Pnas-Label-free detection of DNA hybridization using carbon nanotube network field-effect transistor

Carbon nanotube

Label-freedetectionofDNAhybridizationusingcarbonnanotubenetworkfield-effecttransistors

AlexanderStar*,EugeneTu,JosephNiemann,Jean-ChristopheP.Gabriel,C.SteveJoiner ,andChristianValcke

Nanomix,Inc.,Emeryville,CA94608

EditedbyMildredS.Dresselhaus,MassachusettsInstituteofTechnology,Cambridge,MA,andapprovedDecember2,2005(receivedforreviewMay19,2005)

Wereportcarbonnanotubenetwork eld-effecttransistors(NTNFETs)thatfunctionasselectivedetectorsofDNAimmobiliza-tionandhybridization.NTNFETswithimmobilizedsyntheticoli-gonucleotideshavebeenshowntospeci callyrecognizetargetDNAsequences,includingH63Dsingle-nucleotidepolymorphism(SNP)discriminationintheHFEgene,responsibleforhereditaryhemochromatosis.TheelectronicresponsesofNTNFETsuponsin-gle-strandedDNAimmobilizationandsubsequentDNAhybridiza-tioneventswerecon rmedbyusing uorescence-labeledoligo-nucleotidesandthenwerefurtherexploredforlabel-freeDNAdetectionatpicomolartomicromolarconcentrations.WehavealsoobservedastrongeffectofDNAcounterionsontheelectronicresponse,thussuggestingacharge-basedmechanismofDNAdetectionusingNTNFETdevices.Implementationoflabel-freeelectronicdetectionassaysusingNTNFETsconstitutesanimportantsteptowardlow-cost,low-complexity,highlysensitiveandaccu-ratemoleculardiagnostics.

hemochromatosis SNP biosensor

T

hedevelopmentofnucleicacidsdiagnosticshasbecomethesubjectofintenseresearch,especiallyinthepostgenomeera.Currentmethodshavemainlyfocusedonopticaldetectionusingfluorescence-labeledoligonucleotideswithdyes(1),quantumdots(2),orenhancedabsorptionoflightbyoligonucleotide-modifiedgoldnanoparticles(3).Ontheotherhand,label-freeelectronicmethodspromisetooffersensitivity,selectivity,andlowcostforthedetectionofDNAhybridization(4).Forexample,microfabricatedsiliconfield-effectsensorscanmonitordirectlytheincreaseinsurfacechargewhenDNAwashybridizedonthesensorsurface(5).Nanomaterialspossessuniqueprop-ertiesthatareamenabletobiosensorapplications;theyareone-dimensionalstructuresthatareextremelysensitivetoelec-tronicperturbations,readilyfunctionalizedwithbiorecognitionlayers,andcompatiblewithmanysemiconductingmanufactur-ingprocesses.Thus,one-dimensionalsiliconnanowires(6–8)andindiumoxidenanowires(9)haveshownpromisingperfor-mance,http://doc.xuehai.netingsmallernanowireswithvirtuallyallatomsontheirsurface,suchassingle-walledcarbonnanotubes(SWNTs),willprovideadditionaladvantagesinDNAdetection.Todate,thereareseveralreportsonelectrochemicaldetectionofDNAhybridizationusingmulti-walledcarbonnanotubeelectrodes(ref.10andreferencestherein,andref.11).Whereaselectro-chemicalmethodsrelyonelectrochemicalbehaviorofthelabels,measurementofdirectelectrontransferbetweenSWNTsandDNAmoleculespavesthewayforlabel-freeDNAdetection.SWNT-basedfield-effecttransistors(12)haveexcellentoperat-ingcharacteristics(13),andtheyhavealreadybeenexploredforhighlysensitiveelectronicdetectionofgases(14,15)andbi-omoleculessuchasantibodies(16,17).

Single-strandedDNA(ssDNA)hasbeenrecentlydemon-stratedtointeractnoncovalentlywithSWNTs(18,19).ThessDNAformsastablecomplexwithindividualSWNTsbywrappingaroundthembymeansofthearomaticinteractions

http://doc.xuehai.net cgi doi 10.1073 pnas.0504146103

betweennucleotidebasesandSWNTsidewalls.Double-strandedDNAmoleculeshavealsobeenproposedtointeractwithSWNTsasmajorgroovebinders(20).Here,weemploycarbonnanotubenetworkfield-effecttransistor(NTNFET)devicestoinvestigateinteractionsbetweenssDNAoligonucle-otidesandSWNTsandsubsequentDNAhybridizationprocessesthattakeplaceonthedevicesurface.WehavefoundthatNTNFETscanbeselectivelyfunctionalizedwithDNAoligonu-cleotidesandretainhybridizationspecificity.Thus,NTNFETswithimmobilizedsyntheticoligonucleotideshavebeendemon-stratedtoselectivelyrecognizetargetDNAsequenceswithSNP.Wedemonstratethesinglebasemismatchdiscriminationusingwild-typeversusH63DmutationintheHFEgene,responsibleforhereditaryhemochromatosis.ResultsandDiscussion

DNAImmobilizationandHybridizationonNTNFETs.First,wecon-siderdepositionofssDNAonNTNFETdevices.Asacaptureprobe,ssDNA:5 -CCTAATAACAAT-3 (AlphaDNA)wasselected.Thisoligonucleotidesequencewaspreviouslyusedinsensorsbasedonsiliconnanowires(8).Fig.1Ashowsafluo-rescentimageoftheinterdigitateddevicewithdistancebetweenelectrodesof10 mafterincubationwithCy5-labeledssDNAcaptureprobe,followedbythoroughwashingstoremoveexcessandweaklyboundDNAmolecules.WhereasbareNTNFETdeviceshaveshownnomeasurablefluorescencesignal(imageisnotshown),thedevicesafterincubationwithCy5-labeledssDNAshowclearfluorescence(Fig.1A).Interestingly,fluo-rescenceclearlycomesfromdeviceareascoveredwithcarbonnanotubes;thereisnofluorescencefromthebaresiliconsur-face.ThisobservationsupportsselectiveadsorptionofssDNAmoleculesonthesidewallsofcarbonnanotubes.TheDNAincubationexperimentswererepeatedunderthesameconditionswithunlabeledssDNAcaptureprobe(Fig.1B),topreparetheNTNFETdevicesforsubsequenthybridizationexperimentswithFITC-labeledcomplementaryDNAsequences(Table1).Forfluorescentexperiments,weused50nMtargetDNAtoallowintegrationofthefluorescencesignalin10–20sectominimizephotobleaching.Thechangesinfluorescencebefore(Fig.1B)andafter(Fig.1C)hybridizationconfirmthatDNAhybridiza-tiontakesplaceundertheseexperimentalconditions.

Seriesofcontrolexperimentshavebeenconductedtoverifytheseobservations.Fig.1DshowsfluorescentimageoftheNTNFETdeviceafterincubationwithCy5-labeleddA12oligo-nucleotide.ThisssDNAisadsorbedoncarbonnanotubesina

Con ictofintereststatement:Nocon ictsdeclared.

Thispaperwassubmitteddirectly(TrackII)tothePNASof ce.

Abbreviations:NTNFET,carbonnanotubenetwork eld-effecttransistor;PB,phosphatebuffer;SWNT,single-walledcarbonnanotube.

*Towhomcorrespondencemaybesentatthepresentaddress:DepartmentofChemistry,UniversityofPittsburgh,Pittsburgh,PA15260.E-mail:astar@pitt.edu.

Present

address:DepartmentofChemistryandBiochemistry,UniversityofCalifornia,

LosAngeles,CA90095.

To

whomcorrespondencemaybeaddressed.E-mail:cvalcke@http://doc.xuehai.net.

©2006byTheNationalAcademyofSciencesoftheUSA

PNAS January24,2006 vol.103 no.4 921–926

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