Ligand Customization and DNA Functionalization of Gold Nanorodsvia Round-Trip Phase Transfer Ligand Exchange

Andy Wijaya† and Kimberly Hamad-Schifferli*,‡

Departments of Chemical Engineering, Biological Engineering, and Mechanical Engineering,Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ReceiVed June 18, 2008

Customizable ligand exchange of gold nanorods (NRs) is described. NRs are synthesized with the cationic surfactantcetyltrimethylammonium bromide (CTAB) which is exchanged with thiolated ligands that enable suspension in buffer.Exchange is achieved by a two phase extraction. First, CTAB is removed from the NR-CTAB by extracting the NRsinto an organic phase via the ligand dodecanethiol (DDT). The NR-DDT are then extracted into an aqueous phaseby mercaptocarboxylic acids (MCA), HS-(CH2)n-COOH (n ) 5, 10, and 15). Ligands can be further customizedto thiolated poly(ethylene glycol), PEGMW (MW ) 356, 5000, and 1000). Ligand-exchanged NRs (NR-MCA andNR-PEGMW) are stable in buffer, do not aggregate, and do not change size upon ligand exchange. They can be runin agarose gel electrophoresis with narrow bands, indicating uniform charge distribution and enabling quantitativeanalysis. DNA functionalization of NR-MCA is straightforward and quantifiable, with minimal nonspecific adsorption.

Gold nanorods (NRs) have been attractive for many biologicalapplications such as gene delivery,1 cell imaging,2 and photo-thermal therapy.3 However, their ligand functionalization hasbeen problematic for conjugation chemistries. Au NR synthesisresults in a double layer of cetyltrimethylammonium bromide(CTAB) for passivation (NR-CTAB), which is problematic forbioconjugation, nonspecific adsorption of DNA, cytotoxicity,and stability. These factors have severely limited the use of NRsin biological applications, especially compared to Au nanopar-ticles (NPs).4 In order to tune the biological properties of theNR-DNA conjugate and minimize nonspecific adsorption,exchange must permit ligand customization. For gel electro-phoresis to assay conjugation and DNA conformation on theNRs, NRs must have uniform charge distribution. There arereports of NR ligand exchange that separately permits conjugationto an antibody2,5,6,7 or gel electrophoresis.8 However, there hasnot yet been a ligand exchange method which enables ligandcustomization, biofunctionalization, and gel electrophoresis.Furthermore, customizable ligand chemistry would broadlyenhance the versatility of NRs in biological applications.

We show here a new approach for ligand exchange that utilizes“round-trip” phase transfer for ligand customization and DNAfunctionalization (Scheme 1). Resulting NRs are stable inphysiological buffers and exhibit narrow bands in gel electro-phoresis. DNA functionalization is straightforward, with minimalnonspecific adsorption on the NR.

Au NRs were synthesized by literature methods.9 Transmissionelectron microscopy (TEM) was employed to determine meanNR-CTAB dimensions of 43.5 ( 11.9 nm (Figure 2a). Round-trip ligand exchange first utilizes aqueous-to-organic phasetransfer.10,11 NR-CTAB at high concentration (2 × 10-8-5 ×10-8 M) in water was put into contact with dodecanethiol (DDT)(Scheme 1, left). After addition of acetone, NRs were extractedinto DDT by swirling the solution for a few seconds, upon whichthe aqueous phase became clear, indicating that no NRs remained(Scheme 1, middle).

Next, organic-to-aqueous phase transfer was performed.12,13

Excess DDT was removed by diluting the DDT coated NRs(NR-DDT) in toluene (1×) and an excess of methanol (5×), andthen spun down and resuspended in 1 of mL toluene by brief

* To whom correspondence should be addressed. E-mail: schiffer@mit.edu.

† Department of Chemical Engineering.‡ Departments of Biological Engineering and Mechanical Engineering.(1) Chen, C.-C.; Lin, Y.-P.; Wang, C.-W.; Tzeng, H.-C.; Wu, C.-H.; Chen,

Y.-C.; Chen, C.-P.; Chen, L.-C.; Wu, Y.-C. J. Am. Chem. Soc. 2006, 128, 3709–3715.

(2) Oyelere, A. K.; Chen, P. C.; Huang, X. H.; El-Sayed, I. H.; El-Sayed, M. A.Bioconjugate Chem. 2007, 18(5), 1490–1497.

(3) Huang, X.; Jain, P. K.; El-Sayed, I. H.; El-Sayed, M. A. Nanomedicine2007, 2(5), 681–693.

(4) Murphy, C. J.; San, T. K.; Gole, A. M.; Orendorff, C. J.; Gao, J. X.; Gou,L.; Hunyadi, S. E.; Li, T. J. Phys. Chem. B 2005, 109(29), 13857–13870.

(5) Yu, C.; Irudayaraj, J. Biophys. J. 2007, 93(10), 3684–3692.(6) Li, P.-C.; Shieh, D.-B.; Wang, C.-R.; Wei, C.-W.; Liao, C.-K.; Ding, A.-

A.; Wu, Y.-N.; Poe, C.; Jhan, S. Nat. Preced. 2008. Available from NaturePrecedings, http://hdl.handle.net/10101/npre.2008. 1687.1, 2008.

(7) Liao, H.; Hafner, J. H. Chem. Mater. 2005, 17, 4636–4641.

(8) Hanauer, M.; Pierrat, S.; Zins, I.; Lotz, A.; Sonnichsen, C. Nano Lett. 2007,7(9), 2881–2885.

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Nano Lett. 2002, 2(8), 803–806.(11) Jebb, M.; Sudeep, P. K.; Pramod, P.; Thomas, K. G.; Kamat, P. V. J. Phys.

Chem. B 2007, 111(24), 6839–6844.(12) Gittins, D. I.; Caruso, F. ChemPhysChem 2002, 3(1), 110–113.

Scheme 1. Method for NR Ligand Exchange and DNAConjugation

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10.1021/la8019205 CCC: $40.75 2008 American Chemical SocietyPublished on Web 08/22/2008

sonication. The NR-DDT in toluene was then added to 9 mL of0.01 M mercaptocarboxylic acid (MCA) in toluene at elevatedtemperature and vigorously stirred. Three MCA ligands (mer-captohexanoic acid (MHA), mercaptoundecanoic acid (MUDA),and mercaptohexadecanoic acid (MHDA)) were used. Operatingtemperatures were 95 °C for MHA and 70 °C for MUDA andMHDA.14–16 Reflux and stirring continued until visible ag-gregation was observed (within ∼15 min), and then the solutionwas allowed to settle and cool to room temperature. Aggregationindicated that NRs were successfully coated by MCA (NR-MCA),which are insoluble in toluene. The aggregates were washed 2×with toluene via decantation and then once with isopropanol todeprotonate the carboxylic acid. The aggregates spontaneouslyredispersed in 1× tris-borate-EDTA buffer (TBE) and were nolonger soluble in toluene (Scheme 1, right), suggesting residualDDT on the NR is minimal.

Once resuspended in TBE, NR-MCA could have their MCAcoating optimized, be ligand-exchanged with another species, orbe conjugated to DNA (Scheme 1). We incubated NR-MCAwith 1 mM MCA in H2O or a H2O/ethanol mixture for furtheroptimizing the MCA coating. We also performed further ligandexchange of NR-MHA by incubating them in 1 mM aqueoussolution of poly(ethylene glycol) (PEG)-thiols (HS-PEG356, HS-PEG1000, and HS-PEG5000). Lastly, we conjugated NR-MHA withfluorescently labeled thiolated DNA 40-mers (5′ HS-TTTTTTTTTT TTTTT TTTTT TCGGC CCGTA TAATT-TMR 3′)using the charge screening method for DNA functionalizationof Au NPs.17

The width of the longitudinal surface plasmon resonance canbe used to directly probe the stability and aggregation of NRs.UV-vis spectra of ligand-exchanged NRs with MCA show shiftsof the longitudinal plasmon with no significant broadening,indicative of no aggregation (Figure 1a). The peak shift is linearwith chain length (Figure 1a, inset) as expected from the changein refractive index with increasing alkyl chain length.18–20 Thelongitudinal plasmon of NRs functionalized with PEG-SH (Figure1b) also showed no significant broadening. We found thatthe ligand-exchanged NRs were stable even after 3 or 4 monthsof storage at high concentration (∼2 × 10-8 M), and the plasmonpeaks exhibited no significant changes in peak width or position(Figure 1c).

Heating gold nanoparticles near the boiling point of the solventin the present of surface-active ligands such as alkanethiols overa certain period of time may result in a reduction in the averagesize of the particle due to digestive ripening.21 TEM imagingwas used to probe any change in size of the NR dimensions uponligand exchange (Figure 2). TEM size analysis determined meanNR-MHA dimensions of 43.2 × 11.8 nm (Figure 2b), indicatingno significant size change with ligand exchange.

Fourier transform infrared (FTIR) spectroscopy was per-formed to probe the nature of the ligands on the surface ofgold nanorods before and after the ligand exchange. FTIRspectroscopy of NR-CTAB (Figure 3a, black) showed peak at

958 cm-1 (arrow, 1) due to the quarternary amine stretch ofCTAB (Figure 3a, green). NR-MHA (Figure 3b, red) exhibiteda COO- stretch (1585 cm-1, arrow, 2), shifted from the COOHstretch (1690 cm-1, arrow, 3) for MHA (Figure 3b, blue), whichis due to deprotonation of carboxylic acid. NR-MHA lack aS-H stretch (2613 cm-1, arrow, 4) but still have a C-S stretch(706 cm-1, arrow, 5).22–25

Gel electrophoresis was performed with 0.5% agarose in 0.5×TBE (Figure 4a). NR-CTAB aggregate in buffer and do notmove from the well (lane 1). NR-MHDA (lane 2), NR-MUDA(lane 3), and NR-MHA (lane 4) all run in the positive direction,indicating that NRs are negatively charged. The increasing trendin mobility is most likely due to a decrease in the hydrodynamicradius (RH) of the NRs as a result of decreasing alkyl chain

(13) Aldana, J.; Lavelle, N.; Wang, Y. J.; Peng, X. G. J. Am. Chem. Soc. 2005,127(8), 2496–2504.

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2717–2726.(17) Zhang, J.; Song, S. P.; Wang, L. H.; Pan, D.; Fan, C. Nat. Protoc. 2007,

2(11), 2888–2895.(18) Ehler, T. T.; Malmberg, N.; Noe, L. J. J. Phys. Chem. B 1997, 101(8),

1268–1272.(19) Templeton, A. C.; Pietron, J. J.; Murray, R. W.; Mulvaney, P. J. Phys.

Chem. B 2000, 104(3), 564–570.(20) Sun, Y. G.; Xia, Y. N. Anal. Chem. 2002, 74(20), 5297–5305.

(21) Prasad, B. L. V.; Stoeva, S. I.; Sorensen, C. M.; Klabunde, K. J. Langmuir2002, 18(20), 7515–7520.

(22) Nelson, G. J. Lipids 1968, 3(1), 104.(23) Xing, S. X.; Chu, Y.; Sui, X. M.; Wu, Z. S. J. Mater. Sci. 2005, 40(1),

215–218.(24) Morales-Cruz, A. L.; Tremont, R.; Martinez, R.; Romanach, R.; Cabrera,

C. R. Appl. Surf. Sci. 2005, 241(3-4), 371–383.

Figure 1. UV-vis spectra of NRs upon ligand functionalization. (a)UV-vis spectra of NR-CTAB (black) before and after ligand exchangewith MCA, NR-MHA (red), NR-MUDA (blue), and NR-MHDA (green)[inset: longitudinal surface plasmon peak as a function of ligand lengthfor SH-(CH2)n-COOH]; (b) UV-vis spectra of NR-MHA (black) beforeand after functionalization with PEG-SH, NR-PEG356 (red), NR-PEG1000

(green), NR-PEG5000 (blue); and (c) UV-vis spectra of ligand-exchangedNRs right after exchange (solid line) or after 3-4 months of storage athigh concentration (dashed line).

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length. NR-PEG356 (lane 5), NR-PEG1000 (lane 6), and NR-PEG5000 (lane 7) all ran in the positive direction, which couldbe due to residual MHA on the NR surface. Mobility decreasedwith increasing PEG chain length, most likely due to the increasein RH with longer PEG. Furthermore, bands of all of the ligandfunctionalized NRs were narrow, exhibiting clear mobility shiftswith surface functionalization, enabling reliable quantitativeFerguson analysis to find the hydrodynamic radius8,26 and�-potential.27

After ligand exchange, the resulting NR-MHA was conjugatedto HS-DNA as described above. Figure 5a shows the UV-visspectra of before and after DNA conjugation. DNA conjugation(Figure 5a, red solid line) did not broaden or shift the longitudinalsurface plasmon resonance (SPR) significantly relative to theNR-MHA peak (black dashed line), indicating no aggregationafter DNA conjugation. Furthermore, the UV-vis spectra of theNR-DNA conjugate were taken 7 days after the conjugation wasperformed, indicating its long-term stability. Gel electrophoresiswas performed to confirm the DNA-NR conjugation via Au-Sbonding. NR-DNA conjugates (Figure 4b, lane 10) ran slowerrelative to NR-MHA (lane 8), indicating a RH increase. Bandsare narrow enough to permit Ferguson analysis of the NR-DNA.The narrow band also indicates no aggregation and a uniform

(25) Hostetler, M. J.; Stokes, J. J.; Murray, R. W. Langmuir 1996, 12(15),3604–3612.

(26) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Alivisatos, A. P.Nano Lett. 2001, 1(1), 32–35.

Figure 2. TEM images and size analyses of (a) NR-CTAB before ligandexchange and (b) NR-MHA after ligand exchange.

Figure 3. FTIR spectra of (a) CTAB (green) and NR-CTAB (black)before ligand exchange and (b) MHA (blue) and NR-MHA (red) afterligand exchange.

Figure 4. Gel electrophoresis (0.5% agarose in 0.5× TBE) of (a) beforeand after ligand exchange: lane 1, NR-CTAB; lane 2, NR-MHDA; lane3, NR-MUDA; lane 4, NR-MHA; lane 5, NR-PEG356; lane 6, NR-PEG1000; lane 7, NR-PEG5000. (b) DNA functionalization of NR-MHA(UV image): lane 8, NR-MHA; lane 9, NR-MHA incubated withnonthiolated DNA; lane 10, NR-MHA incubated with thiolated DNA;lane 11, nonthiolated DNA; lane 12, thiolated DNA. All DNA wasfunctionalized with a 3′ TMR (TAMRA).

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charge distribution. A control with fluorescently labeled non-thiolated DNA exhibited no significant mobility shift, indicatingminimal nonspecific adsorption (lane 9). The fluorescent DNAband of NR-DNA (lane 10) decreases in intensity compared toa control sample of the thiolated DNA by itself (lane 12) whilethe nonthiolated DNA does not (lane 9 vs lane 11), also supportingcovalent attachment. Fluorescence spectroscopy also indicatedNR-DNA conjugation. Supernatant fluorescence decreased(Figure 5b, red solid line) from its original value (red dashedline), indicating that DNA was removed from solution byconjugation to the NRs. Nonthiolated DNA showed a smaller

decrease (Figure 5b, black line, solid vs dashed line), indicatingthat covalent conjugation was favored over nonspecific adsorption.The NR concentration was estimated using the extinctioncoefficients of the longitudinal plasmon band peak, ε ) 4.6 ×109 M-1 cm-1.28 Fluorescence spectra quantified ∼41 DNA/NR.

NR-DNA bands are not at the same position as the free DNAbands (lanes 9 and 10), so the band can be cut to extract thepurified sample. Conjugated DNA was also quantified bydisplacement with mercaptohexanol (MCH).29 Purified NR-DNAwere incubated in 1 mM MCH overnight, displacing the DNAfrom the NRs. Free DNA was separated from the NRs bycentrifugation and quantified by fluorescence (Figure 5c, redsolid line). As a control, a fluorescence scan was taken of thesupernatant of the same NR-DNA solution without MCHtreatment (red dashed line). The scan was taken 9 days after theconjugation, indicating there was no significant detachment ofDNA from NR-DNA conjugates over this period of time. Thegel-purified NRs incubated with nonthiolated DNA were alsotreated with MCH, and the supernatant exhibited no significantfluorescent peak (black solid line), indicating minimal nonspecificadsorption. Quantification of fluorescence determined ∼28 HS-DNA/NR. This DNA loading translates to approximately 2 pmolDNA/cm2, which is the same order of magnitude for DNA ongold nanowires30 and nanoparticles.31

These results show that ligand exchange of Au NRs by theround-trip phase transfer method enables ligand customizationand straightforward DNA conjugation. Ligand-exchanged NRshave uniform charge distribution, enabling their stability inphysiological buffers and quantitative analysis by gel electro-phoresis. This method for modifying the surface chemistry ofgold NRs will enhance their versatility in biological applicationssuch as therapy, sensing, and imaging.

Acknowledgment. We would like to thank the MIT Centerfor Materials Science and Engineering for use of their facilities.

Supporting Information Available: Additional TEM imagesand size histograms, and FTIR spectra. This material is available freeof charge via the Internet at http://pubs.acs.org.

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(27) Park, S.; Hamad-Schifferli, K. J. Phys. Chem. C 2008, 112, 7611–7616.(28) Orendorff, C. J.; Murphy, C. J. J. Phys. Chem. B 2006, 110(9), 3990–

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Figure 5. DNA conjugation. (a) UV-vis spectra of NR-MHA (blackdashed line), NR-MHA + nonthiolated DNA (black solid), and NR-MHA + thiolated DNA (red line). (b) Fluorescence spectroscopy ofsupernatants of NRs conjugated with thiolated DNA (red solid line) ornonthiolated DNA (black solid line). Fluorescence spectroscopy ofcontrols (DNA without NRs) of thiolated DNA (red dashed line) ornonthiolated DNA (black dashed line). (c) Fluorescence spectroscopyof supernatants of MCH treated NRs conjugated with thiolated DNA(red solid line) and nonthiolated DNA (black line). Spectrum ofsupernatant of NR conjugated with thiolated DNA without MCHtreatment (red dashed line).

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