Programmed Assembly of Peptide-Functionalized Gold Nanoparticles

Jul 30, 2010 - E-mail: [email protected], [email protected]. ... can be attached to self-organized oligonucleotide templates to formulate well-order...
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Programmed Assembly of Peptide-Functionalized Gold Nanoparticles on DNA Templates Danielle Coomber,† Dorota Bartczak,‡ Simon R. Gerrard,† Sarah Tyas,† Antonios G. Kanaras,*,‡ and Eugen Stulz*,† ‡

School of Physics and Astronomy and †School of Chemistry, University of Southampton, Southampton SO17 1BJ, U.K. Received June 9, 2010. Revised Manuscript Received July 20, 2010

We present a novel nanoparticle building block system based on the interactions between short synthetic oligonucleotides and peptides. Gold nanoparticles coated with DNA-binding peptides can be attached to self-organized oligonucleotide templates to formulate well-ordered structures of nanoparticles. By regulating the amount of DNA-binding peptide attached to the nanoparticle surface and using specifically designed oligonucleotides, the nanoparticle assembly can be controlled to form dimers, trimers, and adjustable-length nanoparticle chains as well as more complex structures.

Introduction The development of new tools for the controlled assembly of inorganic nanocrystals is of great importance in view of enabling a systematic investigation of their physical properties (e.g., plasmon resonance and electron spin) and for the fabrication of mesoscale materials with tunable functions.1 Several approaches have so far been used to control self-assembly, mainly utilizing strategies based on prefabricated templates,2 small-molecule and electrostatic interactions,3,4 novel solution-deposition methods,1a nanowelding,5 and biomolecular tools.6 Each of these methods offers alternative pathways towards the self-assembly and creation of mesoscale structures of different sizes, shapes, and architectures.7 The applications of such arrays range from scaffolds for biological systems to the fabrication of devices for information storage, photodetectors, light-emitting diodes, and photovoltaics.1a,8 Among these strategies, using DNA as a scaffold is particularly attractive and has been employed to arrange nanoparticles *Corresponding authors. E-mail: [email protected], [email protected]. Fax: þ44 (0)23 8059 3910. Tel: þ44 (0)23 8059 2466.

(1) (a) Talapin, D. V.; Lee, J. S.; Kovalenko, M. V.; Svechenko, E. V. Chem. Rev. 2010, 110, 389–458. (b) Claridge, S. A.; Castleman, A. W., Jr; Khanna, S. N.; Murray, C. B.; Sen, A.; Weiss, P. S. ACS Nano 2009, 3, 244–255. (2) Dickerson, M. B.; Sandhage, K. H.; Naik, R. R. Chem. Rev. 2008, 108, 4935– 4978. (3) (a) Puigmartı´ -Luis, J.; Perez del Pino, A.; Laukhina, E.; Laukhin, V.; Rovira, C.; Vidal-Gancedo, J.; Kanaras, A. G.; Nichols, R. J.; Brust, M.; Amabilino, D. B. Angew. Chem., Int. Ed. 2008, 47, 1861–1865. (b) Brust, M.; Kiely, C. J. Colloids Surf., A 2002, 202, 175–186. (4) Warner, M. G.; Hutchison, J. E. Nat. Mater. 2003, 2, 272–277. (5) Figuerola, A.; Franchini, I. R.; Fiore, A.; Mastria, R.; Falqui, A.; Bertoni, G.; Bals, S.; Van Tendeloo, G.; Kudera, S.; Cingolani, R.; Manna, L. Adv. Mater. 2009, 21, 550–554. (6) (a) Claridge, S. A.; Mastroioanni, A. J.; Au, Y. B.; Liang, H. W.; Micheel, C. M.; Frechet, J. M. J.; Alivisatos, A. P. J. Am. Chem. Soc. 2008, 130, 9598–9604. (b) Kanaras, A. G.; Wang, Z.; Brust, M.; Cosstick, R.; Bates, A. D. Small 2007, 3, 590– 594. (c) Kanaras, A. G.; Wang, Z.; Hussain, I.; Brust, M.; Cosstick, R.; Bates, A. D. Small 2007, 3, 67–70. (d) Kanaras, A. G.; Wang, Z.; Bates, A. D.; Cosstick, R.; Brust, M. Angew. Chem., Int. Ed. 2003, 42, 191–194. (7) (a) Baker, J. L.; Widmer-Cooper, A.; Toney, M. F.; Geissler, P. L.; Alivisatos, A. P. Nano Lett. 2010, 10, 195–201. (b) Chen, C. L.; Rosi, N. L. Angew. Chem., Int. Ed. 2010, 49, 1924–1942. (8) Mirkin, C. A. Inorg. Chem. 2000, 39, 2258–2272. (9) (a) Seeman, N. C. Nano Lett. 2010, 10, 1971–1978. (b) Sharma, J.; Chhabra, R.; Cheng, a.; Brownell, J.; Liu, Y.; Yan, H. Science 2009, 323, 112–115. (c) Zheng, J.; Constantinou, P. E.; Micheel, C.; Alivisatos, A. P.; Kiehl, R. A.; Seeman, N. C. Nano Lett. 2006, 6, 1502–1504. (d) Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A. Nature 2008, 451, 553–556. (e) Ceyhan, B.; Alhorn, P.; Lang, C.; Schuler, D.; Niemeyer, C. M. Small 2006, 2, 1251–1255.

13760 DOI: 10.1021/la1023554

(NPs).9 However, the reported approaches either require a high level of sophistication in design and synthesis or they are limited to the fabrication of only very small amounts of material. Herein, we describe a minimal building block system, based on short synthetic oligodeoxynucleotides (ODNs) combined with a DNA-binding peptide, that can form a range of gold NP (Au-NP) arrays in a one-step process. To our knowledge, this is the first demonstration of nanoparticle assembly by virtue of the specific binding of oligopeptides to DNA and has the potential to become a tool for the development of new analytical and diagnostic methods. Manipulation of the assembly size and shape is obtained simply by altering the composition of the peptide coating and varying the architecture of the DNA system. Such exploitation of peptide-DNA binding has not been used before for Au-NP organization, and the simplicity of DNA and peptide synthesis has the potential for scaled-up production.

Experimental Section Materials. All reagents were purchased from the following suppliers and used without further purification: sodium tetrachloroaurate(III) dihydrate, trisodium citrate, sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Sigma-Aldrich. Bis(p-sulfonatophenyl)phenyl phosphine dehydrate dipotassium salt (BSPP) was purchased from StremChemicals, Inc. Milli-Q water was used in all experiments. The peptides were obtained from either Activotec (Cambridge) or GL Biochem (Shanghai) Ltd. Methods. ODN synthesis was carried out on an ABI Expedite 8909 synthesizer using the standard solid-supported phosphoramidite cycle (1.0 μmol scale). Phosphoramidite monomers of natural 20 -deoxynucleosides and reagents for oligonucleotide synthesis were purchased from Link Technologies Ltd. and SAFC Supply Solutions. ODNs were deprotected and cleaved from the solid support in conc. ammonium hydroxide at 35 °C for 15 h and then purified directly by Glen-Pak purification cartridge following the supplied procedure (Glen Research). TEM images were obtained with a Hitachi H7000 transmission electron microscope operating at a bias voltage of 75 kV. All samples were deposited on carbon film 400-mesh copper (50) grids purchased from Agar Scientific Ltd. UV- visible spectra of colloidal gold nanoparticles were collected using a Cary 300 Bio UV-vis spectrophotometer over the range of 400-800 nm.

Published on Web 07/30/2010

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Coomber et al.

Synthesis of Peptide-Coated Nanoparticles. Gold nanoparticles were synthesized according to the well-established citrate reduction method10 and capped with BSPP.11 (See the Supporting Information for physicochemical characterization.) The surface of the gold nanoparticles was further functionalized with CALNN and CALNN-linker-FQGII (C-FQGII; the linker is aminohexanoic acid) peptides. In detail, a mixture of CALNN and C -FQGII (final concentration 0.63 μM) in 0.2 M phosphate buffer at pH 8 was added to an aqueous solution of gold nanoparticles (0.5 mL, 5 nM) while stirring. The sample was sonicated for 20 min and kept overnight at 4 °C to ensure a fine coating of nanoparticles. The particles were purified of excess ligands by three centrifugation steps (16 400 rpm, 15 min), followed by decantation and redispersion in buffer (0.5 mL of 0.1 M phosphate buffer, 0.1 M NaCl, 1 mM EDTA, pH 8) prior to incubation with DNA. To modify the ratio of the peptides on the nanoparticle surface, the amount of C-FQGII in the reaction mixture was increased from 5 to 25% while keeping the final peptide concentration steady at 0.63 μM. Variations in the degree of C-FQGII loading were evident by the different mobility of the nanoparticle samples in an agarose gel (Supporting Information). DNA-Nanoparticle Assemblies. To create the assemblies, the different types of peptide-coated nanoparticles were mixed either with ODN1 and ODN2 or with ODN3. In detail, a solution containing 2 nM peptide-coated particles and 0.5 μM ODN1 and ODN2 or 1 μM ODN3 in buffer (0.1 M sodium phosphate, 0.1 M NaCl, 1 mM EDTA, pH 8) was mixed and stirred. To ensure complete hybridization between the DNA strands, the samples were heated to 85 °C for 2 min and then gradually brought to room temperature (0.5 °C/min). The samples were left to stand for 1 h and then characterized by visible spectroscopy and transmission electron microscopy.

Results and Discussion The Au-NPs were coated with a mixture of CALNN and C-FQGII peptides to give peptide-coated NPs (pep-NPs). CALNN was selected for the preparation of stable gold colloids.12 CALNNcoated NPs are also known to be taken up by cells,13 which could be of benefit for biological applications. Pentapeptide sequence FQGII was shown by Sasaki and co-workers to bind selectively to an AT-rich region of a short target DNA duplex with high binding constants. The nature of the binding can most likely be attributed to hydrophobic interactions of the peptide with the DNA.14 Two DNA systems were used to induce pep-NP assembly: a long DNA duplex formed from two short, staggered oligonucleotides (ODN1 and ODN2) and a short self-complementary sequence (ODN3) (Scheme 1). Self-complementary sequence ODN3 contains two AT steps capable of binding, and staggered duplex ODN1 3 ODN2 has AT regions at regular intervals along the length of the duplex. We expected that the long duplex would form extended chains of Au-NPs whereas the short duplex would rather be bound around the NP with little or no control of NP organization. The added amount of the DNA-binding peptide (C-FQGII) was varied from 0 to 25% (denoted as x% pep-NP) to obtain increasing numbers of DNA binding sites per particle. Each batch of particles was incubated with template 1 or 2, maintaining a constant NP-to-DNA ratio. A heating/cooling (10) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55–75. (11) Schmid, G. Chem. Rev. 1992, 92, 1709–1727. (12) Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076– 10084. (13) (a) Nativo, P.; Prior, I. A.; Brust, M. ACS Nano 2008, 2, 1639–1644. (b) Sun, L.; Liu, D.; Wang, Z. Langmuir 2008, 24, 10293–10297. (14) (a) Alam, M. R.; Maeda, M.; Sasaki, S. Bioorg. Med. Chem. 2000, 8, 465– 473. (b) Alam, M. R.; Maeda, M.; Sasaki, S. Nucleic Acids Symp. Ser. 1999, 42, 173–174.

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Letter Scheme 1. DNA Systems Used to Induce pep-NP Assemblya

a

(a) Sequence of the staggered DNA forming a long duplex (template 1) and of the self-complementary DNA (template 2). (b) Schematic representation of the DNA-NP arrays formed along template 1 and (c) along template 2.

cycle was performed to ensure the formation of DNA-nanoparticle assemblies. To gain an understanding of the morphology of the formed assemblies, the pep-NP DNA complexes were analyzed using transmission electron microscopy (TEM). Figure 1 shows images of x% pep-NP that were obtained from the interaction of the pepNPs with template 1 (Figure 1a-e) or template 2 (Figure 1f-j). A clear organization of discrete pep-NP DNA complexes can be seen in all images where pep-NPs were used. For 5% pep-NPs, the formation of dimers or trimers on both templates is observed (red circles in Figure 1). As the number of DNA binding sites per particle was increased (10% pep-NPs), short chains of NPs were produced on both templates. Longer, single-chain-like arrangements of 15% pep-NPs can be observed on 2, whereas double chains of particles of up to 3 μm in length were formed on the 1 duplex. It is most striking that these unexpectedly long double chains exhibit helicity and the NP lines cross over (indicated by the red arrows in Figure 1). The helical turn in these linear arrays is about 100 nm and could be correlated to the type of oligonucleotides as well as the size and functionality of particles used in these experiments. Much shorter nanoparticle chains are observed when 25% pep-NPs were incubated with 1. In contrast, the same pep-NPs on template 2 resulted in their organization to highly complex 3D particle networks. Statistical analysis of the images showed that for both templates the yield of assembly was gradually increased with the number of binding sites per Au-NP (Supporting Information). Consequently, we can hypothesize that the probability of binding is correlated with the C-FQGII content. Therefore, a higher number of DNA binding sites per particle dramatically changes the morphology of the assemblies. This is possibly due to the higher or lower binding rate of the particles to the DNA template as well as the actual number of binding events. To ensure that the nanoparticle organization is due to the specific DNA templates, we performed a control experiment where we incubated the pep-NPs only with ODN1. In this case, because the ODN1 does not form a template, we did not observe any ordered structures (Supporting Information). The optical properties of assembled Au-NPs depend strongly on the size and shape of the aggregates as well as the distance DOI: 10.1021/la1023554

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Figure 1. TEM images of nanoparticle assemblies; x% pep-NPs incubated with template 1 (a-e) and template 2 (f-j). The red circles show the formation of dimers and trimers when 5% pep-NPs are used (b, g). The red arrows indicate possible helical twists in nanoparticle arrangements (d, e). Long NP arrays are observed for 15% pep-NPs (d, i). When 25% pep-NPs are used, more complex arrays are formed for template 2 (j). Scale bars are 100 nm.

signatures of all NPs: those participating in the assemblies as well as those remaining free in solution. As discussed earlier, the probability of nanoparticle participation in the assemblies increases with the C-FQGII content.

Conclusions

Figure 2. UV-vis spectra of the x% pep-NPs incubated with template 1 (a) and template 2 (b).

between particles. The presence of small structures (e.g., dimers and trimers) is usually indicated by a broadening and/or a decrease in the intensity of the plasmon band, and additional red shifting suggests the formation of larger aggregates. Figure 2 shows the visible spectra of the formed assemblies. Sharp absorption peaks of the 0% pep-NPs indicate the presence of homogeneously dispersed spherical colloids. The visible spectra of small 5 and 10% pep-NP DNA assemblies do not show considerable changes in the plasmon band. A notable decrease in the intensity in conjunction with broadening and red shifting can be observed for the higher C-FQGII loading, indicating the presence of larger assemblies in solution. Red shifts of up to 11 nm were observed for pep-NP-1 systems (Figure 2a) and up to 21 nm for pep-NP-2. Nevertheless, the plasmon bands in the visible spectra of the pep-NP/DNA template solutions represent the average optical

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We have demonstrated that NP-array architectures can be controlled relatively easily using short ODNs and simple peptides and their specific recognition properties. Dimers, trimers, shorter and longer chains of nanoparticles, and even more complex structures can be assembled at will by regulating the number of binding sites on the Au-NP’s surface and/or by engineering the DNA template. By using a minimal building block system and by simply varying the combination of short ODNs and pep-NPs, various morphologies are obtained, which makes this method very attractive for designing Au-NP assemblies. We are now probing this system using peptides with different sequence selectivity. Acknowledgment. The financial support of the EPSRC, the Royal Society, and the University of Southampton (NanoUSRG) is gratefully acknowledged. A.G.K. thanks the Research Council U.K. (RCUK) for a Roberts fellowship. Supporting Information Available: Experimental details, additional TEM images, statistical analysis, and spectroscopic characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(17), 13760–13762