(Fig.3b,rightandbottom).Initially,therewerenoopticallyvisibleassembliesuntiltwistedfilamentswithdiscerniblepitches(yellowtriangles)appearedafter4s(Fig.3candSupplementaryMovie1).Overthenext10s,thesefilamentsfurtherelongatedinbothaxialandlateraldirectionsatthesametimewiththeirpitchesmaintaining.Well-definedmicroscopictwistswithstablewidthandmorphologywereessentiallyformedafter20s,indicatingtheattainmentofequilibriuminthedynamicassemblysystem(Fig.3f).Toquantifythegrowthdynamics,time-resolvedmeasurementsandcurvefittingwereperformedonthewidthandlengthoftheevolvingmicroscopictwists(Fig.3d,eandSupplementaryFigs.19–26).Statisticalanalysisrevealedthatbothdimensionsincreasedlinearlywithtime,describedbyseparatelinearfunctions:W(t)=kt+b and L(t)=k t+b , wheretheslopesk andk representthelateralandaxialgrowthrates,respectively,andtheinterceptsb andb correspondtotheinitialsizesatt=0,reflectingthelimitedsizeforsamplesobservablewithOMunderthismagnification.Thislinearkineticsindicatedthattheassemblywasinterface-controlled,withgrowthrategovernedbytheincorporationofpre-assembledsubunitsatthegrowthfrontofasingletwistratherthanbythediffusionrateofSPGmonomersinsolution52,53.Theconsistencyofgrowthratesacrossdifferenttwistsevidencedbyk andk values,respectively,demonstratedthatinterfacialadditionproceededatanearlyconstantrate,determinedbyauniformenergybarrier.Thismacroscopiclineargrowththusservedasastrongindicatorthatthesystem,withintheobservabletimewindow,operatedasahighlyordered,cooperative,andprogrammableself-assemblyprocess.Crucially,therelativelyinsensitivityofthegrowthratetochangesinbulkconcentrationprovidedadditional,independentsupportforthisinterface-controlledmechanism(SupplementaryFigs.27–31andSupplementaryMovie3).

Theconcurrentaxial-lateralelongationmode(thatcoupledaxialgrowthwithlateralwidening)provideddefinitivetemporalevidenceforthehierarchicalevolutionfromfibrilstomicroscopictwistedribbonarchitectures,whiletheconstantpitchthroughoutelongationdemonstratedthefidelityinchiralitytransferacrossmultipleassemblystates.Giventhattheinsituformationprocessofthemicroscopictwistscloselyresemblesthechiralgrowthpatternsobservedinnaturebotanicsystems,particularlythetwistingmorphogenesisofPersooniahelixleaves,thisgeometricsimilarityhelpedframetheinvestigationintothefunctionaladvantagesofthetwistedassemblies54.Drawinginspirationfromthenaturalmechanismswherebytwistedplantleavesleveragetheirinherentcurvatureandtwistinganglestoachievehighlyefficientdirectionaltransportofvariousfluids,includingwaterandair,thegrowthfeaturesofmicroscopictwistswereexplainedfromabiomimeticperspective.Thepersistenceofchiraltwistedstructuresacrossobservedassemblyhierarchiesfacilitateddirectionalsolventtransportalongthetwistaxisviawell-definedtwistingangles,whileconcurrentlyenhancingthestructuralstiffnessofassemblies,asshowninFig.2a,bandSupplementaryMovie1.Suchstructuralstiffnesseffectivelycounteractedthermalperturbationsfromsolventmolecules,ultimatelycontributingtosupramoleculargelationbyrestrictingsolventflow.Inaddition,uponreachingthemicronscale,thisstiffnessenabledchiraltwiststomaintainarigidlinearmorphologyevenatexceptionallyhighaspectratioswhilesurpassingthesizelimitationsofconventionalsupramolecularassemblies,distinctfromtheflexibleandchain-likenanoscalestructurescommonlyobserved.To-furtherconfirmtheaboveconclusion,theinsituformationprocessofracemicassemblyrac-SPGgelwasalsomonitored(SupplementaryFig.32andSupplementaryMovie2).SEMandAFMimagesdemonstratedthatthemorphologyofrac-SPGgelwaspresentedasachiralmicrobeltsduetoracemizationatthemolecularlevel(SupplementaryFig.33).Real-timeobservationrevealedrapidformationofthesemicrobeltswithintensofseconds,buttheabsenceofintrinsictwistedstructuresrenderedthemsusceptibletosolvent-inducedperturbations,resultinginpronouncedandrandomcurvatureduringgrowth.Thesefindingsprovidedadirectvisualizationoftheassemblydynamicsofmicroscopictwistsanduncoveredthespatiotemporalprofileofchiralexpressionatthatscale.However,bridgingthevisualgapbetweenmolecularandmicroscopicstructuresremainedaformidablechallenge,astheresolutionofinsituOMfellshortofdirectlyresolvingthemolecularandoligomericscale.Tofullyunderstandthedynamicassemblyprocess,particularlythesynergisticNCIsgoverningthepathwayatthemolecularlevel,wethereforeemployedanintegratedapproach.InsightsfrommicroscopywerecomplementedbypowderX-raydiffraction(PXRD),insituspectroscopictechniquesandcomputationalsimulations,whichtogetherprovidedamultiscaleperspectivetoelucidatetheunderlyingmolecularmechanisms.

Dynamicassemblyprocessatamolecularlevel
Spectroscopictechniquesenableprecisedetectionandreal-timetrackingofatomic-andmolecular-levelchanges(e.g.,vibrationalmodes,electronicstates,andenergytransitions),therebyprovidinginsightsintoadiversearrayofNCIs.Asuiteofvariable-temperature(VT)spectroscopicmethodswasemployedtoprobehierarchicalassemblydynamicsscalesscales,frommolecularrecognitiontoemergentsupramoleculararchitectures.VT-UV–visandVT-CDspectroscopiesdeliveredessentialinformationonchiralpolymerizationkinetics(Fig.4a–candSupplementaryTable5)55–57.DuringtheVTprocess,UV–visspectraunveiledaspectraltransitioncharacterizedbysignificantintensityenhancementatλ=353nmandaconcomitantdecreaseintheabsorptionbandatλ=394nm.Thistrade-offrelationshipbetweenthetwobandsandthepresenceofisosbesticpointsconfirmedthedynamicequilibriumbetweenmonomericandaggregatedL-SPGspeciesduringthermaldepolymerization.Inaccordance,VT-CDspectrashowedagradualreductionindifferentialasymmetricalabsorptionwithincreasingtemperature,indicatingthedisassemblyofchiropticalsupramolecularspeciesinbulksolution.Thedegreeofaggregation(α )derivedfromtheVT-UV–visandVT-CDspectrawasplottedagainsttemperaturetoelucidatetheformationmechanismofthesupramolecularpolymericsystems(Fig.4candSupplementaryFig.34).α fromUV–visprimarilyreflectsthepackingdensityoftheπ-conjugatedheadgroups,withtheCDsignalreportingontheformationandexpressionofsupramolecularchiralspecies.Thenormalizedα obtainedfrombothspectraexhibitedclearnon-sigmoidalcurves,implyinganucleation-elongationpolymerizationmodel,inwhichatwo-stageassemblypathwaywasunmaskedthroughthermodynamicanalysis.Thecalculatedelongationtemperature(T )indicatedthatthenucleationoccurredat357.6Kinthistwo-stageassemblypathway,whileaggregateswithsupramolecularchiralityemergedbelowT throughhierarchicalorganizationofSPGbuildingblocks.Thissupramolecularchiralityoriginatedfromtheπ–πstackingofchromophores,leadingtoexcitoncoupling.TherelativelylargevalueoftheGibbsfreeenergy(−33.4kJ/mol,comparedwithL-SPGsol)duringtheformationofL-SPGtwistsindicatedexceptionalthermodynamicstability,servingasakeydrivingforceforthemicroscopictwistassembly.ComplementaryVT-FLandVT-CPLmeasurementsdisplayedrelativelystablesignalsthatpersisteduntilthetemperaturereachedT (357.6K).Beyondthisthreshold,abruptsignalscollapseoccurredandapproachedspectralsilence,aligningwiththedepolymerizationprocessobservedinVT-UV–visspectra(SupplementaryFig.36).

Toprobedeeplyintotheintermolecularinteractionsduringthetwo-stageassemblyprocess,VT-1HNMRspectroscopywasexploited,whichrevealedaT -separatedtwo-stageassemblyprocessevidencedbytemperature-dependentintegrationchangesinarylprotonsignals(Fig.4d,eandSupplementaryFig.39).Theassignmentofamideprotonswasunequivocallyconfirmedviahydrogen-deuterium(H–D)exchangeexperimentsand2DcorrelationNMRspectra(graydottedboxinFig.4d,eandSupplementaryFig.37).Togaininsightintotheformationofhydrogenbondsduringassembly,weemployedaco-solventsystemofDMF-d containingasmallamountofnon-deuteratedH O,effectivelymimickingtheoriginalassemblyconditionswhilepreservingoptimalNMRshimmingandlocking.AsshowninFig.4f,temperature-dependentchemicalshiftanalysis(Δδ/ΔT)oftheamideprotonsH,H ,andH demonstratedthegradualformationofintermolecularhydrogenbondsduringstageI’58.ThisprocesswasspatiallyfacilitatedbytheapproachofSPGmoleculesthroughhydrophobicinteractionbetweenalkylchains,ultimatelyleadingtotheformationofhydrogen-bondedSPGoligomericnucleiwhereinsupramolecularchiralitywasnotyetmanifested(Fig.4bandSupplementaryFig.36b).DuetothehighpolarityofH O,thepositivechargeofconjugatedcationicheadgroupswaslocalizedonnitrogen.Combinedwithgoodsolubilityinthistemperaturerange,electrostaticrepulsionbetweenthecationicmoietiesreducedtheefficientstackingofπ-conjugatedaromaticrings,renderingtheintegrationofH less-temperature-sensitivecomparedtoH andH (SupplementaryFig.38).UponenteringstageII’,furtherdecreaseoftemperaturecausedasharpdeclineinthesolubilityofSPG.Tofacilitatecloserintermolecularpacking,theconjugatedcationicheadgroupsunderwentchargedelocalizationthatmitigatedelectrostaticrepulsion.Subsequently,chirality-directedhydrogenbondingandπ–πstackinginteractionsdrovethedirectionalgrowthofSPGintoNMR-invisibleaggregates.Throughoutthisprocess,theconjugatedheadgroupsadoptedmolecular-chirality-dictatedorientations,whilethethreehydrogenbondswentthroughconformationaloptimization.Itwasnoteworthythatthethreeamideprotonsexhibiteddistinctresponses.H andH shiftedupfieldwithbondelongation,whereasH shifteddownfieldwithbondshortening.Thisdifferentialbehaviorwascorroboratedbythepresenceoftwosplitpeaksat1620–1645cm−1intheattenuatedtotalreflectionFourier-transformedinfrared(ATR-FTIR)spectrum,correspondingtodistinctC=Ostretchingmodes(SupplementaryFig.40a).Furthermore,thedownfieldshiftsofthreecarbonylcarbonsinsolid-state13C NMRspectracomparedtosolution-stateprovideddirectevidenceforhydrogenbondformationwithintheassemblies(SupplementaryFig.40b).

Plausibleassemblymechanismofmicroscopicchiraltwists
ComparedtomolecularlydispersedSPGmonoinsolution,SPGgelexhibitedapronouncedbathochromicshiftof41nminUVspectra,unambiguouslyconfirmingtheformationofJ-aggregates(Fig.4aandSupplementaryFig.13b).PXRDanalysisdisplayedadiffractionpeakat22.9°,correspondingtoad-spacingof0.39nm,whichfellwithinthetypicalrangeofπ–πstackingdistances.Itwasthusproposedthatconformationaladaptationallowedforeffectiveπ–πstackingbetweenchromophores,overcomingtheinherentelectrostaticrepulsionamongcationicheadgroups.Thisπ–πstackinginteractionnotonlyenhancedtheplanarityofπ-conjugatedheadgroups,butalsoeffectivelytriggeredAIEeffect,resultinginabathochromicshiftofFLandasubstantiallyimprovedquantumyield(Fig.4g;SupplementaryFig.13candSupplementaryTable6).Additionally,alamellarpackingmodeforL-SPGxerogelwasevidencedbythediffractionpeaksof3.74(1),1.90(1/2),1.28(1/3),0.95(1/4)and0.74(1/5)nm59.Alatticefringeof3.76nmshownintheinsetcryo-TEMimageofL-SPGtwistsmatchedthePXRDinterlamellarspacing,verifyingthehighlyorderednanoscalearrangementofthetwistedassemblies.Itwasworthnoticingthatthed-spacingof3.74nmforL-SPGassemblieswasshorterthanthemolecularlengthofSPG(4.33nm,SupplementaryFig.S41),whichsuggestedthatSPGmoleculeswerealignedinatiltedbilayerstructurewithinterdigitationofthealkylchains.

Bromidecounterions(Br−)werealsoinstrumentalinstabilizingthesupramolecularassembliesformedbytheSPG+moieties.Zeta(ζ)potentialofthemacroscopictwistswasmeasuredtoexploresurfacechargeinformation(SupplementaryFig.S42andSupplementaryTable7).Inanaqueousmedium,theapparentζpotentialvaluefordispersedassemblieswas58.1±1.1mV(mean±s.d.,n=3),indicatingthatmostBr−ionswereelectrostaticallyboundtothesurfaceoftheassembliesandformedanelectricaldoublelayer.Thishighζpotentialsuggestedexcellentkineticstabilityoftheassemblies,therebysuppressingaggregation-inducedprecipitationandpreventingtheprematuregrowthterminationofSPGoligomersatsmallsizes.

Thepivotalregulatoryroleoff wasfurthersubstantiatedbyspectroscopicandstructuralanalysesasfinalstructuralorderwasfoundtobecriticallydependentonf (SupplementaryFigs.S43–45).Thesystematf =30%achievedanidealbalancebetweenthermodynamicstabilityandkineticadaptability,whichwasevidencedbythemostpronouncedbathochromicshiftinUV–visspectra,thesmallestinterlamellarspacingandπ–πstackingdistanceinPXRD,andthestrongesthydrogenbondingsignatureinATR-FTIR.Thesedatacollectivelyindicatedthetightestmolecularpackingandthemostfavorableenergylandscapeunderthisspecificcondition.Thisoptimizedintermolecularcooperationenabledhighlyefficienttransferandamplificationofmolecularchirality,ultimatelygivingrisetotheobservedwell-definedmicroscopictwists.Incontrast,deviationsfromthisoptimalf disruptedthedelicateenergeticequilibrium,leadingtolessorderedstructureswithcompromisedchiralexpressionandmorphologicalregularity.

Toevaluatethecriticalpartplayedbyhierarchicalassembly,SPGsolcomposedofnanobeltswithwidtharound60nmwaspreparedthroughanantisolventprotocolratherthanaheating-coolingprocedure(SupplementaryFig.S46).Incomparisonwiththenucleation-elongationpolymerizedmodelforSPGgel,VT-UV–visspectrademonstratedanisodesmicpolymerizationmechanismforSPGsol(SupplementaryFig.S35),disclosingtheabsenceofhierarchicalstructuraloptimization.Consequently,SPGsolexhibitednegligiblemicroscopicchiralityandweakchiropticalactivitywitha|g |valueof2.8×10−3(SupplementaryFig.S47),underscoringthepivotalroleofhierarchicalassemblyinpromotingefficientchiralitytransferandasymmetryamplification.StructuralanalysesprovideddirectevidencefortheinferiorchiraltransferefficiencyinSPGsol(SupplementaryFig.S48).IncontrasttothetightlypackedSPGgel,theSPGsoladoptedalooselamellarstructurewithlesseffectiveπ–πstacking,asconfirmedbyPXRD.Notably,intheabsenceofstabilizingπ–πstacking,theintermolecularhydrogenbondinginSPGsolwasweakened,ascorroboratedbythehigherwavenumberofν N–H andν C=O inATR-FTIRandtheupfield-shiftedcarbonylcarbonsignalsolid-state13C NMRspectrum.Moleculardynamics(MD)simulationsfurtherillustratedthattheresultantinterdigitatedandloosepackingmodehinderedeffectiveexcitoncouplingamongthechromophores,whichconsequentlydisruptedlong-rangechiraltransmissionandaccountedforthediminishedchiropticalactivity.

Basedoncomprehensiveanalysis,ahierarchicalassemblymechanismforthemicroscopicchiraltwistswasformulated(Fig.1e).First,theassemblyprocesswasinitiatedbytemperature-drivenhydrophobicassociationofthealkylchains,whichpromotedmolecularproximityandfacilitatedtheformationofSPGoligomersthroughintermolecularhydrogenbondsamongamidegroups.Inthehigh-polaritysolventenvironment,thepositivechargesmaintainedarelativelylocalizeddistribution.UponcrossingthecriticaltemperaturethresholdofT ,intensifiedhydrophobicinteractionsdramaticallyreducedtheintermoleculardistances,triggeringrapidcoalescenceofSPGoligomersdevoidofsupramolecularchirality.Crucially,themolecularchiralcentersthenorchestratedthealignmentofchirality-directedhydrogen-bondnetworksandπ–πstackinginteractions,accompaniedbypositivechargedelocalization,therebyencodingchiralityintothenascentassemblies.Thischiralencodingwasstructurallyrealizedbytheformationofahighlytiltedbilayerdrivenbytheoptimalf =30%condition,whichinturnenabledeffectiveinter-bilayerπ–πstacking.Theresultinginterlockedarrangementoftheheadgroupsservedasapivotalstabilizingfactorfortheentireassembly.Afterthenucleationstage,thegrowthproceededinaninterface-controlledmanner,resultinginarelativelyconstantgrowthrateandcomparableelongationinbothlateralandaxialdimensions,whichwascharacteristicofthelong-rangeorderedchiralJ-aggregatesstabilizedbyBr−counterions.Thisinterlockedπ–πstackingactedsynergisticallywithhydrogenbondingandhydrophobiceffects,constitutingamulti-facetedstabilizationmechanism.Thereal-timeevolutionofstructuralasymmetryfurtherevidenceduninterruptedchiralitytransferthroughouttheinsitutwistingpathway.Thistime-resolvedprocessexcludedartifactsarisingfrompost-assemblyperturbationssuchasbulksolutionshearordrying-inducedstress.Suchgraduatedchiralamplificationthroughhierarchicalconformationalrefinementpromotedexceptionalmicroscalechiralityexpressioninpreciselyalignedmicroscopictwistarchitectures.Consequently,theintegrationofreal-timetrackabilityandhighdissymmetryfactorsintheSPGsystemprovidedanopportunitytomonitorandcontrolchiralassemblydynamics,settingabenchmarkforfuturechiralluminescentsystems.

Inordertobolstertheaforementionedmechanism,theglobalminimumenergystructureforL-SPGdimerwasoptimizedviaconformationalsearch(Figs.4handS49).TheresultantJ-typeaggregationdistinctlyexhibitedthechromophoresalignmentwithacalculateddistancebetweentwoπ-conjugatedaromaticringsof3.8Å,preciselymatchingtheπ–πstackingdistancederivedfromthediffractionpeakinPXRD.NCIanalysisdemonstratedthatbothπ–πstackingandhydrogenbondsplayedapivotalpartinstabilizingL-SPGdimers,whichcouldbereasonablyextrapolated,combinedwithotherexperimentalresults,tounderstandtheformationprocessofthechiralassembly.MDsimulationsindicatedthatthepre-assembledL-SPGbilayerscontaining32moleculesdisplayedatightlypackedarrangementwithacounterclockwise(CCW)rotationdirection,whichfacilitatedthegenerationofright-handedCPLwithahighg value(Fig.4i).

Methods
Materials
Allreagentsandsolventswereobtainedfromcommercialsupplierswithoutfurtherpurification.Thesuppliersforthestartingmaterialswerelistedasfollows:benzaldehyde(99%,Innochem),4-picoline(98%,Innochem),bromoacetylbromide(97%,TCI),EDCI(99%,J&K),HOBt(99%,Innochem),NaHCO (AR, SCR), Boc-Glu-OH (98%, TCI), and C H NH (97%,Alfa).Trifluoroaceticacid(TFA,AR),aceticanhydride(AR),cyclohexane(CYH,AR),toluene(PhMe,AR),trichloromethane(TCM,AR),tetrahydrofuran(THF,AR),ethylacetate(EA,AR),ethanol(EtOH,AR),acetonitrile(MeCN,AR),N,N-dimethylformamide(DMF,AR),anddimethylsulfoxide(DMSO,AR)werepurchasedfromConcordTechnology(Tianjin)LimitedLiabilityCompany.AllaqueoussolutionswerepreparedusingMilli-Qwater(18.2MΩcm)inthisinvestigation.

PreparationofL/D-SPGself-assemblythroughaheating-coolingprotocol
Takef =30%asanexample,L/D-SPG(1.05×10−2mmol)wasdispersedbyultrasonictreatmentin1mLDMF/HOmixturesolvents(f =30%)ina4mLsamplevial.Themixturewasheateduntilatransparentsolutionformed.Afternaturalcoolingtoroomtemperature,theL/D-SPGself-assembly(L/D-SPGgelandmicroscopictwistsforf =30%)wasproduced.

PreparationofL/D-SPGsolthroughananti-solventprotocol
L/D-SPG(1.05×10−2mmol)wasdispersedbyultrasonictreatmentin700μLDMFina4mLsamplevialandheatedtoformatransparentsolution.Then,300μLofwaterwasaddedquicklyintotheorganicphase,formingtheL/D-SPGsol.

PreparationofdilutedL/D-SPGsupramoleculargel
L/D-SPGsupramoleculargelwasfirstpreparedaccordingtothe“preparationofL/D-SPGself-assemblythroughaheating-coolingprotocol”part.ThenthesupramoleculargelwasdilutedbytheDMF/HOmix-turesolvents(f =30%)inaspecificratioatroomtemperature.Thedilutedsystemwasdispersedthroughvigorousshaking.

Ultraviolet–visiblespectroscopy
UV–visspectrawererecordedonJASCOUV-550andHitachiUV-3900spectrometers.Thesampleswerepreparedfortestingonquartzcuvetteswithlightpathof0.1mm,andthewavelengthscanningrangewas250–650nm.

Photograph
PhotographsofthediluteddispersionofL-SPGsupramoleculargelweretakenusingSONYZV-E10Lcameraequippedwithamacrolensat2timesmagnification.Thesampleswerepreparedbydrippingthediluteddispersionofthesupramoleculargelontocoverslipswithoutdrying.

Scanningelectronmicroscopy(SEM)
SEMimageswererecordedonaHitachiS4800withanacceleratingvoltageof10kVandanoperatingcurrentof10µA.Thesampleswerepreparedonsingle-crystalsiliconwafers,driedundervacuum,andcoatedwithathinlayerofPttoincreasethecontrast.Thestatisticalanalysisofthewidthand1/2pitchareperformedwithaNanomeasurersoftware.

Atomicforcemicroscope(AFM)
AFMimageswererecordedonaDimensionFastScan(BrukerNano)instrumentwithasilicontiponsiliconnitridecantileversintheFastscanBmodeunderambientconditions.Thesampleswerepreparedbydrippingthediluteddispersionofthesupramoleculargelontosingle-crystalsiliconwafers.

Fluorescentmicroscopy
FluorescentmicroscopyimageswererecordedonanOlympusX83usinghigh-pressuremercurylampasexcitationsourceforfluorescentimages.Thesampleswerepreparedbydrippingthediluteddispersionofthesupramoleculargelontocoverslipsfollowedbynaturaldrying.FLimageswereacquiredusingaU-DAPI-LPfiltercube(excitationband:325–375nm;emission:>425nm).

Cryo-transmissionelectronmicroscope(cryo-TEM)
Cryo-TEMimageswererecordedontheThemis300(ThermoScientific)instrumentwithanacceleratingvoltageof300kV.Thesamplesweredrippedontoaholeycarbonfilmoncoppergrid(GIG)anddriedundervacuum.

UV–visspectroscopy
UV–visspectrawererecordedonaU-2600(Hitachi).Thesampleswerepreparedfortestingonquartzcuvetteswithalightpathof0.1mm(forassembly)and1mm(forsolution).

Fluorescencespectra
ThefluorescencespectrawererecordedonanF-4500fluorescencespectrophotometer(Hitachi).Thetestingvoltagewas400V,thewidthoftheexcitationandemissionslitswere5nm,thescanningspeedwas1200nm/min.Thesampleswerepreparedfortestingonquartzcuvetteswithalightpathof0.1mm(forassembly)and1mm(forsolution).

Circulardichroism(CD)spectra
CDspectrawererecordedonaJASCOJ-815spectrophotometer.CDspectrawereconvertedtog spectrausingSpectraManager(JASCO)software.Thesampleswerepreparedfortestingonquartzcuvetteswithalightpathof0.1mm(forassembly)and1mm(forsolution).

Circularlypolarizedluminescence(CPL)spectra
CPLspectrawererecordedonaJASCOCPL-200spectrophotometer.CPLspectrawereconvertedtog valuesusingSpectraManager(JASCO)software.

Opticalmicroscopy(OM)
OMimagesandvideoswerecollectedonaLeicaDM2700Mmaterialsmiroscope.

VTspectra
TheVTspectrawererecordedonanF-4500fluorescencespectrophotometer(Hitachi,forFL),aJASCOJ-815spectrophotometer(forUV–visandCD),aJASCOCPL-300spectrophotometer(forCPL),andaBrukerAVIII500WB(for1HNMR).Thesamplesofchiropticalspectrawerepreparedfortestingonquartzcuvetteswithalightpathof0.2mm(forUV–visandCDspectraofL-SPGgel),1mm(forCPL)and10mm(forFL).Thesamplesof1HNMRwerefirstlypreparedandthendilutedafterdryingundervacuumusingdeuteratedsolvent.AllVTspectroscopicmeasurementswereperformedwithaheatingrateof2°C/min.A2-minequilibrationtimewasincorporatedateachtargettemperaturetoensurethesystemreachedfullthermodynamicequilibrium.

PowderX-raydiffraction(PXRD)measurements
PXRDanalysiswasperformedonaRigakuD/Max-2500X-raydiffractometerwithCu/Kαradiation(λ=1.5406Å),whichwasoperatedatavoltageof40kVandacurrentof200mA.Thesampleswerepreparedonsingle-crystalsiliconwafersanddriedundervacuum.

AttenuatedtotalreflectionFouriertransforminfrared(ATR-FTIR)
ATR-FTIRspectrawererecordedonaBrukerTENSOR-27FT-IRspectrometer.Thetestwavenumberrangedfrom400to4000cm−1.

Zetapotential(ζ)measurements
ζvaluesoftheassembliesweremeasuredonaMalvernnanosizeandzetapotentialanalyzer.Thesamplesweredispersedinwaterbysonicationandtestedatroomtemperature(25°C).

Fluorescencequantumyields
FluorescencequantumyieldsweremeasuredonaFluoroMax+(HORIBA)instrumentbyusinganintegratingsphere.

Rheologicalmeasurements
ThereologicalmeasurementswereperformedonaKinexuspro+rotationalrheometerat25°C.Frequencysweepmeasurementswereperformedwithintherangeof0.1–10Hzataconstantstrainwithinthelinearviscoelasticregion.Amplitudesweepmeasurementswereconductedatafixedfrequencyof1Hztodeterminethelinearviscoelasticregion.

NMRspectra
1H NMR, 13C NMR spectrawererecordedonaBrukerAvance400spectrometer(for400M)andBrukerAVIII500WB(for500M).

Massspectra
Matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-FTICR-MS) were performed on a Bruker-Autoflex. Electrosprayionization time of fight mass spectrometry (ESI-FTICR-MS) were performed on a Bruker-Autoflex III.

Conformationalsearch
MolclusprogramwasappliedtoperformconformationalsearchtoobtaintheglobalminimumenergystructuresofdimericL-SPGaggregates60.Atotalof2000configurationswerefirstsampledbyMDmethodbasedonxTBprogramwith100psastotaltimeand1fsasatimestepunder400K,andthenoptimizedbythesemiempiricalquantummechanicalmethodsGFN0-xTBandGFN2-xTB61–63.TheconfigurationswithΔE<5kcalmol−1 (ΔE is the energy difference betweenaselectedconfigurationandtheconfigurationwiththelowesttotalenergy)werefurtheroptimizedandcarriedoutfrequencyanalysesviatheGaussian16softwarepackageatB3LYP-D3(BJ)/6–31G*level64–68.

Interactionregionindicator(IRI)
ToprovideamoreintuitivedescriptionoftheintermolecularinteractionsofdimericL-SPGaggregates,theIRIwasappliedthroughMultiwfn3.8(dev)69,70.IntheIRIfunction,theparameteraissetto1.1,whichensurestheoptimalrepresentationofbothchemicalbondsandweakinteractions.DifferentcolorsareprojectedontotheIRIisosurfacetodistinguishtheintensityandcharacteristicsofinteractionsindifferentregions.Furthermore,variousinteractionforcesindifferentregionswererenderedusingthemolecularvisualizationprogramVMD71.

Moleculardynamics(MD)
TheinitialconfigurationsofthesystemswereconstructedusingPackmolsoftware72.SimulationswereperformedusingLAMMPS-2Aug2023,andvariousinteractionsatplayweredescribedusingthepolymerconsistentforcefield73,74.L-SPG,DMFandH Ohybridsystemswereconstructed,respectively.Periodicboundaryconditionswereappliedinallthreedirections,andthesteepdescentmethodwasusedtominimizetheenergyineachsystem.ThevelocityVerletalgorithmwasadoptedforintegration.ParticlemeshEwaldmethodwithaprecisionof1×10−6wasusedtocalculatethelong-rangeelectrostaticinteractions.Lorentz–BerthelotmixingruleswereadoptedforvanderWaalsinteractionsfordifferentkindsofatoms.Thecut-offdistancesforelectrostaticinteractionsandvanderWaalsinteractionswere1.0nm.Thesystemsarefirstrelaxedfrom373to298Kfor1ns,subsequentlycollectdynamicstrajectoriesforsubsequentdataanalysisfor5nsundertheisothermal-isometricNVTensembleusingVelocity-rescalethermstatwithatimestepof0.5fs75.ConfigurationswerevisualizedusingOVITOsoftware76.

Quantificationofreal-timeobservationimages
Thevideocapturedinsituisusedtoanalyzethegrowthprocessoftheassembly,therebyobtainingitsassemblykineticsinformation.QuantitativeanalysiswasperformedusingsoftwarenamedMolFocus,programmedbyamemberofourgroup.First,weimportedtherecordedmediafilesandscalecalibrationwasconductedwithinthesoftwarebasedonthemicroscope-providedscaleinformationtoestablishtherelationshipbetweenpixelcoordinatesandactuallengthunits.Specifically,basedontheparameterssetduringthemicroscopeimaging,ascalebarispre-addedtothevideo.Alineofthesamelengthasthescalebarisdrawnintheimageforscalecalibration,andthesoftwareautomaticallycalculatesthepixellengthofthisline.Duringcalibration,byinputtingtheactuallengthoftheline,thesoftwarecompletestheconversionbetweenpixellengthandactuallength.Aftercalibration,therecordedmediafileswereplayed,andthefirstpixelatthebottom-leftcornerofeachvideoframewasautomaticallysetasthecoordinatesystemorigin(0,0).Thisconversionenabledaccuratemeasurementofassemblylengthandwidth.Thesoftwarealsoincorporatedanintegratedframeadjustertolocateindividualframes,allowingthetimescaleofassemblygrowthtobetracked.Timeconversionwasappliedusingarateof3framespersecond(3 fps)basedonspecificframes.Atvarioustimepoints,startandendpointsalongthewidthdirectionoftheassemblyweremarkedtogenerateastraightlineforwidthmeasurement.Asimilarprocedurewasfollowedforlengthmeasurement,wherestartandendpointsalongthelengthdirectionwereidentifiedandmeasured.Toensuredatareliability,fourreplicatesampleswereanalyzedforeachphysicalquantity.Lengthandwidthdatacollectedatdifferenttimepointswereusedtoinvestigatethegrowthkineticsoftheassemblies,revealingmorphologicalchangesandtheassemblyprocessacrossdifferentstages.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Comments on the Research article entitled as “Cascading Chirality from Molecule to Twisted Microstructures with Amplified Circularly Polarized Luminescence” is given bellow:\nThe manuscript is well written. However, the manuscript needs some major improvement and after addressing that I recommend it to publish. Below are some points that should be answered by the authors:\n1. ‘…participated in a dynamic interplay with π-π stacking, which contributed to stabilizing the hydrogen bonding orientation’, is there any experimental evidence of this π-π stacking, responsible for the stabilisation of H-bonding.\n2. ‘These microscopic twists interpenetrated to form a three dimensional network structure that immobilized the solvent, affording a stable supramolecular gel.’ How authors confirmed about gel formations? Rheological analysis (Variation of storage modulus-loss modulus and strain-sweep analysis) must be performed in a confirmational support of gel formation.\n3. The phenomenon of ‘enhanced intramolecular charge transfer (ICT)’ responsible for bathochromic shift of SPGgel compared with styrene pyridine must be explained more precisely.\n4. Cryo-TEM microstructures, support the twisted morphology and dimensional characteristics must be placed in main manuscript instead of supplementary section.\n5. ‘The central height of the twisted segment (red line) was measured to be approximately 900 nm’, Error values in central height of the twisted segment must be mentioned.\n6. ‘When excited by ultraviolet (UV) radiation, the microscopic twists displayed periodic alternating bright and dark cyan emission patterns’, what is the exact excitation wavelength or the range?\n7. ‘Ultraviolet-visible (UV-Vis) and fluorescence (FL) spectra exhibited the absorption and emission maxima of the microscopic twists at 394 nm and 493 nm’, Authors must be mentioned for which particular transitions, such absorption or emission maxima are appeared.\n8. The percentage of purity of the employed chemicals must be mentioned at ‘Materials’ section.\n9. Zeta potential strongly depended on temperature and what is the test temperature of zeta potential of assemblies?", "reviewer_2": "Overall Assessment\nThis manuscript reports supramolecular gel systems that exhibit hierarchical helical assemblies and strong chiroptical activity with a notably high |glum| value (~0.11). The work is visually appealing and potentially impactful in the field of chiral photonics. However, the current version remains largely descriptive, with several key mechanistic aspects insufficiently supported by quantitative or experimental evidence. Strengthening the correlation between molecular structure, hierarchical organization, and optical response would considerably enhance the scientific depth and overall impact of the work. Given the intended scope and audience of Nature Communications, I therefore recommend that the authors address these issues before the manuscript can be considered for publication.\nMajor\n1. Mechanistic elaboration of microstructure formation\nWhile the authors emphasize the helical structures, a more systematic discussion of how the microstructure can be tuned would be scientifically valuable. The results and mechanisms related to Supplementary Fig. 5 should be expanded to identify and rationalize the controllable parameters governing the assembly process.\n2. Optical property section too phenomenological\nThe discussion of optical and chiroptical behaviors is largely descriptive and lacks mechanistic depth. Stronger evidence linking molecular structure and supramolecular organization to the observed spectral shifts and CPL activity is needed. Computational modeling, temperature- or solvent-dependent spectroscopy, or quantitative correlations between morphology and |glum| would greatly enhance the credibility of the interpretation.\n3. In-situ optical microscopy study requires mechanistic justification\nThe section describing the real-time optical microscopy observations is useful but remains phenomenological. The claim that “the assembly was interface-controlled” (p. 9) requires quantitative support or experimental validation (e.g., temperature-, concentration-, or viscosity-dependent studies). The authors should also clarify what the fitted kinetic curves (W(t) and L(t)) physically represent—specifically, what are the numbers of those two slops represent. Without a clear mechanistic interpretation of these fits and supporting evidence, the conclusion regarding interface-controlled growth remains speculative.\n4. Missing connection between molecular and microscopic scales\nThe authors present molecular-level analysis and simulations; however, a clear experimental gap exists between the oligomeric assemblies and the microscopic twisted structures. This limitation, likely due to the resolution of the in-situ technique, should be explicitly acknowledged and discussed to provide a more continuous multiscale picture.\n5. Clarification of the physical picture of high |glum|\nSince the manuscript highlights a record |glum|, the authors should contextualize this result by listing comparable values across related systems (e.g., small molecules, aggregates, gels) to quantify the advancement. As the large |glum| is a central claim, a summary table or comparative figure with literature data would significantly strengthen the paper’s impact.\nMinor\n1. Misleading analogy on page 4–5\nThe statement “The two long alkyl chains functioned analogously to DNA base pairs …” is inaccurate. DNA base pairs are complementary and programmable, whereas hydrophobic interactions are nonspecific. This analogy should be revised or removed.\n2. Clarify schematic representation\nThe schematic drawings do not clearly illustrate how molecules pack into chiral structures.\n3. Reference accuracy and context\nEnsure that all kinetic and mechanistic claims (e.g., interface-controlled growth) are supported by appropriate literature references and clearly compared with previous supramolecular systems.\n4. Detailed methodology of optical microscopy analysis\nThe authors should describe the image analysis procedure in more detail—specifically, how the size of assembled structures was measured, what software or custom scripts were used, and how many samples or replicates were analyzed.\n5. Overstated biomimetic analogy\nThe comparison to botanical morphogenesis is primarily morphological. Since biological systems are non-equilibrium and energy-driven, this “biomimetic” framing should be toned down or rephrased as a structural resemblance rather than genuine mechanistic mimicry.\n6. Rearrangement of figures\nFigure 4 provides stronger evidence and simulations at the molecular level compared with Figures 2 and 3. If the latter cannot be substantially improved through deeper analysis or discussion, rearranging the figures to highlight the molecular insights earlier in the manuscript would improve narrative coherence.", "reviewer_3": "This study (NCOMMS-25-81475) reported a rational design strategy to achieve the hierarchical chiral nanostructures using self-assembly of glutamide amphiphiles with an AIE cationic headgroup. By harnessing a delicate orchestration of multiple non-covalent interactions including π-π stacking, hydrogen bonding, and van der Waals interactions, molecular chirality is effectively propagated across length scales to construct microscale twisted superstructures. In this work, the resultant supramolecular gels can exhibit intense CPL emission (gₗᵤₘ up to 0.11). Based on in-depth and comprehensive analysis, these findings advance the fundamental understanding of chirality transfer in soft materials, and open new avenues for designing high-performance CPL materials. Generally speaking, this work is novel and interesting. I think this study can be published in Nature Communications after the following revisions.\n1. Authors need note the obvious morphological and spectral differences between the hierarchical and non-hierarchical SPG assemblies. Further elaboration on the structural and spectral properties of the non-hierarchical assembly is helpful to promote a comprehensive comparison.\n2. This work observed the exceptional CPL performance in the DMF/H2O mixed solvent at fw = 30%. In order to highlight the specificity of this optimized condition, please provide additional background information on the solvent screening process and in other solvent environment to demonstrate the solvent-mediated control on structure and chirality.\n3. Normally, the kinetic analysis based on two real-time imaging datasets can provide a valuable insight into the growth mechanism. Including quantitative analysis from one or two additional independent assembly experiments can greatly enhance the statistical robustness and generalizability of the proposed linear growth kinetics.\n4. Figure 4 and the related discussion indicated that the π-π interlocking between bilayers can play a crucial role in stabilizing the assembly. Authors need further expand the discussion to offer the analysis of this kind of interaction within the context of the synergistic stabilization mechanism alongside hydrogen bonding and hydrophobic effects."}, "author_response": "Answer to Reviewer 1\nComments on the Research article entitled as “Cascading Chirality from Molecule to Twisted Microstructures with Amplified Circularly Polarized Luminescence” is given bellow:\nThe manuscript is well written. However, the manuscript needs some major improvement and after addressing that I recommend it to publish. Below are some points that should be answered by the authors:\nResponse: We sincerely appreciate your positive feedback on our manuscript and your constructive comments. Accordingly, we have made careful revision to the manuscript, and responded to your comments point-to-point as follows.\n1. ‘…participated in a dynamic interplay with π-π stacking, which contributed to stabilizing the hydrogen bonding orientation’, is there any experimental evidence of this π-π stacking, responsible for the stabilization of H-bonding.\nResponse: Thank you very much for this insightful comment. This conclusion is supported by the VT spectroscopies (Figure 4a-f and Supplementary Figure 36). The VT spectroscopic data revealed that π-π stacking and hydrogen bonding occurred concurrently, as evidenced by the long-wavelength shift of the absorption/emission in VT-UV-Vis/VT-FL spectra alongside the hydrogen bonding observed in VT-1H NMR during the elongation stage (T < Te). This synergistic interplay led to the emergence of strong exciton coupling (VT-CD) and strong excited-state supramolecular chirality (VT-CPL). Crucially, in SPGsol where π-π stacking was weak (Figure R1a), the hydrogen-bonding network remained poorly organized (Figure R1b), giving rise to significantly diminished CD and CPL (Supplementary Figure 47b-c). This demonstrated conclusively that π-π stacking was not merely concurrent with but essential for stabilizing the optimized hydrogen-bonding configuration.\nThe PXRD and ATR-FTIR data corresponding to L-SPGsol in Figure R1 have been added to Supplementary Information as Supplementary Figure 48.\nFigure R1. a. PXRD patterns and b. ATR-FTIR spectra of L-SPGgel and L-SPGsol.\n2. ‘These microscopic twists interpenetrated to form a three dimensional network structure that immobilized the solvent, affording a stable supramolecular gel.’ How authors confirmed about gel formations? Rheological analysis (Variation of storage modulus-loss modulus and strain-sweep analysis) must be performed in a confirmational support of gel formation.\nResponse: We appreciate this valuable suggestion. The initial confirmation relied solely on the vial-inversion test and indeed required further substantiation. Accordingly, detailed rheological measurements have been performed following the suggestion. The frequency sweep and amplitude sweep results are presented in Figure R2a and R2b, respectively. As shown in Figure R2a, the storage modulus (G′) remained consistently higher than the loss modulus (G′′) over the entire angular frequency range, which was a hallmark of gel-like behavior. Furthermore, the amplitude sweep conducted at 1 Hz (Figure R2b) demonstrated a linear viscoelastic region where G′ significantly exceeded G′′, followed by a sharp decrease in G′ beyond a critical strain of 58.17%, indicating a gel-to-sol transition. In short, these rheological data conclusively confirmed the formation of a stable supramolecular gel.\nFigure R2 has been added to Supplementary Information as Supplementary Figure 8.\nFigure R2. Rheological studies of L-SPGgel. a. Frequency sweep measurements performed within the range of 0.1 to 10 Hz. b. Amplitude sweep measurements conducted at a fixed frequency of 1 Hz. All measurements were carried out at a constant temperature of 25 °C.\n3. The phenomenon of ‘enhanced intramolecular charge transfer (ICT)’ responsible for bathochromic shift of SPGgel compared with styrene pyridine must be explained more precisely.\nResponse: Thank you very much for raising this important point. A more precise explanation regarding the enhanced intramolecular charge transfer (ICT) is as follows. The styrylpyridine derivative features a canonical D-π-A structure, wherein the phenyl ring serves as the electron donor (D) and the pyridine moiety acts as a weak electron acceptor (Aw). Alkylation of the pyridine nitrogen, which introduces a positive charge to form the pyridinium cation, converts this moiety into a strong acceptor (As). This transformation significantly enhances the push-pull character of the system, thereby promoting more efficient ICT. The DFT calculations unambiguously support this phenomenon (Figure R3). Upon alkylation, a significant decrease in the LUMO energy (-4.17 eV) compared to the HOMO (-3.23 eV) is observed, with the HOMO-LUMO gap reduced from 4.10 eV to 3.16 eV. More importantly, the orbital diagrams confirm a more distinct spatial separation of the HOMO (localized on the phenyl ring donor) and the LUMO (predominantly confined to the pyridinium acceptor) after alkylation, providing direct theoretical evidence for the enhanced ICT character in the alkylated system.\nFigure R3 has been added to Supplementary Information as Supplementary Figure 14.\nFigure R3. Frontier molecular orbitals and energy levels of StPy and StPyMe calculated by DFT at B3LYP/6-311G(d) level. The blue and green electron clouds indicate the distributions of hole and electron, respectively. The alkyl chain is simplified as a methyl group for computational efficiency.\n4. Cryo-TEM microstructures, support the twisted morphology and dimensional characteristics must be placed in main manuscript instead of supplementary section.\nResponse: We thank the reviewer for this helpful suggestion. As recommended, we have moved the cryo-TEM images that support the twisted morphology and dimensional characteristics from the supplementary information to the manuscript (Inserted images in Figure 2a-b). This revision strengthens the evidence presented in the main manuscript.\n5. ‘The central height of the twisted segment (red line) was measured to be approximately 900 nm’, Error values in central height of the twisted segment must be mentioned.\nResponse: Thank you very much for pointing out this critical issue. To address the concern regarding error values, we performed a statistical analysis of nine parallel height measurements across the twisted segment. As shown in Figure R4, the updated result was reported in the manuscript as 813.34 ± 41.60 nm (mean ± s.d.), which provided a robust quantitative assessment of the central height with an associated error margin.\nFigure R4 has been added to Supplementary Information as Supplementary Figure 11.\nFigure R4. Height analysis of the AFM image. a. The annotated AFM image indicating the measured region. b. Nine parallel height profiles collected across the twisted segment. c. Statistical distribution of the measured heights.\n6. ‘When excited by ultraviolet (UV) radiation, the microscopic twists displayed periodic alternating bright and dark cyan emission patterns’, what is the exact excitation wavelength or the range?\nResponse: We thank the reviewer for the precious advice. The excitation wavelength range has been specified in the Fluorescent microscopy subsection of the Methods section as 325–375 nm.\n7. ‘Ultraviolet-visible (UV-Vis) and fluorescence (FL) spectra exhibited the absorption and emission maxima of the microscopic twists at 394 nm and 493 nm’, Authors must be mentioned for which particular transitions, such absorption or emission maxima are appeared.\nResponse: Thank you very much for this constructive suggestion. The absorption maximum at 394 nm is attributed to an ICT-assisted π→π* transition. Upon aggregation, the excitonic coupling among molecules induces a bathochromic shift by further reducing the energy gap of this transition. Accordingly, the emission maximum at 493 nm is assigned to the radiative decay from the S state (π*→π transition), which is enhanced due to the restricted intramolecular motion that suppresses non-radiative pathways and stabilizes the emissive state.\n8. The percentage of purity of the employed chemicals must be mentioned at ‘Materials’ section.\nResponse: Thank you very much for this important remark. The purity grades for all chemicals employed in this study have now been clearly specified in the Materials subsection of the Methods section.\n9. Zeta potential strongly depended on temperature and what is the test temperature of zeta potential of assemblies?\nResponse: Thank you very much for raising this important point. The zeta potential measurements for the assemblies were conducted at room temperature (25 °C). We have now explicitly stated this information in the Zeta potential (ζ) measurements subsection of the Methods section to avoid any ambiguity, as suggested.\nAnswer to Reviewer 2\nOverall Assessment\nThis manuscript reports supramolecular gel systems that exhibit hierarchical helical assemblies and strong chiroptical activity with a notably high |glum| value (~0.11). The work is visually appealing and potentially impactful in the field of chiral photonics. However, the current version remains largely descriptive, with several key mechanistic aspects insufficiently supported by quantitative or experimental evidence. Strengthening the correlation between molecular structure, hierarchical organization, and optical response would considerably enhance the scientific depth and overall impact of the work. Given the intended scope and audience of Nature Communications, I therefore recommend that the authors address these issues before the manuscript can be considered for publication.\nResponse: We are deeply grateful to the reviewer for this exceptionally insightful assessment, which we find truly gets to the heart of the scientific issues. In response, we have undertaken significant efforts to move beyond description towards a more quantitative and mechanistic explanation. We believe the manuscript is now greatly strengthened, and we provide a detailed response to each specific comment below.\nMajor\n1. Mechanistic elaboration of microstructure formation\nWhile the authors emphasize the helical structures, a more systematic discussion of how the microstructure can be tuned would be scientifically valuable. The results and mechanisms related to Supplementary Fig. 5 should be expanded to identify and rationalize the controllable parameters governing the assembly process.\nResponse: Thank you very much for your thoughtful insights and helpful comments. In the original manuscript, our focus on the optimal condition (f = 30%) led to the omission of a systematic discussion of the assembly pathway. Following your suggestion, XRD and ATR-IR spectra were employed to analyze the structural features of assemblies formed at different f values, in order to elucidate how this key parameter governed the resulting morphologies (Figure R5-6). Quantitative analysis of the UV-Vis spectra and the supplementary structural data revealed that the final morphology was cooperatively regulated by the J-aggregation of the headgroups (reflected in the bathochromic shift of the absorption maximum), the interlamellar spacing, the π-π stacking distance, and the strength of hydrogen bonding (Figure R7). Well-defined twisted morphologies emerged only when strong J-aggregation, relatively small interlamellar spacing, tight π-π stacking, and robust hydrogen bonding acted in concert, while even small deviations caused by the variation in f would lead to a progressive loss of the twisted morphology.\nFigure R5-7 have been added to Supplementary Information as Supplementary Figure 43-45.\nFigure R5. PXRD patterns of dried L-SPG assemblies at different f . a. f = 0, b. f = 10%, c. f = 20%, d. f = 40%, e. f = 50%, f. f = 60%.\nFigure R6. ATR-IR spectra of dried L-SPG assemblies at different f . a. f = 0, b. f = 10%, c. f = 20%, d. f = 40%, e. f = 50%, f. f = 60%.\nFigure R7. Structural information of dried L-SPG assemblies at different f based on Supplementary Figure 13b, Figure R5 and R6. a. Absorption maxima wavelength at different f . b. Interlamellar spacing and π-π stacking distance at different f . The π-π stacking distance of f = 10% is labeled \"weak\", emphasizing a low intensity of the π-π stacking peak in the PXRD pattern (Figure R5b). c. Wavenumber of ν at different f .\n2. Optical property section too phenomenological\nThe discussion of optical and chiroptical behaviors is largely descriptive and lacks mechanistic depth. Stronger evidence linking molecular structure and supramolecular organization to the observed spectral shifts and CPL activity is needed. Computational modeling, temperature- or solvent-dependent spectroscopy, or quantitative correlations between morphology and |glum| would greatly enhance the credibility of the interpretation.\nResponse: We sincerely thank the reviewer for this insightful comment regarding the need for deeper mechanistic discussion. We have followed your advice to provide a deeper interpretation of the optical and chiroptical signals. Our revised analysis was structured around two key aspects to establish mechanistic links:\n1) Correlation between aggregation and chirality expression via variable-temperature (VT) spectroscopy:\nFor the optimized assembly at f = 30%, we performed a combined analysis of the VT-UV-Vis and VT-CD spectra. The aggregation degree derived from UV-Vis primarily reflects the packing density of the π-conjugated headgroups, while the CD signal reports on the formation and expression of supramolecular chiral species. As shown in Figure R8, the normalized α obtained from both spectra shared identical transition temperatures and trends. This demonstrated that π-π stacking and the development of supramolecular chirality were synergistic processes. During elongation, the oligomers adopted a specific spatial arrangement and distance upon stacking, facilitating exciton coupling between chromophores, which ultimately led to the cooperative expression of assembly and chiral signals.\n2) Relating morphology to CPL across different solvent systems:\nA solvent screening study has been added to establish the correlation between morphology and CPL (Figures R9-R12). The key finding was that assemblies with well-defined, uniformly twisted morphologies and larger dimensions consistently exhibited the highest |glum| values. This directly showed that the enhanced structural order and microscopic scale in superior morphologies correlated strongly with improved chiroptical performance.\nFigure R8-11 have been added to Manuscript and Supplementary Information as Figure 4c, Supplementary Figure 4-5, 18 and 34.\nFigure R8. Normalized α (monitoring at 353 nm) obtained from a. VT-UV-Vis spectra and b. VT-CD spectra (Figure 4c in manuscript) of L-SPGgel. c. Comparison of the fitting curves obtained from a. and b.\nFigure R9. Morphology characterization of L-SPG assemblies exhibiting |glum| < 0.01 in different solvents. a. CYH. b. PhMe. c. TCM. d. THF. e. EA. f. EtOH. g. MeCN. h. DMSO. i. THF f = 10%. j. EtOH f = 10%. k. DMSO f = 10%.\nFigure R10. Morphology characterization of L-SPG assemblies exhibiting |glum| > 0.01 in different solvents. a. MeCN f = 10%. b. EtOH f = 30%. c. MeCN f = 30%. d. DMSO f = 30%.\nFigure R11. CPL spectra of L-SPG assemblies in different solvents. a. |glum| < 0.01 systems. b. |glum| > 0.01 systems with corresponding glum values.\n3. In-situ optical microscopy study requires mechanistic justification\nThe section describing the real-time optical microscopy observations is useful but remains phenomenological. The claim that “the assembly was interface-controlled” (p. 9) requires quantitative support or experimental validation (e.g., temperature-, concentration-, or viscosity-dependent studies). The authors should also clarify what the fitted kinetic curves (W(t) and L(t)) physically represent—specifically, what are the numbers of those two slops represent. Without a clear mechanistic interpretation of these fits and supporting evidence, the conclusion regarding interface-controlled growth remains speculative.\nResponse: We thank the reviewer for the comprehensive and insightful feedback, especially the guidance on the methodology and data analysis. To provide a mechanistic justification for the observed linear growth, we have performed a quantitative analysis based on the interface-controlled growth model established by Bigioni et al (Nat. Mater. 2006, 5, 265–270). Crucially, the conclusion of interface-controlled growth in that work rested primarily on direct, real-time observation of monolayer island formation and growth. Similarly, in our system, the conclusion of an interface-dominated process is grounded in the following key experimental observations: the growth originates from defined seeds and propagates with a clear, advancing front exclusively at the solution-assembly interface, exhibiting pronounced directionality without random nucleation in the bulk solution. The observed linear growth kinetics are a predicted consequence and quantitative hallmark of such an interface-controlled regime, as opposed to diffusion-controlled processes, where the growth rate typically decays with time. This spatial and temporal specificity of growth mirrors the core evidence in Bigioni et al. for interfacial assembly. Therefore, we applied the established model to quantitatively describe this interface-driven front advancement. We simplified and modeled them as ribbon-like structures evolving over time, characterized by its length L(t) and width W(t). This simplification allowed us to apply the classical interface-controlled growth model to analyze the advancement rate of the growth front. The growth rate of the structure's area, A(t) = L(t)·W(t), is proportional to the total effective capture area (Atotal):\ndA/dt = KAtotal (R1)\nwhere K = f·a is the rate constant, f is the net relative flux of solvent molecules to the interface, and a is the area occupied by a single particle. The total capture area Atotal comprises two contributions: 1. Direct deposition: particles landing directly on the ribbon, with an area Adirect = W(t)·L(t). 2. Interfacial diffusion capture: particles landing within a diffusion length δ of the ribbon edge and diffusing to be incorporated. For a long ribbon (L ≫ W), diffusion capture occurs primarily along two sides, yielding Adiff = 2δ·L(t) (for width elongation) or 2δ·W(t) (for length elongation).\nCase 1: Width Elongation (Constant Length)\nFor the analysis of width growth, our optical microscopy observations indicated that L remained much larger than W throughout the measured time window. Therefore, L could be treated as approximately constant when modeling the width evolution. The total capture area for width elongation is:\nAtotal = Adirect + Adiff = W(t)L(t)+2δW L(t) (R2)\nSubstituting (R2) into (R1) under this condition yields:\nd(WL)/dt = K(W(t)L+2δW L) (R3)\ndW/dt = K(W(t)+2δW) (R4)\nThis differential equation describes the width evolution. In the early stages of growth, where the ribbon width is much smaller than the diffusion capture zone (W(t)≪2δ), (R4) simplifies to:\ndW/dt = 2δW K (R5)\nIntegrating (R5) yields the observed linear relationship:\nW(t) = k1 t+b1 (R6)\nwhere the slope k1 = 2δW K represents the constant propagation velocity of the width front, a product of two key factors: the interfacial diffusion length (δW), which sets the spatial range for particle capture, and the interface reaction rate constant (K = f·a). Here, the particle flux (f) and the area per particle (a) together determine the intrinsic incorporation efficiency (K) at the growth interface. Therefore, the slope physically embodies the collective effect of particle supply, incorporation efficiency, and interfacial diffusion in driving the steady advancement of the growth front. The intercept b1 corresponds to the ribbon width at the start of the measurable linear growth regime, reflecting its size upon entering the optically resolvable window.\nCase 2: Length Elongation (Constant Width)\nAn analogous derivation is applied to model the elongation in length. In this case, our observations indicated that the width W remained effectively constant and was significantly smaller than the evolving length L(t) during the measured time window. This justifies treating W as a constant when modeling the axial elongation. The diffusion capture area for incorporation at the ribbon ends is correspondingly defined as Adiff = 2δL·W. The differential equation for length evolution is:\ndL/dt = K'(L(t)+2δL) (R7)\nUnder the condition where the length is much smaller than the diffusion capture zone (L(t)≪2δ) during the initial growth stage, this simplifies to dL/dt ≈ 2δL K’. Integration then gives the linear relationship for length:\nL(t) = k2 t+b2 (R8)\nwhere the slope k2 = 2δL K represents the constant propagation velocity of the length front, a product of two key factors: the interfacial diffusion length (δL), which sets the spatial range for particle capture, and the interface reaction rate constant (K’ = f’·a’). Here, the particle flux (f’) and the area per particle (a’) together determine the intrinsic incorporation efficiency (K’) at the growth interface. Therefore, the slope physically embodies the collective effect of particle supply, incorporation efficiency, and interfacial diffusion in driving the steady advancement of the growth front. The intercept b2 corresponds to the ribbon length at the start of the measurable linear growth regime, reflecting its size upon entering the optically resolvable window.\nTo clarify that the growth process was controlled by the interface rather than by diffusion, we reduced the assembly concentration from 1.05×10-2 M to 7.36×10-3 M under the same conditions and measured the growth dynamics (Figure R13-17). The elongation in length continued to follow a linear relationship with time under both concentrations. The average linear growth rate showed only a slight decrease from 28.5 μm/s to 28.3 μm/s, a change that was notably small in the context of the approximately 30% reduction in initial concentration. This weak concentration dependence did not align with a diffusion-controlled growth mechanism, which fundamentally predicted a strong, proportional relationship between growth rate and bulk monomer concentration. In contrast, the observed behavior was consistent with an interface-controlled process, where the growth rate L(t) was proportional to the chemical potential difference Δμ as L(t) ∝ Δμ ∝ ln(C∞/Ceq). This logarithmic dependence implied the sensitivity of the growth rate to variations in bulk concentration C∞ is markedly weak, particularly in the higher-concentration regime studied here. The modest slowdown in growth rate despite a significant concentration decrease thus reflected the kinetic signature of an interface-controlled growth mechanism.\nIn conclusion, through combined theoretical derivation and experimental validation, we demonstrated that the growth process was interface-controlled.\nThe formula derivation section has been incorporated into Supplementary Computational Details in Supplementary Information. Figure R12-16 have been added to Supplementary Information as Supplementary Figure 27-31.\nFigure R12. Length measurement images of microscopic twist 1 based on the real-time observation at a lower concentration (7.36×10-3 M).\nFigure R13. Length measurement images of microscopic twist 2 based on the real-time observation at a lower concentration (7.36×10-3 M).\nFigure R14. Length measurement images of microscopic twist 3 based on the real-time observation at a lower concentration (7.36×10-3 M).\nFigure R15. Length measurement images of microscopic twist 4 based on the real-time observation at a lower concentration (7.36×10-3 M).\nFigure R16. The variation in length of a growing microscopic twist as a function of time at a lower concentration (7.36×10-3 M), with the fitted lines elucidating the underlying growth dynamics.\n4. Missing connection between molecular and microscopic scales\nThe authors present molecular-level analysis and simulations; however, a clear experimental gap exists between the oligomeric assemblies and the microscopic twisted structures. This limitation, likely due to the resolution of the in-situ technique, should be explicitly acknowledged and discussed to provide a more continuous multiscale picture.\nResponse: Thank you very much for highlighting this essential point. Directly visualizing the continuous structural evolution from molecular monomers to microscopic twists utilizing a single technique presents a fundamental challenge. This limitation stems from the resolution boundaries of in-situ imaging methods at molecular scales, combined with the solubility and size constraints of the oligomeric intermediates. To bridge this observational gap and reconstruct a coherent multiscale picture, we employed a complementary set of experimental techniques. While real-time imaging was inherently limited in spatial resolution, we employed ex-situ PXRD to definitively establish the long-range molecular packing within the microscopic assemblies. In parallel, we leveraged a suite of in-situ spectroscopic methods (VT-UV-Vis, VT-CD, VT-FL, VT-CPL, and VT-1H NMR) to provide a multi-faceted, real-time view of the dynamic assembly process. These techniques collectively probed the evolution of molecular conformation, supramolecular chirality, and non-covalent interactions across the relevant scales. Thus, although a direct, continuous video-like visualization from molecules to microscopic twists remained unattainable with current in-situ imaging, we strategically integrated computational simulations with layered experimental data—from static molecular packing (PXRD) to dynamic spectroscopic signatures—to logically infer and substantiate the assembly pathway. This perspective on connecting molecular and microscopic scales has been incorporated into the revised manuscript as “However, bridging the visual gap between molecular and microscopic structures remained a formidable challenge, as the resolution of in-situ optical microscopy fell short of directly resolving the molecular and oligomeric scale. To fully understand the dynamic assembly process, particularly the synergistic non-covalent interactions governing the pathway at the molecular level, we therefore employed an integrated approach. Insights from microscopy were complemented by Powder X-ray diffraction (PXRD), in-situ spectroscopic techniques and computational simulations, which together provided a multiscale perspective to elucidate the underlying molecular mechanisms.”\n5. Clarification of the physical picture of high |glum|\nSince the manuscript highlights a record |glum|, the authors should contextualize this result by listing comparable values across related systems (e.g., small molecules, aggregates, gels) to quantify the advancement. As the large |glum| is a central claim, a summary table or comparative figure with literature data would significantly strengthen the paper’s impact.\nResponse: Thank you very much for this valuable suggestion. To better contextualize our results, we expanded our comparative analysis to include a wider range of chiral systems, such as axial/planar small organic molecules (SOMs), helicenes and helicoid derivatives, polymers, metal-organic frameworks (MOFs), cages, and clusters. We summarized this comprehensive comparison in Table R1. Furthermore, Figure R18 provides a clear visual representation of our results within this broader context.\nTable R1 and Figure R18 have been added to Supplementary Information as Supplementary Table 2 and Supplementary Figure 50.\nTable R1. |glum| values in different systems.\nType Molecule(s) |glum| Ref.\n(R,R)-3 1.2×10-2 1\nBNPh2-BNaph/F- 10-2 2\nAxial (R)-1 6.1×10-3 3\n(R)-BM2 6×10-3 4\n(R)-3 1.3×10-3 5\n(R)-2 6×10-3 6\nSmall (Sp)-4 2.7×10-3 7\nOrganic Planar (Sp)-9 2.2×10-3 8\nMolecules (SOMs) (Rp)-9 1.7×10-3 9\n(Rp)-CP5 1.2×10-3 10\nS-PCQ32-b 3.8×10-2 11\nHelicenes and DNH 2.5×10-3 12\nhelicenoid 5 1.7×10-3 13\nderivatives M-1 10-3 14\nS-SPAn 2.4×10-4 15\nSPG 0.11 This work\nSingle- L/DGG-Nap 0.1 16\ncomponent {Ag(—)tG}n 0.07±0.01 17\nAssemblies Gel\nNDI-2 4.6×10-2 18\nChol-CN-Py 3.0×10-2 19\nAg-SP Ⅰ 7.9×10-3 20\nPEB 5.3×10-3 21\nDG/LG-DPAC 7.7×10-4 22\nFtpy/ATP 0.2 23\nFtpy/GTP/Ag+ 0.15 24\nGMP/TO/PY 0.15 25\nMulti- TPEHis/FmocAla 0.14 26\ncomponent SC/CBS/Ca2+ 0.1 27\nIonic oligomer/Rhodamine B 0.06 28\nG4-1/Eu(Ⅲ) 10-3-10-2 29\nα-NapHis/BA 3×10-3 30\nBCP/MA/PCA 0.1 31\nCM+CAB/P1 3.1×10-2 32\nPolymers COR@PEN 1.23×10-2 33\nPLBA-PPEA 10-2 34\nP-poly-1 2.4×10-3 35\nL-UCMOF⊃DAECc 7.8×10-2 36\nMetal-organic\nAg6/BTA/phol 6×10-2 37\nframeworks\nAu2Cu2/3F3I/R/S5FI 3.6×10-2 38\n(MOFs), cages\nTFPB/HC 1.3×10-3 39\nand clusters\nS-2 10-3 40\nFigure R17. |glum| values in different systems based on Table R1. Type I-VII refer to axial SOMs, planar SOMs, helicenes and helicenoid derivatives, single-component gel, multi-component gel, polymers, and metal-organic frameworks (MOFs), cages and clusters.\nMinor\n1. Misleading analogy on page 4–5\nThe statement “The two long alkyl chains functioned analogously to DNA base pairs …” is inaccurate. DNA base pairs are complementary and programmable, whereas hydrophobic interactions are nonspecific. This analogy should be revised or removed.\nResponse: We appreciate the reviewer for pointing out the inaccuracy in our analogy. The comparison was indeed inappropriate because the long alkyl chains mediate non-specific hydrophobic interactions, which lack the complementary and programmable characteristics of DNA base pairs. Consequently, we have deleted this sentence from the relevant section in the revised manuscript.\n2. Clarify schematic representation\nThe schematic drawings do not clearly illustrate how molecules pack into chiral structures.\nResponse: We thank the reviewer for this important insight. To improve the schematic representation, we have relocated the illustration of the detailed hierarchical assembly process (originally in Figure 4j) to Figure 1e. Furthermore, the schematic in Figure 1d has been optimized to delineate the assembly hierarchy. Together, these changes provide a clearer pathway from molecular arrangement to chiral structures.\n3. Reference accuracy and context\nEnsure that all kinetic and mechanistic claims (e.g., interface-controlled growth) are supported by appropriate literature references and clearly compared with previous supramolecular systems.\nResponse: We thank the reviewer for this valuable suggestion to strengthen the literature context. Our mechanistic interpretations are firmly supported by prior, well-established work. The kinetics of interface-controlled growth are modeled based on Bigioni et al. (Nat. Mater. 2006, 5, 265–270), which provides a precedent for linear growth behavior under interfacial control. For the polymerization kinetics, we rely on the foundational models of nucleation-elongation and isodesmic aggregation (J. Am. Chem. Soc. 2008, 130, 606-611; Chem. Eur. J. 2009, 16, 362-367; Nat. Commun. 2020, 11, 5460). All supporting calculations were consolidated in the Supplementary Computational Details.\n4. Detailed methodology of optical microscopy analysis\nThe authors should describe the image analysis procedure in more detail—specifically, how the size of assembled structures was measured, what software or custom scripts were used, and how many samples or replicates were analyzed.\nResponse: Thank you very much for your constructive suggestion. The initial description of the software in the Methods section was not sufficiently precise and could lead to misunderstanding. Following your suggestion, we have provided a more detailed description of the methodology in the revised version of the manuscript. The updated description now reads as follows:\nQuantification of real-time observation images. The video captured in-situ is used to analyze the growth process of the assembly, thereby obtaining its assembly kinetics information. Quantitative analysis was performed using software named MolFocus programmed by a member of our group. First, we imported the recorded media files and scale calibration was conducted within the software based on the microscope-provided scale information to establish the relationship between pixel coordinates and actual length units. Specifically, based on the parameters set during the microscope imaging, a scale bar is pre-added to the video. A line of the same length as the scale bar is drawn in the image for scale calibration, and the software automatically calculates the pixel length of this line. During calibration, by inputting the actual length of the line, the software completes the conversion between pixel length and actual length. After calibration, the recorded media files were played, and the first pixel at the bottom-left corner of each video frame was automatically set as the coordinate system origin (0,0). This conversion enabled accurate measurement of assembly length and width. The software also incorporated an integrated frame adjuster to locate individual frames, allowing the time scale of assembly growth to be tracked. Time conversion was applied using a rate of 3 frames per second (3 fps) based on specific frames. At various time points, start and end points along the width direction of the assembly were marked to generate a straight line for width measurement. A similar procedure was followed for length measurement, where start and end points along the length direction were identified and measured. To ensure data reliability, four replicate samples were analyzed for each physical quantity. Length and width data collected at different time points were used to investigate the growth kinetics of the assemblies, revealing morphological changes and the assembly process across different stages.\nAdditionally, contributions related to the software development have been noted in the Author Contributions section.\n5. Overstated biomimetic analogy\nThe comparison to botanical morphogenesis is primarily morphological. Since biological systems are non-equilibrium and energy-driven, this “biomimetic” framing should be toned down or rephrased as a structural resemblance rather than genuine mechanistic mimicry.\nResponse: Thank you very much for this critical insight. We fully agree that the \"biomimetic\" analogy overreached by implying a mechanistic mimicry of non-equilibrium biological processes. Following the suggestion, we have rephrased this discussion throughout the manuscript to frame it as a structural and functional resemblance. The comparison now focuses on the geometric similarity of the chiral growth patterns and the ensuing advantages in liquid transport, without claiming a genuine mimicry of the biological mechanism. The specific sentence has been revised as \"Given that the in-situ formation process of the microscopic twists closely resembles the chiral growth patterns observed in natural botanic systems, particularly the twisting morphogenesis of Persoonia helix leaves, this geometric similarity helped frame the investigation into the functional advantages of the twisted assemblies\".\n6. Rearrangement of figures\nFigure 4 provides stronger evidence and simulations at the molecular level compared with Figures 2 and 3. 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Ge C, et al. Self‐Assembly of an Unlikely Occurring Quadrangular Tube by Modulating Intramolecular Forces. Angew Chem Int Ed 63, e202411401 (2024).\nAnswer to Reviewer 3\nThis study (NCOMMS-25-81475) reported a rational design strategy to achieve the hierarchical chiral nanostructures using self-assembly of glutamide amphiphiles with an AIE cationic headgroup. By harnessing a delicate orchestration of multiple non-covalent interactions including π-π stacking, hydrogen bonding, and van der Waals interactions, molecular chirality is effectively propagated across length scales to construct microscale twisted superstructures. In this work, the resultant supramolecular gels can exhibit intense CPL emission (g up to 0.11). Based on in-depth and comprehensive analysis, these findings advance the fundamental understanding of chirality transfer in soft materials, and open new avenues for designing high-performance CPL materials. Generally speaking, this work is novel and interesting. I think this study can be published in Nature Communications after the following revisions.\nResponse: Thank you for your positive feedback and thoughtful evaluation of our manuscript. We are grateful for your recognition of the significance and novelty of our work. We have meticulously revised the manuscript and provided a point-by-point response to your comments below.\n1. Authors need note the obvious morphological and spectral differences between the hierarchical and non-hierarchical SPG assemblies. Further elaboration on the structural and spectral properties of the non-hierarchical assembly is helpful to promote a comprehensive comparison.\nResponse: Thank you very much for your constructive suggestion. As recommended, we have conducted a detailed characterization of the non-hierarchical SPGsol assembly (Figure R18). In contrast to SPGgel, the L-SPGsol assembly formed a relatively loose lamellar structure with ineffective π-π stacking among the headgroups (Figure 4g and Figure R18a). Correspondingly, ATR-FTIR spectra indicated weaker hydrogen bonding in L-SPGsol (Supplementary Figure 40 and Figure R18b), which was consistent with the upfield shift of the carbonyl carbon in the solid-state 13C NMR spectrum (Figure R18c). Furthermore, the solid-state NMR spectrum of L-SPGsol showed fewer split signals, suggesting a simpler molecular packing pattern compared to the hierarchical L-SPGgel assembly. Molecular dynamics (MD) simulations corroborated these findings, revealing that the SPG molecules in L-SPGsol did not form a typical bilayer but adopted an interdigitated arrangement instead (Figure R18d). This loose and interdigitated packing mode was inefficient for facilitating long-range chiral transfer across scales, which ultimately accounts for the weak CPL activity observed in this non-hierarchical assembly.\nFigure R18 has been added to Supplementary Information as Supplementary Figure 48.\nFigure R18. Structural information of L-SPGsol. a. PXRD pattern of L-SPGsol. b. ATR-IR spectrum of L-SPGsol. c. Solid-state 13C NMR of L-SPG xerogel (red line) and L-SPGsol (blue line). d. MD simulation results of L-SPGsol, in which a clockwise (CW) rotation is clearly presented.\n2. This work observed the exceptional CPL performance in the DMF/H O mixed solvent at f = 30%. In order to highlight the specificity of this optimized condition, please provide additional background information on the solvent screening process and in other solvent environment to demonstrate the solvent-mediated control on structure and chirality.\nResponse: Thank you very much for your constructive suggestion. To better elucidate the influence of the solvent, we have supplemented additional solvent screening data as suggested (Figures R9-R12). The key finding was that assemblies with well-defined, uniformly twisted morphologies and larger dimensions consistently exhibited the highest |glum| values. This directly showed that the enhanced structural order and microscopic scale in superior morphologies correlated strongly with improved chiroptical performance.\nFigures R19-21 have been added to Supplementary Information as Supplementary Figure 4-5 and 18.\nFigure R19. Morphology characterization of L-SPG assemblies exhibiting |glum| < 0.01 in different solvents. a. CYH. b. PhMe. c. TCM. d. THF. e. EA. f. EtOH. g. MeCN. h. DMSO. i. THF f = 10%. j. EtOH f = 10%. k. DMSO f = 10%.\nFigure R20. Morphology characterization of L-SPG assemblies exhibiting |glum| > 0.01 in different solvents. a. MeCN f = 10%. b. EtOH f = 30%. c. MeCN f = 30%. d. DMSO f = 30%.\nFigure R21. CPL spectra of L-SPG assemblies in different solvents. a. |glum| < 0.01 systems. b. |glum| > 0.01 systems with corresponding glum values.\n3. Normally, the kinetic analysis based on two real-time imaging datasets can provide a valuable insight into the growth mechanism. Including quantitative analysis from one or two additional independent assembly experiments can greatly enhance the statistical robustness and generalizability of the proposed linear growth kinetics.\nResponse: We thank the reviewer for this valuable suggestion. Accordingly, we have conducted two additional replicate experiments to quantitatively analyze the growth of width and length (Figure R22-26). The updated data strengthen the statistical robustness and convincingly confirm the linear kinetics.\nFigures R24-28 have been added to Manuscript and Supplementary Information as Figure 3d-e and Supplementary Figure 21-22 and 25-26.\nFigure R22. Width measurement images of microscopic twist 3 based on the real-time observation. The inset images provide a magnified view of the measurement area to show details.\nFigure R23. Width measurement images of microscopic twist 4 based on the real-time observation. The inset images provide a magnified view of the measurement area to show details.\nFigure R24. Length measurement images of microscopic twist 3 based on the real-time observation.\nFigure R25. Length measurement images of microscopic twist 4 based on the real-time observation.\nFigure R26. The variation in a. width and b. length of a growing microscopic twist as a function of time, with the fitted lines elucidating the underlying growth dynamics.\n4. Figure 4 and the related discussion indicated that the π-π interlocking between bilayers can play a crucial role in stabilizing the assembly. Authors need further expand the discussion to offer the analysis of this kind of interaction within the context of the synergistic stabilization mechanism alongside hydrogen bonding and hydrophobic effects.\nResponse: We thank the reviewer for this insightful suggestion. Following your advice, we have expanded the discussion in the manuscript to explicitly address the synergistic stabilization mechanism. The relevant additions in the main text are as follows: \"... This chiral encoding was structurally realized by the formation of a highly tilted bilayer driven by the optimal f = 30% condition, which in turn enabled effective inter-bilayer π-π stacking. The resulting interlocked arrangement of the headgroups served as a pivotal stabilizing factor for the entire assembly.\" and \"...This interlocked π-π stacking acts synergistically with hydrogen bonding and hydrophobic effects, constituting a multi-faceted stabilization mechanism.\" These additions clarify the critical role of π-π interlocking within the cooperative framework of non-covalent interactions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Accept in current format", "reviewer_2": "The authors have addressed all my comments. I support its publication in Nature Communications.", "reviewer_3": "[Note from the Editor: The reviewer submitted only comments to the editor, but raised no further technical concerns]"}, "author_response": "Response: We sincerely appreciate your positive feedback on our manuscript and your strong support."}]

Decision letter