Bioconjugate Chem. 2005, 16, 497−500
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The Peptide Route to Multifunctional Gold Nanoparticles Zhenxin Wang,† Raphae¨l Le´vy,†,‡ David G. Fernig,‡ and Mathias Brust†,* Centre for Nanoscale Science, Department of Chemistry and School of Biological Sciences, The University of Liverpool, Liverpool, UK. Received February 21, 2005; Revised Manuscript Received April 8, 2005
Extremely stable, peptide-capped gold nanoparticles with two different biomolecular recognition motifs expressed on their surface have been prepared, and their specific and selective binding to artificial, DNA-modified target particles and to DNA and protein microarrays has been demonstrated. Stabilization and biofunctionalization has been achieved in a single preparative step starting with citrate-stabilized gold hydrosols and a derivatization cocktail of peptide-capping ligands, which carry the functionalities of choice.
Exploiting the optical properties of nanoparticles for the development of ultrasensitive detection and imaging methods in the biomedical sciences is becoming increasingly important (1-3). Particularly attractive is the use of Au and Ag nanoparticles in resonant light scattering (RLS) and photothermal microscopy, which are able to image single particles (4-7). The latter technique is capable of detecting particles at least as small as 2 nm in complex biological environments. For these applications, attaching the biomolecular recognition motif of interest to the nanoparticles has to be readily achieved, and, most importantly, the probes must not bind nonspecifically to each other or to anything else present in the system under investigation. In addition, introducing multiple functionalities would be of great value, as it provides more flexibility for multiplexing in bioanalytical applications and new tools to control the bottom-up assembly of nanostructures. Here we demonstrate that multiply functional peptidestabilized gold nanoparticles are readily obtained in a one-step surface coating procedure and that the respective surface functionalities can be selectively addressed on a microarray. The particles are of the stability typical for peptide capping (8) and show no indication of nonspecific binding as established by transmission electron microscopy (TEM), UV-vis spectroscopy, and microarray imaging even under testing conditions of ionic strength (see Supporting Information). In contrast to most previously reported approaches, stabilization and functionalization of the particles are independent of each other, while both are achieved in a single step. Functionality is simply introduced by including a proportion of stabilizing peptide to which a functionality of choice has been attached. Therefore, the number of recognition functions present on each particle could be altered and even reduced to a single or very few moieties without compromising the stability of the particles. We have previously reported the preparation of peptide-capped gold nanoparticles and their functionalization with biotin (see also Supporting Information) (8). We now take advantage of the generality of the peptide route and * Corresponding author. E-mail:
[email protected]; Fax: (+44) 151-794-3588. † Centre for Nanoscale Science, Department of Chemistry. ‡ Centre for Nanoscale Science, School of Biological Sciences.
Figure 1. Nanoparticle binding scenarios and sequences of DNA and functionalized peptides. (a) 13 nm DNA-functionalized small particles with high DNA loading (>3%) binding to 40 nm DNA-stabilized large particles to form extended aggregates. (b) 13 nm DNA functionalized small particles with intermediate DNA loading (0.3-1%) binding to DNA-stabilized 40 nm large particles at a small particle to large particle ratio of (i) 10:1, (ii) 30:1, and (iii) 100:1. (c) Sequences of DNA and functionalized peptides used. Peptides are in the conventional N- to C-terminal orientation.
demonstrate that it provides a fast and simple approach to DNA functionalization and to bifunctional nanoparticles carrying both DNA and biotin moieties. These functionalities were chosen for being already well established in nanoparticle systems and thus predictable in their recognition properties (1-7, 9-26). Thirteen nanometer gold nanoparticles were prepared via the classical citrate reduction route (27, 28). Stabilization and DNA functionalization was achieved by adding an aqueous mixture of the CALNN (cysteine, alanine, leucine, asparagine, asparagine) capping peptide and a CALNN-DNA conjugate (18 base single-stranded DNA) followed by centrifugation to purify the material.
10.1021/bc050047f CCC: $30.25 © 2005 American Chemical Society Published on Web 04/26/2005
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Figure 2. Dependence of the aggregation of small (probe) and large (target) particles on the experimental conditions. Varying both the DNA loading of the small particles and the small particle to large particle ratio affects the perceived color of the solutions (top left panel) and the type of aggregates formed (see selected TEM images, labels correspond to those in the top left panel). Blue color indicates the formation of extended aggregates (a and b), red indicates either no aggregation or the formation of large particles labeled by a small number of small particles (c and d), and purple indicates the formation of a dense shell of small particles around individual large particles (e).
The specific recognition properties of these particles were investigated by binding to standard Mirkin-type (9) 40 nm gold nanoparticles via a three-strand system. The 40 nm particles were stabilized by a shell of thiol-modified (ss) DNA, which was then hybridized to a linker strand of 42 bases to leave 18 bases as an overhanging recognition sequence. This sequence was chosen to be complementary to the DNA on the peptide-capped particles. DNA hybridization then leads to particle aggregation, demonstrating the successful DNA functionalization of the peptide-capped particles. In an attempt to control the number of DNA molecules per nanoparticle, we systematically varied the proportion of CALNN-DNA in the stabilization mixture. We also changed the relative abundance of 40 and 13 nm particles in the hybridization buffer. Possible binding scenarios for these different experimental conditions are schematically suggested in Figure 1, and the range of different nanostructures found experimentally by TEM is shown in Figure 2. It was found that the formation of very different aggregated structures was controlled by these two parameters. At a glance, this is best confirmed by optical spectroscopy or simply by visual inspection of the aqueous reaction mixtures. The formation of extended aggregates is observed already for relatively low DNA loadings, i.e., whenever the concentration of CALNN-DNA in the stabilizing mixture reaches 3% in total ligands (40 nM)
or more. This aggregation behavior has been confirmed from a one-to-one ratio to a 100-fold excess of complementary particles in the solution. The transition from a well-dispersed system to complete aggregation happens rather suddenly. Close to the proportion of CALNN-DNA in the stabilization mixture required for the formation of precipitates, structures, in which two or more large particles were bound to each other via small particles were regularly observed. The formation of extended aggregates is readily monitored by a clearly perceptible color change of the solutions from red to blue followed by the slow precipitation of the material. On the contrary, at a CALNN-DNA concentration of 1.4 nM (0.1% of total number of ligands) or less no specific binding was observed in the system, even with a 100-fold excess of small particles. The parameter space in which pairs between large and small particles were obtained was remarkably narrow. Isolated aggregated structures of a discrete number of small particles bound to a single large particle were obtained only for intermediate concentrations (4-14 nM, i.e., 0.3-1% of total number of ligands) of CALNN-DNA in the stabilizing mixture. Within this narrow range the binding number of small particles per large particle is chiefly determined by the relative proportion of small particles in the system, as illustrated by the bar diagrams in Figure 3. Depending on the relative excess of small particles, aggregates with binding
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Figure 3. Dependence of small particle to large particle binding numbers on the applied small particle to large particle ratio. Experimentally found binding number distributions (by TEM) are shown for small particle to large particle ratios from 1:1 to 100:1 for a DNA loading of the small particles of (a), 0.3% and (b), 1%.
Figure 4. Schematic representation of DNA and protein microarrays labeled with bifunctional gold nanoparticles followed by silver enhancement (a). Light scattering images of protein microarray (b) and DNA microarray (c) after silver enhancement. In b, avidin was spotted on column 1, 2, and 4, while protein A was spotted as a nonspecific control on column 3; in c, perfectly matched DNA was spotted on column 1, three-base-mismatched DNA was spotted on column 2, two-base-mismatched DNA was spotted on column 3, and one-base-mismatched DNA was spotted on column 4.
numbers from 1 to ca. 100 small particles per large particle could be prepared. The limiting case is the formation of a complete shell of small particles around each large particle as shown in Figure 2e. Aggregates with such high binding numbers also led to a perceptible color change of the solution from red to purple without subsequent precipitation. Core-shell nanostructures of large particles surrounded by small particles are stable in solution for weeks and are readily isolated from excess small particles by centrifugation. These experiments may mimic the attachment of gold nanoparticle probes to target structures in biological systems and map out the conditions required to achieve effective labeling. More importantly, they provide guidance for the preparation of nanoparticles with two or more different specific recognition groups attached. Such multifunctionality of nanoparticles has been reported previously for nonbiomolecular functionalities on monolayer protected clusters (MPCs) (18, 29-31). Niemeyer et al. have shown that multifunctionality can be achieved by the use of different DNA sequences on one particle and also reported that a relatively low loading of the particles with DNA is sufficient to achieve very efficient specific binding (19). Here we demonstrate that different types of biomolecular recognition groups, i.e., biotin and
DNA, can be readily incorporated in a single stabilization/ functionalization step via our peptide route. Guided by the results obtained for the DNA only system we have chosen to include 70 nM (5% of total number of ligands) CALNN-DNA (18 base recognition sequence) and 70 nM (5% of total number of ligands) CALNNGK(biotin)G in the stabilization mixture. This should lead to high reactivity of the particles toward both complementary DNA and avidin. To demonstrate the binding specificity of these bifunctional particles, we prepared a protein microarray containing spots with avidin as well as control spots with protein A, and a DNA microarray containing spots with complementary DNA, as well as control spots with DNA possessing mismatches of one, two, and three bases. After binding of the particles the microarrays were exposed to a silver enhancement step, i.e., electroless deposition of metallic silver on the gold particles, which act as nucleation sites (32). The microarrays were read by measuring the intensity of scattered light using a Qiagen High-light System. The results of specific binding of the bifunctional particles to these microarrays are shown in Figure 4. It was found that the particles bind to both avidin and to complementary DNA with high specificity. Importantly, no evidence for significant nonspecific binding was found (detailed data in Supporting
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Information). DNA with a one base mismatch is still weakly detected, which is consistent with the expected remaining recognition properties of this DNA. No binding to DNA with a mismatch of two or more bases is detected. In conclusion, we have developed a straightforward route to stable gold nanoparticles, both with single and with dual biological functionality. The particles exhibit the specific recognition properties of the biological functionalities they carry without any indication of nonspecific binding or particle aggregation. While the stability of the particles does not depend on the number of functional groups introduced, functionalization and stabilization are achieved in a single step where the relative abundance of functional peptides in the stabilization mixture appears to control the number of functionalities introduced into the peptide ligand shell of the particles. The large difference in the chemical nature of the recognition motifs used in this study suggests that this approach should be easily extended to a variety of peptide and peptide conjugate labels, including the large number of peptides recognizing with high specificity and affinity biological and nonbiological structures. ACKNOWLEDGMENT
The authors thank the BBSRC (Centre for BioArray Innovation), the Cancer and Polio Research Fund, the European Union, and the North West Cancer Research Fund for financial support. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Alivisatos, A. P. (2004) The use of nanocrystals in biological detection. Nature Biotechnol. 22, 47-52. (2) Liz-Marza´n, L. M. (2004) Nanometals: formation and color. Mater. Today 7 (2), 26-31. (3) Katz, E., and Willner, I. (2004) Integrated NanoparticleBiomolecule Hybrid Systems: Synthesis, Properties, and Applications. Angew. Chem., Int. Ed. 43, 6042-6108. (4) Yguerabide, J., and Yguerabide, E. E. (2001) Resonance light scattering particles as ultrasensitive labels for detection of analytes in a wide range of applications. J. Cell. Biochem. 37 (Suppl.), 71-81. (5) Sonnichsen, C., Franzl, T., Wilk, T., von Plessen, G., Feldmann, J., Wilson, O., and Mulvaney, P. (2002) Drastic reduction of plasmon damping in gold nanorods. Phys. Rev. Lett. 88, art. no. 077402. (6) Boyer, D., Tamarat, P., Maali, A., Lounis, B., and Orrit, M. (2002) Photothermal imaging of nanometer-sized metal particles among scatterers. Science 297, 1160-1163. (7) Berciaud, S., Cognet, L., Blab, G. A., and Lounis, B. (2004) Photothermal heterodyne imaging of individual nonfluorescent nanoclusters and nanocrystals. Phys. Rev. Lett. 93, art. no. 257402. (8) Levy, R., Thanh, N. T. K., Doty, R. C., Hussain, I., Nichols, R. J., Schiffrin, D. J., Brust, M., and Fernig, D. G. (2004) Rational and combinatorial design of peptide capping ligands for gold nanoparticles. J. Am. Chem. Soc. 126, 10076-10084. (9) Mirkin, C. A., Letsinger, R. L., Mucic, R. C., and Storhoff, J. J. (1996) A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382, 607609. (10) Alivisatos, A. P., Johnsson, K. P., Peng, X., Wilson, T. E., Loweth, C. J., Bruchez, M. P., Jr., and Schultz, P. G. (1996) Organization of ‘nanocrystal molecules’ using DNA. Nature 382, 609-611. (11) Mucic, R. C., Storhoff, J. J., Mirkin, C. A., and Letsinger, R. L. (1998) DNA-directed synthesis of binary nanoparticle network materials. J. Am. Chem. Soc. 120, 12674-12675.
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