Fabrication of single-walled carbon nanotubes dotted with Au nanocrystals: Potential DNA delivery nanocarriers

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Fabrication of single-walled carbon nanotubes dotted with Au nanocrystals: Potential DNA delivery nanocarriers Dae-Hwan Jung a,b, Byung Hun Kim a, Yong Taik Lim c, Jaiwook Kim a, Sang Yup Lee a, Hee-Tae Jung a,* a

Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea b Bio and Nano Tech Industries, Ministry of Knowledge Economy, 88 Gwanmoonro, Gwacheon-si, Gyunggi-do 427-723, South Korea c Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, South Korea

A R T I C L E I N F O

A B S T R A C T

Article history:

Single-walled carbon nanotubes (SWCNTs) dotted with Au nanocrystals (Au-SWCNTs)

Received 10 September 2009

were fabricated by using a two-phase reduction of hydrogen tetrachloroaurate in the pres-

Accepted 15 November 2009

ence of thiol groups anchored to SWCNTs for their potential applications in DNA (deoxyri-

Available online 16 December 2009

bonucleic acid) delivery. To allow a surface reaction on SWCNTs during the metal nucleation and growth processes, Au nanocrystals were grown using a two-phase system. Raman, XPS and transmission electron microscopy results show that the Au nanocrystals were grafted primarily to the sidewalls of the SWCNTs. DNA probes were immobilized on Au-SWCNTs by the conjugation of DNA functionalized at the 3 0 end with a thiol group with Au dots of SWCNTs, followed by hybridizion of complementary oligonucleotides, as verified by fluorescence-based measurements. To investigate whether the target DNA hybridized to DNA probes immobilized on Au-SWCNTs, 618-base-pair fragments of amplified DNA were prepared by polymerase chain reaction using plasmid pET-22b as a template. Atomic force micrograph (AFM) images show that the nanorod-bound DNA is recognizable with excellent specificity, indicating the potential use of such material as a versatile gene delivery carrier in gene-based disease therapy.  2009 Elsevier Ltd. All rights reserved.

1.

Introduction

Exploration of the biological and medical applications such as drug delivery and gene therapy using carbon nanotubes (CNTs) is a rapidly expanding field of research, because of their flexible surface chemistry, large surface areas and excellent electronic properties. CNTs can be used to introduce foreign genes into somatic cells to supplement defective genes or to provide additional biological functions [1–6]. To realize such applications, the surface activation of CNTs is essential to attaching guest biomolecules on CNT surfaces, since the pristine CNTs are generally chemical-inert. Various

approaches have been used to provide appropriate surface functionalities on nanotubes for their medical applications [7–12]. Most of these methods can be used for the direct or indirect modification of CNTs with biomolecules except the methods for the fluorination of CNTs [12]. The most common method for the covalent functionalization of CNTs involves the reaction with carboxylic acid (–COOH) residues on CNTs. The carboxylic acids are usually generated by oxidation using strong acids, and they occur predominantly at the more reactive end or defect sides of SWCNTs [7–11]. In some cases, the CNTs were directly reacted with biomolecules (e.g., aminesite or thiol-site of the target biomolecules) or an additional

* Corresponding author: Tel.: +82 42 869 3931; fax: +82 42 869 3910. E-mail address: [email protected] (H.-T. Jung). 0008-6223/$ - see front matter  2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2009.11.028

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biofunctional spacer is first reacted with the functionalized CNTs and the resulting construct then linked to the biomolecules of interest [13–17]. In the present study, a single-walled carbon nanotube dotted with Au-nanocrystals (Au-SWCNTs) was developed by the two-phase reduction of hydrogen tetrachloroaurate in the presence of thiol groups anchored to SWCNTs. In addition to the excellent biocompatibility of Au nanocrystals, such materials offer a versatile surface chemistry for conjugation of various biomolecules including DNA, antibodies, and enzymes. Au-SWCNTs can play a role as an important new functional material for applications such as the conjugation of biological materials. The two-phase system was used to allow the surface reaction (on SWCNTs) to take place during the metal nucleation and growth. If the hydrogen tetrachloroaurate is reduced on aqueous solution, the gold nucleation and growth will be occurred on the solution as well as on the surface of SWCNTs. During the Au nanocrystals formation process, we found that the sidewall of SWCNTs had a greater reaction tendency than did the SWCNT ends and the SWCNT ropes retained their shape, without any distortion. Raman, XPS and transmission electron microscopy results showed that the Au nanocrystals were grafted primarily to the sidewalls of the SWCNTs. The unique properties of SWCNT regions were harmonized with the specific molecular-recognition features of DNA by coupling of Au-SWCNTs to thiol-terminated DNA, followed by hybridization with 618-base-pair DNA fragments. The amplicon included 20base-pair single-stranded ‘sticky’ ends that were complementary to the DNA probe sequence. Atomic force microscopy (AFM) was used to investigate whether target DNA hybridized to the Au-SWCNTs-immobilized DNA probe. Our new nanomaterial, termed the ‘‘Versatile nanorod,’’ possesses remarkable chemical stability, wide biological compatibility, and is easy to prepare, because of the superior reaction properties of SWCNTs and Au. Using the versatile surface chemistry of gold nanocrystals with various biomolecules, the Au-SWCNTs can be used as a potential gene delivery systems to allow the transfer to and expression of therapeutic genes in a target organ or tissue [5,18–21].

2.

Experimental details

2.1.

Materials

The as-prepared SWNTs (AP-SWNTs) purchased from CarboLex Inc., which were purified, shortened and polished by the method of Smalley and co-workers [22]. Subsequent exposure to 1 M HCl produced abundant carboxyl end-groups [23]. Hydrogen tetrachloroaurate (0.01% w/v), potassium phosphate buffer (KH2PO4), succinic anhydride (C4H4O3), 1methyl-2-pyrrolidinone (NMP), sodium borate (NaBO2Æ4H2O, 99%), SSC (standard saline citrate) buffer, sodium dodecyl sulfate (SDS), and N,O-bis(trimethylsilyl)acetamide (BSA) were purchased from Sigma. 2-Aminoethanethiol (95% by volume) was obtained from TCI. Ethanol (absolute), 1,3-dicyclohexylcarbodiimide (99% by volume), sodium borohydride (99% by weight), tetraoctylammonium bromide (98% by volume) and toluene (certified ACS reagent) were obtained from Aldrich

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and used as received. Poly-L-lysine-coated slide glasses (25 · 75 mm) were the product of Shin-Won Science. Mica sheets were purchased from Electron Microscopy Sciences. The thiolated single-stranded DNA, abbreviated ssDNA-SH, was a 20-base oligonucleotide with the following sequence (GenoTech): 5 0 -TGT GCC ACC TAC AAG CTG TG – Thiol-3 0 (A: adenine, C: cytosine, G: guanine, T: thymine). The thiol group was at the 3 0 -end. The complementary single-stranded DNA used for hybridization, abbreviated C-ssDNA, was a 20-base oligonucleotide with the following sequence: 5 0 -CAC AGC TTG TAG GTG GCA CA – FITC (fluorescein isothiocyanate)-3 0 . The non-complementary single-stranded DNA, abbreviated NC-ssDNA, was a 20-base oligonucleotide with the following sequence: 5 0 -TGT GCC ACC TAC AAG CTG TG – FITC-3 0 . For hybridization studies, the complementary and non-complementary single-stranded oligonucleotides were labeled with FITC at the 3 0 -ends. The plasmid template for PCR was pET22b(+) (Novagen). Deionized water (18 MX/cm) was obtained from a Millipore system.

2.2.

Preparation of Au-SWCNTs

Au-SWCNTs were fabricated in a two-phase liquid–liquid system. This method takes advantage of the ionic nature of Au as well as the affinity of the metal toward the thiol group. In the present study, reactions were performed as described by Brust and colleagues, except that SWCNTs were functionalized with thiol anchoring groups derived from an amide linkage (Fig. 1) [24]. To form SWCNTs with thiol anchoring groups employing this linkage, carboxyl-terminated SWCNTs were further thiol-derivatized by reaction with 2-aminoethanethiol in an ethanol suspension with the aid of a condensation agent, dicyclohexylcarbodiimide, for 24 h at room temperature [25]. Au nucleation sites in defects and the open ends of SWCNT ropes, formed by Au–S chemical bonding, were derived as follows. Twenty-five milliliters of a bright yellow 0.01% (w/v) hydrogen tetrachloroaurate solution was poured into an empty glass reaction vessel. To this solution was added 16.5 mL of a 0.01665 mmol N(C8H17)4Br solution (in toluene), with rapid stirring. Immediately, two phases were apparent. This mixture was vigorously stirred for several minutes to achieve complete removal of color from the aqueous phase. To the organic phase was added 10 mL of a suspension containing 2 mg of SWCNT-CONH-(CH2)2-SH in toluene, followed by addition of 20.5 mL of a 0.0825 mmol NaBH4 solution to the slowly stirred mixture. The reaction mixture was left open to the atmosphere for 20 h, with rapid stirring. When the reaction was complete, the organic phase was separated from the aqueous phase and filtered through a polyvinylidenefluoride (PVDF) membrane filter of pore size 0.1 lm. The product was washed several times with toluene. The filtrate was suspended in ethanol, using sonication. The mixture was centrifuged at 2000 rpm for 60 min. After centrifugation, the supernatant was decanted and the dark brown precipitate was washed several times with ethanol. The product was retained by filtration through a PVDF membrane. The filtrate was washed several times with deionized water. The product was passed through a membrane filter and dried on a vacuum line.

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Fig. 1 – Overall scheme for fabrication of Au-SWCNTs, and the immobilization and hybridization of DNA. (a) A cut SWCNT with carboxylic acid groups, (b) aminoethanethiols anchored by amide linkages to a SWCNT, (c) Au nucleation sites created in the thiol groups of SWCNTs, (d) Au nanocrystals fabricated by a two-phase redox reaction with the hydrogen tetrachloroaurate, (e) DNA probes immobilized onto the Au nanocrystals of SWCNTs, (f) hybridization of FITC-labeled complementary DNA, and (g) hybridization of a long-chain oligonucleotide with a sequence complementary to that of probe DNA.

2.3. Immobilization of thiol-derivatized DNA probes to Au-SWCNTs Ten milligrams of Au-SWCNTs were suspended in 0.948 mL 1.0 M KH2PO4 buffer by sonication. The ssDNA-SH solution was prepared at a concentration of 100 lM in 1.0 M potassium phosphate buffer. To the suspension of the AuSWCNTs was added 0.948 mL of ssDNA-SH solution, followed by slow stirring for 12 h at room temperature.

2.4. Attachment of the Au-SWCNT-immobilized DNA probes to a glass slide Au-SWCNT-immobilized DNA probes were attached to a poly-L-lysine-coated glass slide (Shin-Won Science) using the procedure employed in DNA microarray experiments [26]. First, concentrated AU-SWCNT-immobilized DNA probes were washed several times with deionized water to remove free DNA strands, using a cross-flow filtration system and a 200 nm PTFE (polytetrafluoroethylene) filter. The filtered product was recovered and concentrated by centrifugation. The concentrated solution was spotted onto the poly-L-lysine-coated glass slide and dried in a high-purity argon stream. Au-SWCNT-immobilized DNA probes spotted onto glass slides were rehydrated by placement over warm 1 · SSC solution for approximately 5 s. Glass slides were dried in a 95 C oven for approximately 5 s, followed by cross-linking, using

UV irradiation with a total energy of 65 mJ (Spectrolinker XL-1500 UV cross-linking instrument). Glass slides were transferred to a 95 C water bath for 2 min and next quickly immersed in 95% (v/v) EtOH for 1 min. Glass slides were dried by bench centrifugation.

2.5. Blocking reaction to prevent nonspecific binding of ssDNA oligonucleotide Free amine-functional groups on poly-L-lysine-coated glass slides were blocked by the following process [27]. Five grams of succinic anhydride and 35 mL of 0.2 M sodium borate (pH 8.0) were successively added to 315 mL NMP. Each glass slide spotted with Au-SWCNT-immobilized DNA probes was immediately placed into the solution and remained therein for 15 min with gentle agitation, were next transferred to a 95 C water bath for 2 min, then quickly immersed in 95% (v/v) ethanol for 1 min, and dried by bench centrifugation [28].

2.6. Hybridization probes

of

Au-SWCNT-immobilized

DNA

Hybridization was performed using complementary single-stranded DNA (C-ssDNA). Non-complementary singlestranded DNA (NC-ssDNA) was employed as a negative control. Each blocked glass slide was incubated in a mixture of 3.5 · SSC buffer, 0.1% (w/v) SDS, and 10 mg/mL BSA, for

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30 min at 50 C. Lyophilized C-ssDNA and NC-ssDNA were diluted with 3 · SSC and 0.3% (w/v) SDS to yield a final concentration of 100 lM DNA. The C-ssDNA (or NC-ssDNA) was denatured at 95 C and snap-cooled on ice. The C-ssDNA (or NC-ssDNA) solution (40 lL) was deposited onto the surface of a freshly-prepared chip using a micropipette. For hybridization, each glass slide chip was placed under a cover slip (a 22 · 22 mm Hybrid-Slip) and incubated for 10 h at 55 C in a hybridization chamber. The cover slip was removed by immersing the glass slide chip in 2 · SSC solution, and the chip was then washed five times with 0.1 · SSC and 0.1% (w/v) SDS solution at room temperature, immersed in 0.1 · SSC solution for 5 min, and dried.

2.7. Preparation of long-chain DNA fragments by PCR, and hybridization thereof For preparation of long-chain DNA fragments with 618 base pairs, a segment of pET-22b(+) was used as template in PCR [29]. The sequences of the primers were 5 0 -CAC AGC TTG TAG GTG GCA CAG AGC AGA CAA GCC-3 0 and 5 0 -CTG AGA TAC CTA CAG CGT G-3 0 (the underlined bases hybridize to the DNA probe immobilized to Au-SWCNTs). PCR amplification was performed for 25 cycles. The PCR product was verified using 0.8% (w/v) agarose gel electrophoresis and next purified using a QIAquick gel extraction kit (Qiagen). Finally, the DNA was eluted with deionized water (18 MX/cm) in a final volume of 20 lL. Hybridization was performed to probe DNA immobilized on the Au-SWCNTs under the following conditions. First, the PCR product was heated at 95 C for 5 min to ensure separation of double-stranded DNA. Next, a mixture of l lL of the probe DNA immobilized to the Au-SWCNTs, 5 lL 10 · PCR buffer (TakaRa), 4 lL PCR product, and 40 lL distilled deionized water was prepared and incubated at 55 C for 2 h.

2.8.

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Characterization

Raman spectra were collected using a Renishaw Raman microscope with an air-cooled charge-coupled device (CCD) detector, an Olympus microscope, and a He–Ne laser operating at 632.2 nm. Samples were examined in a cylindrical tube of diameter 3 mm. X-ray photoelectron spectroscopy (XPS) spectra were obtained using a VG Scientific ESCALAB MK II spectrometer equipped with a Mg Ka X-ray source (1253.6 eV photons) and a hemispherical energy analyzer. The X-ray source was run at 12 kV, a filament current of 10 mA, and a take-off angle of 75 to the sample surface. The pressure in the analysis chamber was maintained at 108 mbar or lower during measurements. All binding energies were referenced to the C1s peak of benzene for calibration, which was assigned the value of 284.6 eV. For transmission electron microscopy (TEM) studies, AuSWCNTs were dispersed in water, and the floating material was retrieved onto carbon-coated copper microscope grids. TEM images were taken with a Philips CM20 instrument at an accelerating voltage of 120 kV. Bright field, phase-contrast TEM micrographs were obtained using low-dose procedures on either film or a GATAN CCD camera at appropriate under-focus conditions, to obtain lattice images.

AFM was used to explore hybridization of long-chain DNA to the DNA probe immobilized on Au-SWCNTs. An aminopropyl mica (AP mica) surface was obtained by vapor deposition of aminopropyltriethoxysilane (APTES) under ambient conditions, for 1 h. The mica square (1 · 1 cm) was cleaved with tape immediately before use. To prepare samples for visualization, 10 lL amounts of the DNA probe-attached AuSWCNTs were placed on samples of AP mica for 3 min, rinsed with deionized water, and argon-dried. The AFM images in air were taken with a NanoScope III MultiMode system equipped with a D-scanner (Digital Instruments), operating in the tapping mode. A NanoProbe TESP probe instrument (Digital Instruments) was used for imaging in air. The typical tapping frequency for such imaging was 240–280 kHz, and the scanning rate was 0.83 Hz. Images were taken in the topographic mode. Detection of fluorescence signals was achieved using a ScanArray 5000 unit (Packard BioScience; BioChip Technologies LLC) and analyzed with the QuantArray 3.0 software package (GSI Lumonics).

3.

Results and discussion

Fig. 1 illustrates the overall scheme for fabricating AuSWCNTs by two-phase reaction methods, and DNA attachment on A-SWNTs. To allow a surface reaction on SWCNTs thiol-derivatized with 2-aminoethanethiol to proceed during the metal nucleation and growth processes, Au nanocrystals were grown using a two-phase system. For the biphasic reduction of hydrogen tetrachloroaurate, it was necessary to select appropriate redox reagents active at the phase interface. In the  present case, AuCl4 was transferred from an aqueous solution to toluene using tetraoctylammonium bromide as the phase-transfer reagent and was then reduced with aqueous NaBH4 in the presence of thiol-derivatized SWCNTs (SWCNT-CONH-C2H4SH). The two-phase redox reaction is shown below, in which the electron source was BH 4 . The conditions of the reaction determine the ratio of thiol to gold, i.e. the ratio n/m: 

AuCl4 ðaqÞ þ NðC8 H17 Þþ 4 ðtolueneÞ 



! NðC8 H17 Þþ 4 AuCl4 ðtolueneÞmAuCl4 ðtolueneÞ þ nSWCNT-CONH-C2 H4 SHðtolueneÞ þ 3me 

! 4mCl4 ðaqÞ þ ðAum ÞðSWCNT-CONH-C2 H4 SHÞn ðtolueneÞ To verify Au–S bonding and the amide linkage of the anchoring group on SWCNT ropes, we used surface-enhanced Raman spectroscopy (SERS) for each intermediate product (Fig. 2). This method allows direct investigation of the vibrational characteristics of species at the solution–metal interface. After chemical attachment of the thiol group to Au, the S–H bond of the alkanethiol was cleaved [30]. Thus, the chemical formation of Au–S can be monitored by examining the relative intensity of the m(S–H) band in free thiols (pure mixing of the thiol-derivatized SWCNTs and Au particles) and thiol/Au-nanocrystals (after chemical attachment). Strong peaks at 2590–2540 cm1, corresponding to SH-stretching modes [m(S–H)], were clearly visible in the free-thiol system, but were not seen in thiol/Au-nanocrystals, indicating

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Fig. 2 – Raman spectra of Au-SWCNTs (solid curve) and pristine SWCNTs (dashed curve). The inset shows Raman bands at 1650 [m(C@O)] and 1540 [m(CNH)] cm1 associated with the amide linkage of 2-aminoethanethiol anchored to SWCNTs.

chemical attachment of Au to the SWCNTs. The group mode involving a Au–sulfur stretching [m(Au–S)] characteristic was observed at both 478 cm1 and 482 cm1. Two Raman bands at 524 cm1and 498 cm1 correspond to C–S stretching modes [m(C–S)] [31,32]. In addition, a low-frequency band at 226 cm1 was assigned to a C–S bending mode [d(C–S)]. Thus, it was confirmed that Raman bands at 1650 [m(C@O)] and 1540 [m(CNH)] cm1 are associated with the amide linkage caused by reaction of the carboxyl-terminated SWCNTs with the amine group of 2-aminoethanethiol [33], confirming that Au nanocrystals were chemically attached to SWCNT ropes. The strong peak (G-line) in the range 1500–1600 cm1 indicates a high degree of crystalline order in the nanotubes [34]. Additionally, a peak at ca. 1340 cm1 signifies residual ill-organized graphite, and is known as the D-line. Interestingly, the strong peak (G-line) becomes considerably weaker upon chemical attachment of the Au nanoparticles, suggesting that well-organized graphite regions, such as the sidewalls of the nanotubes, are studded with Au nanocrystals formed from nucleation sites. In addition, the Raman band of m(C@O) disappeared when SWCNT ropes functionalized with carboxylic acid groups [–COOH, m(C@O) of 1753 cm1] were chemically treated to form the amide linkage and the subsequent Au–S bonds. This indicates that most potential grafting sites on the SWCNT ropes were covered with Au nanocrystals. XPS further confirm amide linkages (–CONH) created by 2aminoethanethiol anchored to SWCNT carboxylic acid groups (Fig. 3). XPS in the N 1s and S 2p regions after 2-aminoethanethiol became anchored to the SWCNTs. After reaction of carboxylterminated SWCNTs with the amine group of 2-aminoethanethiol, an N 1s signal, which can be attributed to the amide linkage, shows a peak at a higher binding energy of 399.5 eV, compared to the peak at 398.5 eV assigned to the existence of primary amino groups [35]. In thiol-derivatized SWCNTs obtained after the anchoring of 2-aminoethanethiol, an S 2p

Fig. 3 – XPS spectra of N 1s (399.5 eV) and S 2p (163.6 eV) corresponding to the amide linkage and the thiol group, respectively, of 2-aminoethanethiol anchored to SWCNTs.

signal attributable to a free thiol group appears at a peak binding energy of 163.6 eV. Analysis of the N 1s and S 2p signals from thiol-derivatized SWCNTs thus revealed the existence of both the amide linkage and the free thiol group of 2-aminoethanethiol.

Fig. 4 – XPS spectrum of the doublet for 4f5/2(87.9 eV) and 4f7/2(84.2 eV) in the Au nanocrystals on SWCNTs. The majority of Au is in the Au0 form.

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After the two-phase reduction of hydrogen tetrachloroaurate in the presence of thiol groups anchored to SWCNTs, XPS analysis was performed to determine the oxidation state of the Au dotted onto the SWCNT ropes. The binding energies of the doublet for Au 4f5/2(87.9 eV) and Au 4f7/2(84.2 eV) are characteristic of Au0, whereas the typical binding energy for AuI oxidized in a Au octanethiol complex is 84.9 eV (Fig. 4) [36]. Hence, the Au atoms in nanocrystals must be present largely as Au0. This is a significant result, as Au nanocrystals are formed through reduction from the Au nucleation sites in the hydrogen tetrachloroaurate solution [24]. Small Au particles have been used as markers in various microscopic techniques and to study Au grafting sites on SWCNTs. Fig. 5 shows a TEM image of an SWCNT rope reacted with hydrogen tetrachloroaurate after formation of thiol groups (Fig. 5a). A higher magnification is also shown

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(Fig. 5b). Remarkably, Au nanoparticles bound primarily to the sidewall of the SWCNT, although a small number of particles were observed at the open ends. As an oxidizing acid attacks the surface of SWCNT tubes, leaving holes in the tube side after sonication, it may be concluded that chemical functionalization occurred primarily at the sidewalls and that functionalization of the nanotube ends contributed only slightly to binding, resulting in a high density of Au nanocrystals in the sidewalls. Because it is well known that the tips of the nanotubes are more reactive than the side-walls, gold nanocrystals could be formed on the ends of nanotubes if the bundles of SWCNTs are separated into individual SWCNT after long time sonication. More detailed experiments on that condition will be conducted in future research. By measuring the number of Au particles in a wide range of micrographs, it was indeed confirmed that the

Fig. 5 – TEM photographs of (a) An SWCNT rope reacted with hydrogen tetrachloroaurate after formation of the thiol group, (b) a higher magnification image of the rope. The Au nanoparticles, which appear as dark spots, are observed primarily on the sidewall of the SWCNT rope.

Fig. 6 – High-resolution TEM images of Au nanoparticles on the SWCNT rope. (a) The SWCNT rope, consisting of several tens of nanotubes of about 1.2 nm diameter arranged in parallel, and, (b) a high-magnification view of the Au lattice attached to ˚ ). the rope (the d-spacing of the Au lattice is 2.36 A

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Fig. 7 – Scanned fluorescence images of oligonucleotides hybridized with probe DNA immobilized to Au-SWCNTs. (a) Hybridization of the FITC-labeled complementary oligonucleotide with probe DNA immobilized to Au-SWCNTs (above); hybridization of the non-complementary oligonucleotide (below). (b) Hybridization of the FITC-labeled complementary oligonucleotide to probe DNA immobilized to Au-SWCNTs (above); hybridization of the complementary oligonucleotide to Au-SWCNTs without probe DNA (below).

SWCNT sidewalls had more grafting sites than did the ends. Approximately 20 Au nanoparticles per 100 nm of SWCNT rope were observed. The structural changes in SWCNTs and Au became clearer at higher magnification (Fig. 6) of a region of Fig. 5a. The width of the SWCNT rope was approximately 10–20 nm, indicating that SWCNTs became regularly arranged into ropes consisting of several tens of parallel nanotubes in mutual contact [37]. The measured regular d˚, spacing of the observed lattice planes was 2.36 ± 0.02 A which corresponds to the {1 1 1}, plane of Au (literature value: ˚ ). Therefore, versatile dotted SWCNT nanorods 2.355 A showed a uniform distribution of Au nanocrystals, without any distortion of the rope. To evaluate binding specificity, experiments comparing the binding of probe DNA immobilized to Au-SWCNTs were performed using both perfectly complementary DNA and mismatched material. To illustrate general hybridization specificity, two pairs of spots of Au-SWCNTs containing immobilized probe DNA were applied to a surface to form two circular areas each approximately 5 mm in diameter (Fig. 7a). A solution containing fluorescently tagged C-ssDNA was applied to the upper half of the surface and NC-ssDNA was applied to the lower half. After hybridization, the surface was washed and fluorescence signals measured. Fig. 7a shows the fluorescence images, indicating that binding of CssDNA occurred in the region containing Au-SWCNTs with immobilized ssDNA probe, and not to the NC-ssDNA sequence. To test whether fluorescently tagged ssDNA was adsorbed by Au-SWCNTs without probe DNA, a pair of spots of Au-SWCNTs containing immobilized probe DNA, and a pair of spots of Au-SWCNTs without probe DNA, were applied to the surface (Fig. 7b). As described above, a solution containing fluorescently tagged sequence C-ssDNA was then applied to the surface. As seen in the lower part of Fig. 7b, no binding to C-ssDNA was observed. Thus, Au-SWCNTs with immobilized probe DNA hybridized efficiently with complementary

strands, and showed minimal nonspecific interaction with non-complementary sequences. This observation supports the notion that probe DNA present on Au-SWCNTs can recognize DNA with a complementary sequence and that no unrelated ssDNA can adsorb to such Au-SWCNTs. AFM was used to study the conformation of Au-SWCNTs tethered to the long-chain DNA, which would represent the configuration of Au-SWCNTs used as a carrier to deliver DNA. Fig. 8 shows AFM images of several strands of a 618base-pair DNA molecule containing probe-complementary sequence hybridizing with probe DNA immobilized to AuSWCNTs. A zoomed image displayed in three-dimensions shows that the height of the rope-type Au-SWCNTs was much greater than that of the 618-base-pair DNA and that hybridized 618-base-pair DNA molecules were clearly visible (Fig. 8). From observations of many samples, it was concluded that DNA attachment occurred predominantly at Au nanocrystals located on the sidewalls of SWCNT ropes. In addition, only several strands of DNA were hybridized to each rope, although many Au nanocrystals were present on the SWCNT rope, indicating the possibility of steric hindrance by longchain DNA. The 618-base-pair DNA strand was calculated to be 210 nm in length (618 · 0.34  210 nm), assuming that the bases are nearly perpendicular to the helix axis in a helical structure and because adjacent bases are separated by 0.34 nm [38]. AFM measurements of hybridized DNA yielded a mean length of 210 ± 17 nm, which is very close to the expected size. The error in the length measurement of hybridized DNA strands was attributable to a tip convolution effect, which led to 10 nm broadening of DNA strand length under the conditions employed. AFM images showed that AuSWCNTs were densely tethered to the long-chain DNA strands, resulting in a worm-like shape. This experimental results suggest that a single Au-SWCNT can carry DNA strands, which may be very valuable in the field of gene delivery for medical applications such as gene therapy.

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mid DNA, or synthetic oligodeoxynucleotides. This will open new therapeutic and preventive opportunities to combat disease.

Acknowledgments This work was supported by the National R&D Project for Nano Science and Technology and the Brain Korea 21 Program.

R E F E R E N C E S

Fig. 8 – AFM images of the 618-base-pair DNA complementary fragment hybridizing with probe DNA immobilized to Au-SWCNTs on AP mica, using the tapping mode (top). A zoomed image is displayed in threedimensions by tilting the mica substrate (bottom). The sample was deposited onto AP mica, argon-dried, and imaged in air under ambient conditions.

4.

Conclusion

In conclusion, Au-SWCNTs was developed by a two-phase reduction of hydrogen tetrachloroaurate in the presence of thiol groups anchored to SWCNTs for DNA delivery nanocarriers. Using a two-phase system, gold nanoparticles were grown on SWCNTs during the metal nucleation and growth processes. Using various characterization methods, we found that the Au nanocrystals were grafted primarily to the sidewalls of the SWCNTs. Finally, DNA probes were immobilized on Au-SWCNTs and followed by hybridizion of complementary oligonucleotides. Our experimental results suggest that the Au-SWCNTs developed in this study can possibly used as a versatile gene delivery carrier in gene-based disease therapy. Recently, considerable advances have been made in the field of nanotechnology. Our versatile nano-rod with DNA recognition properties has potential applications as a delivery system for diverse molecules such as peptides, proteins, plas-

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