将 DNA 折纸精确放置在图案化硅片表面 Precision Placement of DNA Origami onto Patterned Silicon Wafer Surfaces

PrecisionPlacementofDNAOrigamiontoPatternedSilicon

WaferSurfaces

LeoHuang

YunjeongPark,Ed.

GrigoryTikhomirov,Ed.

ElectricalEngineeringandComputerSciencesUniversityofCalifornia,Berkeley

TechnicalReportNo.UCB/EECS-2025-76

/Pubs/TechRpts/2025/EECS-2025-76.html

May15,2025

Copyright©2025,bytheauthor(s).

Allrightsreserved.

Permissiontomakedigitalorhardcopiesofallorpartofthisworkfor

personalorclassroomuseisgrantedwithoutfeeprovidedthatcopiesare

notmadeordistributedforprofitorcommercialadvantageandthatcopiesbearthisnoticeandthefullcitationonthefirstpage.Tocopyotherwise,torepublish,topostonserversortoredistributetolists,requirespriorspecificpermission.

PrecisionPlacementofDNAOrigamiontoPatternedSiliconWaferSurfaces

LeoHuang

ResearchProject

SubmittedtotheDepartmentofElectricalEngineeringandComputerSciences,

UniversityofCaliforniaatBerkeley,inpartialsatisfactionoftherequirementsforthedegreeofMasterofScience,PlanII.

ApprovalfortheReportandComprehensiveExamination:

Committee:

ProfessorGrigoryTikhomirovResearchAdvisor

5/14/2025

(Date)

*******

ProfessorBoubacarKantéSecondReader

05/14/2025

(Date)

PrecisionPlacementofDNAOrigamiontoPatternedSiliconWaferSurfaces

by

LeoHuang

Athesissubmittedinpartialsatisfactionofthe

requirementsforthedegreeof

MastersofScience

in

ElectricalEngineeringandComputerScience

inthe

GraduateDivision

ofthe

UniversityofCalifornia,Berkeley

Committeeincharge:

ProfessorGrigoryTikhomirov,Chair

ProfessorBoubacarKant´e

Spring2025

PrecisionPlacementofDNAOrigamiontoPatternedSiliconWaferSurfaces

Copyright2025

by

LeoHuang

1

Abstract

PrecisionPlacementofDNAOrigamiontoPatternedSiliconWaferSurfaces

by

LeoHuang

MastersofScienceinElectricalEngineeringandComputerScience

UniversityofCalifornia,Berkeley

ProfessorGrigoryTikhomirov,Chair

StructuralDNAnanotechnologyoffersapromisingrouteforconstructingnanometer-scalecomponentswithhighspatialprecision,whiletop-downphotolithographictechniquesre-mainessentialforproducingpatternedsubstratesatscale.Previouswork—mostnotablybyGopinathetal.—hasdemonstratedpreciseplacementofDNAorigamiusingelectronbeamlithography,butthisapproach’slowthroughputposeschallengesforbroaderapplica-tion.Here,weextendthisapproachbyexploringtheuseoffractal-assembledDNAorigamitilesforsite-specificdepositionontophotolithographicallypatternedsiliconsurfaces.Thisworkinitiatesasystematicexplorationofhowtilegeometry,surfacechemistry,andbind-ingconditionsinfluencetheintegrationofDNAnanostructureswithscalablefabricationplatforms,specificallytheirimpactonplacementyieldandquality.Ourworkcompareselectrostaticallyandthermodynamicallydrivenbindingstrategiesasasteptowardsamoregeneralizableframeworkforhybridbottom-up/top-downnanofabricationmethods.Ween-visionthismethodtocomplementexistingapproachesandexpandtheroleofDNAorigamiinapplicationssuchasbiosensingandprogrammablenanosystems.

i

Tomyfriendsandfamily,

ii

Contents

Contentsii

ListofFiguresiii

ListofTablesv

1Introduction1

2BackgroundandRelatedWork3

2.1IntroductiontoDNAOrigamiandNanotechnology 3

2.2PreviousWorkonDNAOrigamiPlacement 3

2.3FractalAssemblyandLarge-ScaleDNAOrigamiPatterns 4

2.4TriangularDNAOrigamiTilesand3DStructures 6

3Methods7

3.1DNAOrigamiDesignandSynthesis 7

3.2StructuralVerificationandYieldAnalysis 10

3.3MaskDesignforSurfacePatterning 12

3.4PlacementTechniques 15

4ExperimentsandResults21

4.1DNADesignandYieldOptimization 21

4.2LithographyandSubstrateOptimization 25

4.3Oligo-FacilitatedBindingviaGPTMSFunctionalization 27

4.4ElectrostaticBindingviaMagnesiumIonBridging 31

5ConclusionandDiscussion39

Bibliography40

iii

ListofFigures

2.2AdaptedfigurefromGopinatheta.[2]illustratingself-assemblyofDNAorigami

onlithographicallypatternedsurfaces 4

2.3AdaptedfigurefromKershneretal.[4] 5

2.4AdaptedfigurefromTikhomirovetal.[7]illustratingfractalassemblyofDNA

tiles 5

2.5AdaptedfigurefromTikhomirovetal.[8]showingtriangulartiledesign 6

3.192x92nmDNAorigamimonomertile 7

3.2184x184nmDNAorigamitetramertile 8

3.3AdaptedfigurefromTikhomirovet.al 9

3.4Examplegelexhibitingstrongmonomerbands,verifyingsuccessfulassembly 11

3.5MaskV1isstructuredtoevaluateDNAorigamidepositionacrossmultiplepattern

sizesandshapeswithoutmaskreplacement.Figureshowsthehierarchallayers

ofthemask 13

3.6MaskV2containssix5x5arraysofcircularpatternswithsizesrangingfrom

100nmto580nm,allowingtargetedassessmentofdepositionyieldandorigami

alignment 13

3.7MaskV3consistsofsix5x5arraysofcircularpatterns,eachregioncontaining

uniformpatternsizes.SmallPRarrowmarkerswereaddednearthepatterned

regionstoassistinlocatingspecificareasduringAFMimaging 14

3.8WorkflowforGPTMSfunctionalizationandDNAorigamideposition 15

3.9PotentialbindingmodesofDNAorigamitosurface-boundoligonucleotides.(a)

Vertical,stilt-likebinding.(b)Horizontal,zipper-likebinding 17

3.10WorkflowofMagnesiumIonBridgeDepositionandDNAorigamiplacement 18

3.11MechanismofelectrostaticDNAorigamibindingviaMg2+ionbridging 20

4.1Proposedconnectorvariationsfordouble-layersynthesis 21

4.2Gelelectrophoresisanalysisofasynthesisexperiment.Absenceofdistinctbands

correspondingtosingle-layeranddouble-layer2x2tilesindicatespooryield 22

4.3AFMimagesofdouble-layersynthesis.Double-layertilesareidentifiablebytheir

brighter,tallerprofilesbutarepresentinlowconcentrations,indicatingpooryield.23

iv

4.4Comparisonofhexagonaltilestabilityandyield.(a)NormalHexexhibitslower

yieldandstructuralintegrity.(b)StrongHexdemonstrateshigheryieldand

robustness 24

4.5OptimizedhexagondesignincorporatingpolyC14andpolyT20extensionstomit-

igatestackingandaggregation 24

4.6Hypothesizedmodelofsurfaceroughness.(a)Highroughnesscouldencourage

origamifolding.(b)Smoothersurfaceminimizesunwantedfoldinginteractions 25

4.7AFMcomparisonofsurfaceroughness.(a)Thermallygrownlayerexhibitsrough-

nessof10angstroms.(b)Chemicaloxidelayerachievesroughnessof1.7angstroms.26

4.8SEMimagesshowingtheeffectofPRexposuredosageonfeaturesize 27

4.9ComparisonofsurfacebindingbeforeandafterBSAtreatment.(a)WithoutBSA

application,DNAorigaminonspecificallyadherestothebackground.(b)With

BSAapplication,nonspecificbindingissignificantlyreduced,improvingbinding

specificity 28

4.10WorkflowforPMMA-OHbrushandDNAorigamiplacement 29

4.11DNAorigamidepositiononPMMA-OHtreatedsurfaces.(a)Reducedback-

groundbindingisobserved.(b)Smallerregionrevealsfoldingoforigamiwithin

patterns 29

4.12AFMimagesofPMMA-OHtreatedchipdepositedwithmonomerorigami 32

4.13AFMimagesofdepositiononsmoothedsurfaces.Thebindingsitescanbeob-

servedtobegenerallymoreuniforminheight,indicatingflatter,unfoldedorigami.33

4.14AFMimagesofstackingduringorigamideposition.Intherightimage,multiple

origamicanbeseenbindingtoasinglebindingsite,oftenstackingovereachother.33

4.15AFMimagesshowingoptimizationbetweenpatternsizeandhexagonaltiles.(a)

Tileshapemodificationsfitpatternedareamoreeffectively.(b)OptimizedPR

exposurecloselymatchespatternsizetohexagonsize,reducingmultiplebinding

persite 34

4.16Effectsofdryingonorigamiplacement.Weobservedlowoccupancyanddetach-

ment(left),foldingvialift-off(middle),andaggregationinthedryingdirection

(right) 35

4.17IllustratingeffectofMg2+ondeposition(100pMDNA4hr) 36

4.18IllustratingeffectofNaClondeposition(10mMMg100pMDNA4hr) 37

4.19Illustratingeffectofincubationtimeondeposition(6mMMg100pMDNA) 37

4.20Comparisonbetweeninitialdepositionresultsandcurrentoptimaldeposition 38

v

ListofTables

3.1AFMImagingParametersforStructureVerificationandPlacementEvaluation.11

3.2OligonucleotideSequencesTestedforSurfaceFunctionalization 17

4.1TestedParametersforGPTMSFunctionalizationandOligoDeposition 30

4.2CurrentOptimizedBufferandDepositionParameters 37

vi

Acknowledgments

IwanttothankProfessorGrigoryTikhomirovforhiscontinuedsupportandfeedbackthroughoutmyyearsworkingwithhim.Iwouldalsoliketothankmymentors,Profes-sorLinDuandDr.YunjeongPark,whohaveofferedmeincredibleguidanceandhelpthroughoutmyresearchjourney.Finally,Iwouldliketothankmyfriendsandfamily,whosewarmthandsupporthavepushedmetobewhoIamtoday.

1

Chapter1

Introduction

DNAorigamihasrapidlyadvancedasapromisingplatformforconstructingnanometer-scalestructureswithexceptionalgeometricprecisionanddesignflexibility.Byleveragingtheprogrammablebase-pairingofDNAstrands,itispossibletoassemblearbitrary2Dand3Darchitectureswithsub-nanometercontrol,enablingapplicationsinmolecularcomputa-tion,nanoscalepatterning,andbiosensing.However,despitesignificantprogressincreatingincreasinglycomplexstructures—fromsingle-unitdesignstolargepixel-addressablearraysthroughhierarchicalandfractalassembly—thebroaderintegrationofDNAorigamiintoscalablefabricationworkflowsremainsanopenchallenge.AsDNAnanotechnologymovestowardmoreintricatesystems,reliablypositioningandaligningthesestructuresonsolidsubstratesbecomesincreasinglycritical,particularlyforthoserequiringhigh-throughput,site-specificdeposition.

Recentadvancesinscalableassemblystrategies,suchasfractalandhierarchicaltiling,havesignificantlyexpandedthedesignspaceofDNAorigami.Theseapproachesenablethegenerationoflarge,pixel-addressablearraysfromasmallsetofmodularcomponents,allowingforincreasinglycomplexandspatiallyextensiveDNAnanostructures.Thisabilitytoproduceintricate,programmableassembliespositionsDNAorigamiasaversatileplatformformolecularcomputation,nanoscalepatterning,andsensing.However,asthesestructuresgrowinscaleandfunctionaldiversity,thechallengeshiftsfromassemblytointegration,specifically,howtotransfertheseassembliesontosolidsubstrateswithhighspatialfidelityandreproducibility.

Beyondprogrammableshapes,methodsforreliablypositioningDNAnanostructuresonconventionalsubstrateswithhighyield,spatialaccuracy,andpatterndiversitycanfurtherextendtheapplicationsofDNAnanotechnology.OnepromisingapproachistoguideDNAorigamitobindsite-specificallyontochemicallydefinedregionsofasiliconsurface.Priorworkhasdemonstratedthatlithographicallypatternedbindingsites,particularlythosede-finedviaelectronbeamlithography,canachievehigh-precisionplacementoforigamistruc-tures.Thiscapabilityhasenabledcompellingproof-of-conceptdevices,suchasnanophotonicresonatorswithemitter-origamicouplingandlarge-scaleDNAnanoarraysformolecularpat-terning.However,relianceonelectronbeamlithographypresentsascalabilitybottleneck.

2

CHAPTER1.INTRODUCTION

Itslimitedthroughput,highcost,andserialnaturemakeitpoorlysuitedforintegrationwithwafer-scaleorcommercialmanufacturingprocesses.

ThisthesisexploresanalternativestrategyforDNAorigamiplacementthatcombineslarge-scalefractalassemblyofDNAnanostructureswithhigh-throughputphotolithographicpatterningofsiliconsubstrates.Fractal-assembledorigamienablestheconstructionoflarge,addressableDNAarraysfrommodularcomponents,servingasascalablebottom-upfab-ricationstrategy.Meanwhile,photolithographyprovidesanaccessible,industry-standardmethodfordefiningplacementsitesacrosslargeareas.Together,theseapproachespresentnewopportunitiesforhybridbottom-up/top-downfabrication,bridgingthenanoscalepre-cisionofDNAassemblywiththescalabilityofsemiconductormanufacturing.

Here,wesystematicallyinvestigatehoworigamitilegeometry,surfacechemistry,andbindingmodalityaffectplacementperformance,specificallyyieldandbondquality.Wecompareelectrostaticallymediatedbindingtothermodynamicallycontrolledhybridizationschemesandevaluatehowdifferenttiledesignsinteractwithphotolithographicallypat-ternedfeatures.WeaimtoestablishamoregeneralizableframeworkforintegratingcomplexDNAnanostructureswithscalablesubstratefabricationtechniques.Ultimately,weenvisionthismethodologycomplementingexistingapproachesandcontributingtowardthebroaderadoptionofDNA-basedcomponentsinbiosensing,nanoscalepatterning,andprogrammablemolecularsystems.

3

Chapter2

BackgroundandRelatedWork

2.1IntroductiontoDNAOrigamiand

Nanotechnology

DNAorigami,firstintroducedbyRothemund[6],enablesthefoldingofalongsingle-strandedDNAintowell-definednanoscaleshapesusingcomplementaryshortstaplestrands,resultinginpreciseprogrammablenanoscaleassemblieswithhighyieldandgeometrichomogeneity.Thistechniquehasevolvedtoproduce2Dand3Dstructurescapableofcomplexmolecularorganization,servingasscaffoldsforfunctionalmolecules,nanoparticles,andbiomolecules.Applicationsspanfrombiosensingandmolecularcomputingtotargeteddrugdeliverysys-tems,showcasingtheversatileprogrammabilityofDNAorigami[3].StructuralDNAnan-otechnologythusbridgesmolecularself-assemblywithtop-downlithographictechniques,offeringnewavenuesfornanoscalepatterninganddevicefabrication.

(a)AdaptedfigurefromBabatundeetal.[1]illustratingDNAorigamidesign.

(b)AdaptedfigurefromZhimeietal.[3]

2.2PreviousWorkonDNAOrigamiPlacement

BothAshwinGopinathetal.[2]demonstratedarobustmethodforpreciselyplacingDNAorigamistructuresusingelectronbeamlithographytodefinebindingsitesonsiliconni-

4

CHAPTER2.BACKGROUNDANDRELATEDWORK

tridesurfaces.Thismethodachievedupto94%placementyield,enablingthecouplingofmolecularemitterstophotoniccrystalcavities(PCCs)forenhancedlight-matterinterac-tions.Theapproacheffectivelyutilizedcarboxylate-functionalizedbindingsites,allowingforthedirectedself-assemblyofCy5-labeledDNAorigami,achievingspatialcontrolcrucialfornanophotonicandquantuminformationsystems.However,thescalabilityofthistechniqueremainsconstrainedbythethroughputlimitationsinherentinelectronbeamlithography.

Figure2.2:AdaptedfigurefromGopinatheta.[2]illustratingself-assemblyofDNAorigami

onlithographicallypatternedsurfaces.

Additionally,Kershneretal.[4]developedatechniqueforplacingandorientingindivid-ualDNAorigamistructuresonlithographicallypatternedsurfaces.Byemployingelectron-beamlithographyanddryoxidativeetching,theycreatedbindingsitesonsubstrateslikeSiO2anddiamond-likecarbonthatmatchedtheshapeoftheDNAorigami.Thisapproachachievedhighselectivityandorientationcontrol,with70—95%ofthesitesoccupiedbysingleDNAorigamistructuresalignedwithin±10。ondiamond-likecarbonand±20。onSiO2.SuchprecisioniscrucialforintegratingDNAnanostructuresintonanoelectronicandnano-opticaldevices,asitensuresconsistentpositioningandorientationnecessaryfordevicefunction-ality.Thisworkunderscoresthepotentialofcombiningtop-downlithographictechniqueswithbottom-upDNAself-assemblytofabricatecomplexnanodevices.

2.3FractalAssemblyandLarge-ScaleDNAOrigamiPatterns

GrigoryTikhomirovetal.[7]advancedthescalabilityofDNAorigamiassemblybyintroduc-ingfractalassembly.Thishierarchicalassemblymethodconstructslarge-scalepatternsusingsmallerDNAorigamitilesasmodularbuildingblocks.Byencodingbindinginteractionsateachassemblystage,thismethodfacilitatedthegenerationofmicrometer-scalepatterns

5

CHAPTER2.BACKGROUNDANDRELATEDWORK

Figure2.3:AdaptedfigurefromKershneretal.[4].

withupto8,704addressablepixels,expandingthepotentialforDNAnanostructurestointegratewithlargersubstrateareaswhilemaintainingnanoscaleprecision.Thefractalassemblyframeworkalsodemonstratedrobustnessingeneratingcomplexpatternswithoutcompromisingspatialresolution,underscoringitsapplicabilityincreatingprogrammableDNA-basedmaterials.

Figure2.4:AdaptedfigurefromTikhomirovetal.[7]illustratingfractalassemblyofDNA

tiles.

6

CHAPTER2.BACKGROUNDANDRELATEDWORK

2.4TriangularDNAOrigamiTilesand3DStructures

Infurtherwork,Tikhomirovetal.[8]exploredusingtriangularDNAorigamitilesfortwo-dimensionalandthree-dimensionalassemblies.Unlikeprevioussquaretiledesigns,thesetriangulartilesprovidedadditionalstructuralflexibility,allowingforcontrolledtransitionsbetweenplanararraysandpolyhedralstructures.Theresearchersachievedtunableassem-blymodesbyadjustingparameterssuchastileconcentrationandmagnesiumioncontent,producingbothextended2Darraysandcompact3Drhombictriacontahedrons.Thisap-proachintroducednewgeometricconfigurationsforDNAorigami,enablingthedevelopmentofmorecomplex,reconfigurableDNAnanostructuresthatcouldinterfacewithlithographi-callypatternedsurfaces.

Figure2.5:AdaptedfigurefromTikhomirovetal.[8]showingtriangulartiledesign.

7

Chapter3

Methods

3.1DNAOrigamiDesignandSynthesis

Thissectionoutlinesthedesign,synthesis,andassemblyprocessforthreeDNAorigamistructuresutilizedinthisstudy:the92x92nmsquaretile,the184x184nmsquaretile,andthe270nmhexagonaltile.Eachsubsectionincludesthecomputationaldesign,assemblyprotocol,andpurificationsteps.

SquareMonomerOrigami

(a)SchematicofNESWdesignationsandedgetypes.AdaptedfigurefromTikhomirovet.al.[7].

(b)Blueprintshowingscaffoldpathandstaplelayout.

Figure3.1:92x92nmDNAorigamimonomertile.

8

CHAPTER3.METHODS

DesignandComputationalAnalysis

Thesquaremonomertileisthefundamentalbuildingblockforsubsequentassembliesusedinthiswork.ThetiledesignedbyTikhomirovetal.[7]isfoldedfromanM13mp18scaffoldstrand(7,249nt)andcomprises10heliceswith32basepairsperhelix,formingasquaremeasuring92x92nm.Thetileisorientedusingcardinaldirections(North,East,South,West),witheachedgedesignedto”give”or”receive”throughcomplementarystickyendsequences.Thisedgeassignmentenablescontrolleddirectionalassemblyandminimizesmisalignment.

SynthesisandAssemblyProtocol

•Mixing:Single-strandedM13mp18scaffoldstrands(10nM)arecombinedwithasetof206staplestrands(75nM)in1xTEbuffercontaining12.5mMMgCl2.

•Annealing:Thereactionmixtureisinitiallyheatedto90。Cfor2min,thengraduallycooledto20。Cat6secper0.1。C.

•Negation:Followingannealing,afive-foldexcessof44negationstrandsisaddedtothemixture.Thesampleissubsequentlycooledfrom50。Cto20。Cat2secper0.1。C.

•Purification:AssembledmonomersarepurifiedusingAmiconUltra-0.5centrifugalfilters(100kDaMWCO)toremoveexcessstaplestrandsandunboundscaffoldDNA.

SquareTetramerOrigami

(a)Schematicoftileorientationandlayout.(b)AFMimageof2x2squaretiles.

Figure3.2:184x184nmDNAorigamitetramertile.

9

CHAPTER3.METHODS

DesignandComputationalAnalysis

Thetetramerstructureisassembledfromfour92×92nmsquaremonomers,followingthedesignstrategyintroducedbyTikhomirovetal.[7].Eachmonomerisconnectedthroughcomplementaryedgesequencestoforma2×2arraymeasuring184×184nm.TheNESWorientationsfacilitatecontrolledinteractionsateachedge.Theassignmentof”giving”and”receiving”edgesenforcesdirectionalassembly.

SynthesisandAssemblyProtocol

•Mixing:Monomerswiththeirrespective”giving”and”receiving”endsaremixed.

•Annealing:Thereactionmixtureisannealedfrom55。Cto45。Cat2minper0.1。Candthenfrom45。Cto20。Cat6secper0.1。C.

•Purification:Theassembledtetramerispurifiedusingthesameultrafiltrationpro-cedureasthemonomertoremoveexcessDNAstrands.

HexagonHexamerOrigami

Figure3.3:Schematicofhexagontileconstruction.AdaptedfigurefromTikhomirovet.al.

[8].(a)Edgeinteractionsthatcomposetheassembledhexagon.(b)AFMimagesof270nmhexagontiles.

10

CHAPTER3.METHODS

DesignandComputationalAnalysis

Thehexagontileisassembledfromsixtriangularsubunits,basedonthedesignmethodologyoutlinedbyTikhomirovetal.[8].Twodistincttriangulartileswithdesignatedgivingandreceivingedgesguidespecifichybridization,directingtheformationofahexagonwithadiagonalofapproximately270nm.Thestrategicarrangementofstickyendsequencesensuresproperorientation,minimizingmisalignmentsandpromotingaccurateassembly.

SynthesisandAssemblyProtocol

•Mixing:Twoseparatetubescontainingsingle-strandedM13mp18scaffoldstrands(10nM)aremixedwithasetof216staplestrands(15nM)in1xTEbufferwith12.5mMMgCl2.Eachtubecorrespondstooneofthetwodistincttriangulartilesnecessaryforhexagonassembly.

•Annealing:Thereactionmixturesareinitiallyheatedto90。Cfor2minutesandthencooledto20。Catarateof0.1。Cper6seconds.

•Negation:Followingtheinitialannealing,aten-foldexcessof48negationstrandsisadded.Themixturesarethencooledfrom50。Cto20。Cat2secper0.1。C.

。。

•Purification:Thetwotilemixturesarecombinedandthencooledfrom50Cto20C

at2minper0.1。C.

•Purification:Nopurificationstepisperformedfollowingassembly.

3.2StructuralVerificationandYieldAnalysis

Followingassembly,verifyingthesuccessfulfoldingofdesiredDNAorigamispeciesisoftennecessary.Thissectionoutlinestheverificationmethodsemployedwithinthiswork,alongwiththecriteriaforfoldingyieldanalysis.Theprotocolsdescribedhere,developedincollab-orationwiththeTiLab,incorporatemodificationsbasedonestablishedpracticesinDNAnanotechnology.

GelElectrophoresis

AgarosegelelectrophoresisisemployedtoverifytheassemblyofDNAorigamistructuresandconfirmthepresenceoftargetspeciesbycomparingbandintensitiesagainstaknownDNAladder.Gelswerepreparedusing0.5xTBEbufferwith12.5mMMgCl2andstainedwithethidiumbromide(EtBr).Electrophoresisconditionswereoptimizedbasedonstructuralcomplexityasfollows:

•StandardStructures(Monomers,2x2Arrays):1%(w/v)agarosegel,75V,90minutes.

11

CHAPTER3.METHODS

•DelicateStructures(Hexagons,4x4Arrays):0.6%(w/v)agarosegel,45V,2hours.

Figure3.4:Examplegelexhibitingstrongmonomerbands,verifyingsuccessfulassembly.

AFMMeasurement

AtomicForceMicroscopy(AFM)isemployedtoverifythestructuralintegrityofDNAorigamiassembliesandtoassesstheperformanceofplacementtechniquesbyevaluatingbothoccupancyyieldandbindingqualitytothesubstratesurface.AFMimagingisconductedusingaBrukerAFMsystemwithdistinctmodes,settings,andtiptypes,dependingontheapplication.ThespecificAFMparametersforeachmodeareoutlinedinTable3.1.

Table3.1:AFMImagingParametersforStructureVerificationandPlacementEvaluation

Parameter

AirMode

FluidMode

ExperimentType

ScanAsystAirHR

ScanAsystFluidHR

ScanTip

ScanAsystAirHR

ScanAsystFluid

ScanRate

3.82Hz

3.03Hz

FeedbackGain

4—6.2

3.42—22

PeakForceSetpoint

746pN

460—820pN

PeakForceAmplitude

100nm

6nm

LiftHeight

64.8nm

12nm

TheselectionofspecificAFMmodesandparametersisdeterminedbytheexperimentalobjective,asoutlinedbelow:

•StructureVerification:DNAsamplesaredepositedonmicachipstoverifycorrectassemblyandquantifysynthesisyield.Thismethodistypicallyperformedinfluidmodeforoptimalresolution,thoughairmodeenablesfasterscanning.

12

CHAPTER3.METHODS

•PlacementEvaluation:DNAstructuresareimageddirectlyonsiliconsubstratestoassesssurfacebindinganddistribution.Initially,scanswereconductedinairmode.However,welaterfoundthatfluidmodeimagedsurfaceinteractionmoreaccurately,sotheprotocolwasadjusted.

FoldingYieldAnalysis

FoldingyieldisdeterminedbyanalyzingAFMimagestoquantifythenumberofcorrectlyfoldedstructuresrelativetounfoldedormisfold