Synthesis of Fluorescent Chitosan and Its Application in Noncovalent

Yongho Joo , Gerald J. Brady , Matthew J. Shea , M. Belén Oviedo , Catherine Kanimozhi , Samantha K. Schmitt , Bryan M. Wong , Michael S. Arnold , an...
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Notes Synthesis of Fluorescent Chitosan and Its Application in Noncovalent Functionalization of Carbon Nanotubes Qiang Yang, Li Shuai, and Xuejun Pan* Department of Biological Systems Engineering, University of Wisconsin-Madison, 460 Henry Mall, Madison, Wisconsin 53706 Received August 29, 2008 Revised Manuscript Received October 24, 2008

1. Introduction Carbon nanotubes (CNTs) have extraordinary electrical and mechanical properties, which make them ideal candidates in various applications.1-4 For example, integrating the CNTs into polymeric matrix has attracted considerable attentions, aimed at creating highly conductive composite films for biosensors5,6 and developing novel composite materials with extraordinary mechanical properties.7,8 However, the lack of solubility or dispersibility in most solvents or polymeric systems has impeded the application of the as-produced CNTs. Significant progress has been made in improving the solubility or dispersibility of CNTs. The current efforts and approaches include the covalent grafting of chemical groups through reactions onto the π-conjugated skeleton of CNTs and the noncovalent adsorption or wrapping of various functional molecules.9,10 Chitosan (CH) is a linear β-1,4-linked polysaccharide obtained from partial deacetylation of chitin, which is an antibacterial, biocompatible, environment-friendly and biodegradable material.11 More recently, CNTs-chitosan composites have been employed in the construction of a bioelectrochemical platform.12-14 By sonication, chitosan and its derivatives were able to cover or wrap the surface of CNTs, resulting in provisionally stable suspension of CNTs in acidic or neutral aqueous solutions.15-18 However, long-term stable CNTs suspension has not been achieved due to desorption of chitosan. To overcome the desorption problem, it was reported that chitosan was chemically grafted onto the surface of CNTs.19,20 However, chemical grafting of chitosan has the risk of disrupting the excellent electrical property of CNTs. Noncovalent attachment of modified chitosan is proven a promising method to disperse the CNTs as it does not disrupt the sp2 structure and conjugation of the CNTs.21 We have been working on dispersing the CNTs into solvents with pyrene-labeled polysaccharides by a sidewall functionalization method. In the previous work, we reported that multiwalled carbon nanotubes (MWNT) were well dispersed in various solvents after functionalization with pyrene-labeled hydroxypropyl cellulose.22 In the present paper, chitosan was modified by introducing fluorescent pyrene moieties. The synthesized fluorescent chitosan was then used to “wrap” the CNTs noncovalently for preparing chitosan-CNTs hybrids. * To whom correspondence should be addressed. Tel.: 608-262-4951. Fax: 608-262-1228. E-mail: [email protected].

The CNTs hybrids showed good dispersibility and stability in acetic acid aqueous solution.

2. Experimental Section 2.1. Materials. Multiwalled carbon nanotubes (MWNT; O.D., 20-50 nm; wall thickness, 1-2 nm; length, 0.5-2 µm), single-walled carbon nanotubes (SWNT; CarboLex AP-grade; carbon content, 50-70%; diameter, 1.2-1.5 nm; length, 2-5 µm; bundle dimensions), chitosan (CH; product number, 448869; 75-85% deacetylated; brookfield viscosity, 20-200 cp at 1% concentration in 1% acetic acid), 1-pyrenebutyric acid N-hydroxy succinimide ester (PSE), sodium hydroxide (NaOH), and acetic acid were obtained from Sigma-Aldrich (St. Louis, MO) and used as received. Solvents of tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) were also purchased from SigmaAldrich and dried with 4 Å molecular sieves before use. Deuterated water (D2O) and acetic acid (CD3COOD) for 1H NMR measurements were obtained from Sigma-Aldrich as well. 2.2. Synthesis of Fluorescent Chitosan (FCH). Typically, 0.20 g CH was dissolved in a mixture of 15 mL of DMF and 20 mL of 1.4% acetic acid aqueous solution. Then, 37.10 mg (96.3 µmol) PSE dissolved in 5 mL of DMF was dropwise added into the solution above. The reaction was carried out at 50 °C for 24 h under a nitrogen atmosphere with stirring using a magnetic bar. At the end of the reaction, 3 M NaOH was added until the precipitation occurred. The sediment was filtered out, redissolved in 1.4% acetic acid, and then reprecipitated by adding 3 M NaOH. The resultant light yellow sediment was separated by filtration, washed with THF until there was no free PSE detected in the filtrate by UV, and then dried at vacuum. The yield of the fluorescent chitosan (FCH) was 95%. 1H NMR (360 MHz, in D2O/CD3COOD, δ in ppm):19 8.4-7.8 (br, H1), 4.61 (s, H1′), 4.47-3.39 (br, H1, H3, H5, H6), 3.38-3.01 (br, H2, H2′), 2.45 (H8, H9, H10), 2.07 (s, H7). 2.3. Preparation of FCH-SWNT Hybrid. Typically, 2 mg SWNT was dispersed in a solution of 10 mg FCH in 2 mL of 1.4% acetic acid by sonication for 45 min. The produced black suspension was centrifuged for 30 min at 14000 rpm to remove nonsuspended SWNT. The black supernatant was filtered and washed with 1.4% acetic acid aqueous solution and then dried under vacuum, resulting in a FCHSWNT hybrid. 2.4. Preparation of FCH-MWNT Hybrid. Typically, 2 mg MWNT was dispersed in a solution of 10 mg FCH in 2 mL of 1.4% acetic acid by sonication for 45 min. The resulting suspension was centrifuged for 30 min at 14000 rpm to separate the sediment. The sediment was washed using 1.4% acetic acid aqueous solution until there was no FCH detected in the supernatant by UV. The resultant black sediment was dried at vacuum, producing the FCH-MWNT hybrid. 2.5. Characterization. 1H NMR. 1H NMR spectra of FCH and FCHCNTs hybrids were taken on a Bruker DRX-360 NMR spectrometer (360.13 MHz, Bruker, Rheinstetten, Germany) fitted with a 5 mm 1Hbroadband gradient probe with inverse geometry using a mixture of D2O and CD3COOD (3:1, v/v) as solvent. Fluorescence Spectroscopy. Fluorescence spectra of FCH and FCHCNTs hybrids in 1.4% acetic acid aqueous solution were recorded on a MOS-250 fluorescence spectrometer (Biologic, Claix, France). The excitation wavelength was 345 nm. Raman Spectroscopy. The Raman spectra of CNTs and their hybrids were collected on a Horiba Jobin-Yvon LabRAM ARAMIS Raman confocal microscope (632.8 nm, Aramis CRM, Horiba Jobin Yvon, Edison, NJ). A 50× objective was used to focus 18.5 mW of He-Ne

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Notes (632.8 nm) laser light onto the sample surface with a spot size of about 1 µm. On the spot, the actual laser power on the samples is 6.9 mW for He-Ne (632.8 nm) light. Confocal Microscopy. Confocal image of fluorescent chitosan solid was taken on a Zeiss Axiovert 200 M motorized inverted microscope (Thornwood, NY) equipped with 10× conventional and 20× DIC objectives and 40×, 63×, and 100× DIC oil-immersion objectives. UV-Vis Spectroscopy. Absorption spectra of fluorescent chitosan and CNT hybrids in acetic acid aqueous solutions were collected using a viable-temperature UV-vis spectrophotometer (Cary 50 Bio, Varian, Inc.). The content of pyrene moieties (Con, wt %) in the fluorescent chitosan samples was determined using the equation described in literature:23 Con ) (Sc/So) × 100% (Sc, the slope of the correlation curve of absorbance at 328 nm versus concentration of the fluorescent chitosan in 1.4% acetic acid aqueous solution; So, the slope of the correlation curve of absorbance at 328 nm versus concentration of 1-pyrenebutyric acid N-hydroxy succinimide ester in 1,4-dioxane).

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Scheme 1. Preparation Routes of the Fluorescent Chitosan (FCH) and FCH-CNTs Hybrid

3. Results and Discussion 3.1. Synthesis of Fluorescent Chitosan. Pyrene is a typical fluorescent compound, which has been widely used to prepare the pyrene-based fluorescent materials.24 The unique structure of chitosan is the presence of primary amine at the C-2 position of the glucosamine residues. Many derivatization reactions can be carried out at the amino groups of chitosan.25-27 Previous reports showed that the amino groups of chitosan could easily react with succinic anhydride in neutral or acidic aqueous solvents.23,28,29 In the present research, we intended to synthesize fluorescent chitosan through a substitution reaction between amino group of the chitosan and 1-pyrenebutyric acid N-hydroxy succinimide ester,30,31 as illustrated in Scheme 1. The substitution reaction was carried out in the mixed solvents of acetic acid aqueous solution and DMF. As chitosan is soluble in aqueous acetic acid but not in DMF, while PSE is soluble in DMF but not in aqueous acetic acid, changing the volume ratio of acetic acid aqueous solution to DMF will form a homogeneous or heterogeneous reaction system, which significantly affected the reaction results, as discussed below. The synthesized fluorescent chitosan was verified and characterized by 1H NMR, as shown in Figure S1 in Supporting Information. The peaks at 7.8∼8.4 ppm in the 1H NMR spectrum were assigned to the protons of pyrene moiety in the fluorescent chitosan. The degree of substitution (DS) of pyrene moieties at the C-2 amino group in the fluorescent chitosan was calculated from 1H NMR data, and the results are listed in Table 1.32 The results in Table 1 indicate that volume ratio (R) of aqueous acetic acid to DMF was a crucial factor affecting the content of pyrene moieties in the fluorescent chitosan. In general, high R value gave low DS. Specifically, when the R value increased from 1 to 4, the DS decreased from 0.20 to 0.05. Considering that PSE and chitosan are soluble in DMF and aqueous solution, respectively, the R value decided their solubilities in the mixture of the solvents. When the R value was above 1, the solubility of PSE in the mixed solvents became poor, thereby resulting in a heterogeneous reaction system (two phases). Under this reaction condition, the substitution reaction was limited on the interfaces of the two phases and led to lower DS. In contrast, when the R value was 1 (even lower if aqueous acetic acid was enough to dissolve chitosan), the substitution reaction was carried out in homogeneous system, which produced higher DS. Figure 1 shows the UV-vis absorption spectra of the fluorescent chitosan samples with varied DS at the same concentration in aqueous acetic acid. All fluorescent chitosan samples exhibited five characteristic absorption peaks of pyrene

Table 1. Preparation of Fluorescent Chitosan sample

R (v/v)a

con (wt %)b

DS (%)c

FCH-1 FCH-2 FCH-3

1 1.25 4

1.51 0.87 0.42

0.20 0.11 0.05

a R (v/v) ) volume ratio of 1.4% aqueous acetic acid to DMF. b con (wt %) ) weight percentage of pyrene moieties in the fluorescent chitosan, determined by UV-vis at 328 nm. c DS (%) ) degree of substitution, calculated from the 1H NMR spectrum by comparing the integrated area of pyrene proton (7.8∼8.4 ppm) to that of C-2 proton (3.16 ppm).

moieties at 265, 276, 314, 328, and 344 nm, respectively.33 The absorption intensities of the peaks were proportional to the DS. The results indicated that the UV-vis spectra could be used to determine the contents of pyrene moieties in the fluorescent chitosan. Curves of absorbance value at 328 nm versus concentration of 1-pyrenebutyric acid N-hydroxy succinimide ester and the fluorescent chitosan samples were plotted. The results indicated that the absorbance intensity at 328 nm of the fluorescent chitosan was linearly correlated with the concentration (Figures S2, S3, and S4 in Supporting Information). According to the literature method,23 weight percentage of pyrene moieties (con, wt %) in the modified chitosan was calculated and listed in Table 1. When the R value increased from 1 to 4, weight percentage of pyrene moieties in the

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Figure 1. UV-vis absorption spectra of the fluorescent chitosan samples (concentration: 1 mg/mL in 1.4% aqueous acetic acid).

Notes

Figure 3. UV-vis absorption spectra of FCH, FCH-SWNT, and FCHMWNT.

Figure 2. (A) Emission spectrum of the fluorescent chitosan (λexcitation ) 345 nm); concentration: FCH-1, 0.05 mg/mL in 1.4% aqueous acetic acid. (B) Confocal fluorescence image of FCH-1 (λexcitation ) 326 nm). The scale is 2 mm.

Figure 4. Emission spectra of FCH, FCH-SWNT, and FCH-MWNT (λexcitation ) 345 nm). Concentration: FCH-2, 0.05 mg/mL in 1.4% aqueous acetic acid.

modified chitosan decreased proportionally from 1.51 to 0.42, which was consistent to the NMR calculation results above. The fluorescence characteristic of the synthesized fluorescent chitosan was verified by fluorescence emission spectrum, as shown in Figure 2A. The fluorescent chitosan exhibited the fluorescent characteristic excimer peaks at 376 and 394 nm, respectively.34 In addition, confocal fluorescence microscope was used to directly verify the fluorescent characteristics of the fluorescent chitosan. As clearly shown in Figure 2B, the fluorescent chitosan samples excited at 326 nm and exhibited blue. 3.2. Preparation of FCH-CNTs Hybrids. The fluorescent chitosan contains two moieties of pyrene and free primary amine that can interact with CNTs. On one hand, pyrene moieties of the fluorescent chitosan can interact with the CNTs by π-π stacking, which can be verified by 1H NMR.22 1H NMR spectra of the fluorescent chitosan and FCH-CNTs hybrid were com-

pared in Figure S5 in Supporting Information. Specifically, proton peaks of pyrene moieties in the hybrid became broad and shifted to upfield due to the π-π stacking with CNTs.19 On the other hand, it is known that the electron-donating primary amine interacts strongly with the electron-accepting CNTs.15,35 However, the electron-donating ability of the primary amine decreases when protonated in acetic acid aqueous solution.15 In other words, the amine-induced interaction with CNTs is minimized in acidic solution. Furthermore, when hydrogen of the amino group is substituted with the pyrene moiety, the interaction between the secondary amine and the CNTs is impaired by the large steric hindrance around the nitrogen atom.15 Thus, the interaction between FCH and CNTs in acid solution is dominated by the π-π stacking, as schematically depicted in Scheme 1. The π-π stacking between FCH and CNTs can be implemented by sonication in acetic acid aqueous solution.31

Notes

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Figure 5. Raman spectra of SWNT, FCH-SWNT, MWNT, and FCH-MWNT (λexcitation ) 532 nm). Table 2. Raman ID/IG Intensity Ratios and D- and G-Band Shifts of Pristine CNTs and FCH-CNTs Hybridsa sample

SWNT

FCH-SWNT

MWNT

FCH-MWNT

ID/IG D-band (cm-1) G-band (cm-1)

0.24 1335 1586

0.41 1335 1590

0.72 1347 1575

0.82 1357 1585

a

Excitation at 532 nm on the solid.

UV-vis absorption spectra of the fluorescent chitosan and FCH-CNTs hybrids in aqueous acetic acid were compared in Figure 3. As a whole, absorption spectra of the CNT hybrids were similar to that of the fluorescent chitosan, excepting that their absorption intensities became weaker in the same wavelength regions.36 Furthermore, fluorescence spectra of the fluorescent chitosan and CNTs hybrids recorded in acetic acid aqueous solution were compared in Figure 4. The results clearly showed that when the fluorescent chitosan was attached onto the CNTs surface, its fluorescence was completely quenched, possibly due to the disruption of π-conjugation by a conformational change37 and the weak electronic transfer induced by electro-donating of primary amine.38,39 Raman spectroscopy is a powerful technique to investigate the structure changes of carbon nanotubes after hybridization. Raman spectra of pristine SWNT and MWNT and their corresponding FCH hybrids, recorded on a confocal Raman spectrometer using the laser excitation at 532 nm, are shown in Figure 5. The pristine SWNT had two characteristic peaks at 1335 and 1586 cm-1, corresponding to D-band (C-C, the disordered graphite structure) and G-band (CdC, sp2-hybridized carbon), respectively.40 The ID/IG ratio, which was defined as the intensity ratio of the D-band to G-band of CNTs, directly indicated the structure changes of CNTs. As listed in Table 2, the ID/IG ratio of pristine SWNT was 0.24, while the ID/IG of the hybridized SWNT with FCH increased to 0.41. In addition,

slight blue-shift (4 cm-1) of G-band was observed in the functionalized SWNT. The higher ID/IG ratios and the blueshift of G-band were attributed to the increase in the numbers of the sp3-hybridized sidewall carbons caused by the functionalization.41 Likewise, the pristine MWNT had two characteristic peaks at 1347 and 1575 cm-1, corresponding to D-band and G-band, respectively. The ID/IG ratio of pristine MWNT was 0.72. After being hybridized by FCH, the ID/IG ratio was enhanced to 0.82. Meanwhile, the G-band was divided into G-band and G′-band, representing enhancement of the disorder extent. Additionally, it was noted that large blue-shifts (10 cm-1) for D-band and G-band, respectively, were observed in the MWNT due to the increased density of disorder and defect.41 Because of their strong hydrophobic graphite structures, the pristine CNTs are very difficult to form a stable suspension in solvents. However, after being “wrapped” noncovalently with the fluorescent chitosan, the CNTs hybrids were successfully dispersed in acetic acid aqueous solution and formed a very stable suspension. As shown in Figure S6 in Supporting Information, no sediments were observed in the bottle bottoms after two-month storage at ambient temperature. The stability of CNTs hybrid suspension was attributed to static repulsive force from cationic chitosan. However, the stability of CNTs hybrid suspension may change when temperature increases. It was reported that binding energy of the π-π stacking between pyrene moiety and graphite is ∼55 pN.42,43 Therefore, higher temperature can break the bonding formed between pyrene moiety and CNTs, resulting in desorbing of the pyrenecontaining chitosan from the CNTs surface. To verify the hypothesis, the effect of temperature on stability of the FCH-nanotube hybrids was investigated in acetic acid aqueous solution. When the FCH-nanotube hybrids suspensions were heated at 90 °C for two hours, conspicuous aggregates were observed, indicating that the FCH-nanotube hybrids were de-

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composed into FCH and the CNTs. However, the temperatureinduced hybrid decomposition appears reversible. The aggregates disappeared and were mostly redispersed in the solution when the suspensions were cooled to ambient temperature and sonicated for 20 min.

4. Conclusion Fluorescent chitosan was successfully synthesized by a simple substitution reaction at the amino groups of chitosan with 1-pyrenebutyric acid N-hydroxy succinimide ester. The pyrene moieties introduced to the synthesized fluorescent chitosan were dominated by the volume ratios of aqueous acetic acid to DMF, and could be directly determined by UV-vis spectra as there is a linear relationship between the absorbance and concentration of pyrene moieties. Availing of the strong π-π stacking force, the CNTs were functionalized by a noncovalent method using the synthesized fluorescent chitosan. The chitosan-CNTs hybrids had excellent dispersibility and stability in acetic acid aqueous solution at ambient temperature. The hybrids have potential applications in biosensors, gene and drug delivering fields. Acknowledgment. The authors thank Mr. Jinjin Zhou and Dr. Hongquan Jiang for their assistances in taking confocal microscopy and Raman microscopy, respectively. The authors gratefully acknowledge the supports from the Department of Biological Systems Engineering and the College of Agriculture and Life Science at University of Wisconsin-Madison and USDA McIntire-Stennis Fund. Supporting Information Available. 1H NMR spectra, UV-vis absorbance spectra of FCH at varied concentrations, correlation curves of absorbance at 328 nm versus concentration of the FCH and corresponding linear regression equations, and images of FCH-CNTs suspension. This material is available free of charge via the Internet at http://pubs.acs.org.

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