Preparation of Nucleic Acid
Functionalized Carbon Nanotube Arrays
Cattien V.Nguyen,*,†Lance Delzeit,Alan M.Cassell,†Jun Li,†Jie Han,†and
M.Meyyappan
Center For Nanotechnology,NASA Ames Research Center,
Moffett Field,California94035
Received July9,2002;Revised Manuscript Received August6,2002
ABSTRACT
A vertically aligned carbon nanotube(CNT)array is fabricated as a nanoelectrode platform for biosensor development.Prior to chemical functionalization,metal catalyst particles at the ends of CNT are removed and the closed ends are opened.We find that the oxidative treatment for generating the chemical functional groups at the opened ends of the CNT compromise the mechanical stability of the
nanotubes,often leading to total collapse of the aligned CNTs.To solve this problem,we have developed a new approach for filling the gaps between CNTs with a spin-on glass(SOG).Results from the coupling of nucleic acids to the CNT arrays suggest that the SOG enhances the reactivity by providing structural support to the CNTs.The SOG also covers the length of the sidewalls of CNTs,leading to a less hydrophobic interface and thus may aid in improving the chemical reactivity.
Carbon nanotubes(CNTs)have been proposed as transducer materials for application as biosensors.1One of the challenges facing the implementation of CNT arrays as selective and ultrasensitive biosensors is the addition of chemical func-tionalities while preserving their inherent chemical and electrical properties.2For multiwalled CNT arrays,maintain-ing the desired vertical orientation facilitates their imple-mentation as electrochemical sensor transducers.Several nanoscale materials exhibit enhanced electrochemical sen-sitivity over their larger scale analogues.For example,gold nanoelectrode ensembles are capable of detection limits as much as3orders of magnitude lower than conventional gold disk microelectrodes(>10nm diameter).3,4As such,it is expected that further materials development and electrode downsizing from the micro to nanoscale will increase biosensor detection sensitivity limits.
Electrochemical studies using CNTs as electrode elements have been reported5as well as electroche
mical-initiated chemical functionalization of the sidewall of CNTs.6There are also few examples of small molecules reacting with carboxylic acid groups at the oxidatively opened ends of single wall CNT,including primary alkylamine in CNT dispersion7and benzylamine,as well as ethylenediamine group for the CNT as chemical force microscopy tips.8An interesting CNT ring structure was observed and attributed to the coupling between the terminal carboxylic acid groups at the two open ends of carbon nanotube.9
Here we present an approach for the attachment of nucleic acids to oxidatively opened ends of CNT arrays,an important step in the development of a general platform for detecting biological macromolecules.In addition to necessary steps such as opening the closed CNT ends and removing metal catalyst at the CNT tips,a novel and critical step is the deposition of a spin-on glass(SOG)film inside hydrophobic CNT arrays.
The multiwalled CNT arrays were grown using a plasma enhanced chemical vapor deposition process described previ-ously.10,11Briefly,a10nm film of iron catalyst was deposited on silicon substrate by ion beam sputtering technique.Growth was accomplished at a substrate temperature of900°C for 10min with20sccm of C2H4and80sccm of H2,under3 Torr of total pressure and with100W of inductive and200 W of capacitive power.Electron microscopy(SEM and TEM)analysis shows that the CNTs are relatively
straight and that the ends are closed with metal-encapsulated catalyst and covered by amorphous carbon.After extensive oxidative pretreatment to render the CNT ends with carboxylic acid end groups for nucleic acid attachment,we found some problems.Notably,the mechanical stability of the aligned CNTs was often compromised,and quite frequently,total collapse of the CNTs arrays did occur.Additionally,even after extensive oxidative pretreatment,the CNT arrays were not highly reactive to nucleic acids under aqueous conditions. The mechanical instability also precludes using the vertically oriented CNT array as an ultrasensitive nanoelectrode platform for biosensors.This led us to explore the integration of gap-filling materials to improve the mechanical rigidity as well as to enhance nucleic acid coupling efficiency. Figure1shows the fabrication steps necessary to achieve chemically active,yet mechanically stable CNT arrays for
*Corresponding author.E-mail:*****************.v †ELORET Corporation.
2002
10 10791081
nanoelectrode sensor development.After CNT growth,a film of spin-on glass was used to fill the gaps between the individual CNTs in the array.The SOG film (Honeywell Microelectronics Auccuglass 512B,
with 15wt %methyl groups bonded to Si atoms in the Si -O backbone)was deposited at a spin speed of 3000rpm and underwent subsequent thermal curing at 400°C for 4h under a positive pressure of argon .Figure 2shows SEM images of (a)vertically aligned CNT array,(b)collapsed CNT array without SOG,(c)CNT array with SOG after 430°C air oxidation and acidic treatment,showing CNTs with opened ends,and (d)high resolution TEM image of an open-ended CNT.We found that the SOG film provided structural support to the carbon nanotubes,enabling them to retain their vertical configuration during the purification and tip opening process.The SOG film also serves as a dielectric material,which electrically insulates the individual nanotubes from their neighbors.
Nucleic acid attachment was accomplished (Figure 3)using standard water soluble coupling reagents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
and N -hydroxysulfo-succinimide (sulfo-NHS)(Pierce).In this reaction,the EDC reagent activates the terminal -COOH groups on the ends of the CNTs,forming a highly reactive o -acylisourea active intermediate.The EDC-derived ester intermediate rapidly undergoes hydrolysis;however,in the presence of excess sulfo-NHS a more water stable sulfo-succinimidyl intermediate is formed.Subsequently,the succinimidyl intermediate undergoes nucleophilic substitution reaction with the primary amine linker on oligonucleotide,resulting in the formation of an amide linkage.12It was calc
ulated that the array contains about 1010CNTs,assuming each CNT occupies 100nm 2.Reaction was performed with the CNT array on a silicon chip submerged in 50mL deionized water.Freshly prepared aqueous solutions of sulfo-NHS and EDC (2mL each containing 1000equivalents)were added first.A 2mL aqueous solution of nucleic acid (100equivalents)was then added to the reaction and the solution was stirred.On the basis of fluorescent intensity,it appeared that the reaction was essentially completed after 2h.
Confirmation of nucleic acid attachment was accomplished using a 532nm laser fluorescent microarray scanner to detect the Cy3-labeled DNA molecule.This provides a simple surface characterization technique to probe the efficiency of the coupling reaction.Results for the CNT electrodes with an integrated spin-on siloxane film and without the film are shown in Figure 4.The brighter intensity for the array with the integrated siloxane film (panel 4b)indicates that the efficiency of DNA coupling reaction is higher compared to the case without the siloxane film (panel 4a).Both arrays underwent the same aqueous washing at 90°C following the coupling reaction,which should remove any noncovalent,weakly bound DNA molecules.We confirmed that the coupling of nucleic acid did not occur on the SOG surface by performing a reaction with just SOG film containing no CNTs,in which no fluorescence signal was detected.For detailed discussion on the chemical coupling of nucleic acid to si
licon surfaces,the reader is referred to ref 13.Hybridiza-tion to the CNT coupled nucleic acid was also investigated.In this case,CNT array with SOG was coupled to PNA (H 2N(CH 2)6-GCCGATGCACC)and when hybridized with the Cy3-labeled complementary DNA exhibited strong fluorescent signal as seen in panel 4c.These results and other control experiments are summarized in Table 1and strongly indicate that the coupling occurred via the amide bond formation.The increased reactivity is also imparted
by
Figure 1.Fabrication and pretreatment of carbon nanoelectrode arrays for
functionalization.
Figure 2.Electron microscope images.(A)Vertically aligned multiwalled CNT arrays with length about 1um.(B)Collapsed CNT arrays after purification process.(C)CNT arrays with SOG after purification and tip opening process.(D)High-resolution transmission electron microscope image of an opened CNT
end.
Figure 3.Coupling of nucleic acid to the functionalized CNT arrays.
covering the length of the CNT sidewalls with the SOG,which renders the CNT interface hydrophilic and thus more compatible for coupling chemistry in aqueous solvent.14This is confirmed by contact angle measurements,in which SOG-filled CNT array exhibited a contact angle of 11°(vs 80°for the CNT
array without the SOG film).The hybridization experiments were also done with DNA with similar success.PNA is preferable for in vivo biosensors as it is not biodegradable.Finally,it is also possible to use SiO 2as gap-fill material,instead of SOG,as we demonstrated recently for electrode fabrication.15
In summary,we have shown that the integration of a SOG film with vertically aligned CNT array provides structural support to the CNT during oxidative treatment for the opening of closed CNT ends.In turn,the improved CNT structure enhances the chemical coupling of nucleic acids to the CNT array.
Acknowledgment.This work was supported by a NASA-NCI collaborative contract.Work by ELORET personnel was funded by a NASA contract.References
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NL025689F
Figure 4.Fluorescently scanned images of CNT arrays.(A)Without SOG.(B)With SOG.(C)Hybridization of Cy3-labeled complementary DNA to coupled PNA on CNT array with SOG.Table 1:Summary of Fluorescence Data Supporting the Higher Coupling and Hybridization Efficiency with the SOG-Filled CNT Array
with SOG
without SOG DNA with linker and Cy3strong weak DNA with Cy3,no linker
weak weak DNA with linker +cDNA with Cy3strong weak DNA with linker +noncDNA with Cy3
weak
weak

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