Thiol-Specific and Nonspecific Interactions between DNA and Gold

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Thiol-Specific and Nonspecific Interactions between DNA and Gold Nanoparticles Marite´ Ca´rdenas,†,§ Justas Barauskas,† Karin Schille´n,*,† Jennifer L. Brennan,‡ Mathias Brust,‡ and Tommy Nylander† Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund UniVersity, Lund, Sweden, and Centre for Nanoscale Science, Department of Chemistry, UniVersity of LiVerpool, LiVerpool, UK ReceiVed NoVember 11, 2005. In Final Form: January 20, 2006 The contribution of nonspecific interactions to the overall interactions of thiol-ssDNA and dsDNA macromolecules with gold nanoparticles was investigated. A systematic investigation utilizing dynamic light scattering and cryogenic transmission electron microscopy has been performed to directly measure and visualize the changes in particle size and appearance during functionalization of gold nanoparticles with thiol-ssDNA and nonthiolated dsDNA. The results show that both thiol-ssDNA and dsDNA do stabilize gold nanoparticle dispersions, but possible nonspecific interactions between the hydrophobic DNA bases and the gold surface promote interparticle interactions and cause aggregation within rather a short period of time. We also discuss the adsorption mechanisms of dsDNA and thiolssDNA to gold particles.

Introduction Gold nanoparticles modified by thiol-derivatized singlestranded (ss) DNA (thiol-ssDNA) are promising building blocks for nanoscale materials1-3 and are used in various applications in bioanalysis.4-6 Many recent studies deal with various analytical methods to quantify the number of oligonucleotides on a nanoparticle surface,7 to separate gold nanoparticles with different numbers of oligonucleotides attached,8 or to identify the binding affinity of different oligonucleotides.9 A key property of these types of functionalized particles is their ability to hybridize with complementary DNA.10-12 Herne and Tarlov13 found that the hybridization efficiency largely depends on the surface coverage of the thiol-ssDNA. The authors also pointed out that thiolssDNA adsorbs to the gold surface not only via the thiol group * To whom correspondence should be addressed. E-mail: [email protected]. † Lund University. ‡ Liverpool University. § Present address: Malmo ¨ University, Biomedical Technology 20506 Malmo¨, Sweden. (1) Kanaras, A. G.; Wang, Z.; Bates, A. D.; Cosstick, R.; Brust, M. Angew. Chem., Int. Ed 2003, 42, 191-194. (2) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. J. Phys. Chem. B 2002, 106, 11758-11763. (3) Pedano, M. L.; Martel, L.; Desbrieres, J.; Defrancq, E.; Dunny, P.; CocheGuerente, L.; Labbe´, P.; Legrand, J.-F.; Calemczuk, R.; Rivas, G. A. Anal. Lett. 2004, 20, 10086-10092. (4) Storhoff, J. J.; Mirkin, C. A. Chem. ReV. 1999, 99, 1849-1862. (5) Cao, Y, W.; Jin, R.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 79617962. (6) Dean, H. J.; Fuller, D.; Osorio, J. E. Comput. Immunol. Microb. 2003, 26, 832-837. (7) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A., III; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535-5541. (8) Zanchet, D.; Michel, C. M.; Parak, W. J.; Gerion, D.; Alivisatos, A. P. Nano Lett. 2001, 1, 32-35. (9) Storhoff, J. J.; Elghanian, R.; Mirkin, C. A.; Letsinger, R. L. Langmuir 2002, 18, 6666-6670. (10) Moses, S.; Brewer, S. H.; Lowe, L. B.; Lappi, S. E.; Gilvey, L. B. G.; Sauthier, M.; Tenent, R. C.; Feldheim, D. L.; Franzen, S. Langmuir 2004, 20, 11134-11140. (11) Park, S.-J.; Lazarides, A. A.; Storhoff, J. J.; Pesce, L.; Mirkin, C. A. J. Phys. Chem. B 2004, 108, 12375-12380. (12) Olofsson, L.; Rindzevicius, T.; Pfeiffer, I.; Ka¨ll, M.; Ho¨o¨k, F. Langmuir 2003, 19, 10414-10419. (13) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920.

interactions but also by nonspecific interactions. Furthermore, in a later neutron reflectivity study, it was shown that coadsorption of mercaptohexanol onto ssDNA-functionalized gold surfaces induces desorption of nonthiol-mediated ssDNA binding contacts and causes the remaining ssDNA to be bound to the surface only through their thiol group.14 It was also found that nonspecific interactions contribute to adsorption of both thiol-ssDNA and even double-stranded (ds) DNA to gold surfaces.15 Recently, Rant and co-workers16 studied the structural properties of oligonucleotide monolayers on gold surfaces. They used fluorescence quenching measurements and found fairly good agreement with a model in which the tethered ssDNA strands extend as rigid rods toward the bulk solution. They found that the model failed for oligonucleotides with 24 bases in length or longer, where the oligonucleotides could no longer be treated as rods but should be considered as a polymer with semiflexible chains. The aim of the present study is to reveal the contribution of nonspecific interactions of thiol-ssDNA and dsDNA to their overall interaction with gold nanoparticles, as well as providing insights into the conformation of the DNA on the particle surface. For this purpose, a systematic study using dynamic light scattering (DLS) has been performed to directly measure changes in the particle size during the course of a typical thiol-ssDNA-modified gold nanoparticle preparation procedure.1 For comparison, the study has also been performed using nonthiolated dsDNA in place of the thiolated ssDNA. The DLS technique has earlier proved to be a powerful method to investigate the size changes of colloidal particles due to DNA adsorption.17,18 In addition, cryogenic transmission electron microscopy has been used for direct imaging of the particles and the morphology of the formed particle aggregates in aqueous dispersions. (14) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (15) Sandstro¨m, P.; Boncheva, M.; Åkerman, B. Langmuir 2003, 19, 75377543. (16) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Langmuir 2004, 20, 10086-10092. (17) Ca´rdenas, M.; Schille´n, K.; Nylander, T.; Jansson, J.; Lindman, B. Phys. Chem. Chem. Phys. 2004, 6, 1603-1607. (18) Ca´rdenas, M.; Schille´n, K.; Pebalk, D.; Nylander, T.; Lindman, B. Biomacromolecules 2005, 6, 832-837.

10.1021/la0530438 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/03/2006

Thiol Interactions between DNA and Au NPs

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Experimental Section

t is the lag time and β (e1) is a coherence factor which accounts for deviation from ideal correlation and the experimental geometry. For polydisperse particle sizes, g(1)(t) may be described by

Materials. Short mononulesomal double-stranded DNA (dsDNA) with 146 base pairs (bp), as determined by 7.5% polyacrylamide gel analysis, was kindly provided by D. McLoughlin (Universite´ Paris Sud) and was dialyzed against 10 mM NaBr solution prior to use. The single-stranded DNA (ssDNA) with 46 bases (Alta Bioscience, Birmingham, UK) contained a 5′-thiol modification (OPO3(CH2)6S-S-(CH2)6OH) and was used as received. The oligonucleotide base sequence was 5′-AAA-AAC-TCT-TGC-TAA-GCA-TCCGAT-ATC-ACT-CCG-GAA-TTC-CAT-TCG-3′. Gold nanoparticles were prepared in aqueous solution by the classical citrate reduction route following standard procedures.19,20 Sodium bromide (Aldrich, 99.99%), sodium chloride (Merck, >99.5%), disodium hydrogen phosphate anhydrous (Kebo lab, proanalysis 99%), and sodium dihydrogen phosphate monohydrate (Merck, 99%) were used as received. Water purified by a Milli-Q system (Millipore Corp., Bedford, MA) was used in all measurements. Only the buffer solutions, used in the preparation of the DNA-modified gold nanoparticles, were filtered through 0.2 µm filters (Whatman) to avoid loss of material. The DNA concentration was measured spectrophotometrically by its absorbance at 260 nm, using an extinction coefficient of 33 µg/mL for ssDNA and 50 µg/mL for dsDNA. The ratio of absorbance at 260 and 280 nm was ca. 1.81.9, and the absorbance at 320 nm was negligible, indicating the absence of protein contamination. Also the gold particle concentration was determined by absorption spectroscopy. For particles of this size, the extinction coefficient is 4.2 × 10-8 (M cm)-1 at 520 nm.21,22 Preparation of DNA-Modified Gold Nanoparticles. In a typical preparation,1 citrate-stabilized gold nanoparticles were derivatized with thiolated oligonucleotides by incubating the gold dispersion (500 µL, 6 nM) with disulfide-protected oligonucleotides (thiolssDNA) (25 µL, 40 µM) in aqueous solution, overnight (Step 1). Afterward the dispersion was brought to a final volume of 1 mL in NaCl (0.1 M) and sodium phosphate buffer (5 mM, pH 7) and incubated for another 2 h (Step 2). The volume was then slowly reduced to 150 µL, by vacuum centrifugation using a RotaVap apparatus (Labex Instruments AB, Helsingborg, model RVC2-18), at 40 °C over 3-4 h (Step 3). The concentrate was resuspended in NaCl (0.3 M) and sodium phosphate buffer (10 mM, pH 7), and the unbound oligonucleotides were removed by repeated centrifugation using a Sigma 1-13 Centrifuge (13 000 rpm × 2, 25 min) and redispersion of the pellet. In addition, the concentration of DNA in the supernatant separated after centrifugation was too low to measure, which implies that the loss of material was minor. The DNAcomplexed gold nanoparticles (ssDNA-thiol-NP) were stored in NaCl (0.3 M) and sodium phosphate buffer (10 mM, pH 7) for further use (Step 4). The same procedure was also used to prepare dsDNA-modified gold nanoparticles. Dynamic Light Scattering (DLS). The experimental goniometer setup (ALV-GmbH, Langen, Germany) to perform DLS measurements has been described previously with the difference that cisdecahydronaphthalene is used instead as the refractive index matching liquid.23 The light source is a 532 nm diode pumped Nd:YAG solidstate Compass-DPSS laser (COHERENT, Inc., Santa Clara, CA). In this work, the temperature was controlled at 25 ( 0.1 °C, and the scattering angle (θ) was varied from θ ) 50 to 130°. Two multiple τ digital correlators (with a total of 320 exponentially spaced channels) were utilized to produce the time (and pseudo-cross) correlation function of the scattered intensity with an initial real-time sampling time of 12.5 ns. The normalized intensity correlation function, g(2)(t), is related to the normalized time correlation function of the electric field [g(1)(t)], by Siegert’s relation: g(2)(t) - 1 ) β|g(1)(t)|2, where (19) Turkevich, J.; Stevensson, P. S.; Hilliwe, J. Discuss. Faraday Soc. 1951, 11, 55-75. (20) Frens, G. Nature 1973, 241, 20-22. (21) Link, S.; Wang, Z. L.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 3529-3533. (22) Mulvaney, P.; Giersig, M.; Henglein, A. J. Phys. Chem. 1992, 96, 1041910424. (23) Jansson, J.; Schille´n, K.; Olofsson, G.; Silva, R. C. d.; Loh, W. J. Phys. Chem. B 2004, 108, 82-92.

g(1)(t) )

∫

∞

-∞

τA(τ) exp(-t/τ)d ln τ

(1)

where τ is the relaxation time, and A(τ) is the distribution of relaxation time. The experimental intensity correlation functions (g(2)(t) - 1) were analyzed by regularized inverse Laplace transformation (RILT) to obtain the relaxation time distribution. The RILT analysis uses the calculation algorithm REPES,24,25 incorporated in the GENDIST analysis package.26,27 We display the results from the DLS analysis as relaxation time distributions expressed as τA(τ) versus log(τ/µs), and which are normalized with the maximum peak height. From the DLS data, the apparent size of the particles can be determined. In the limit of small scattering vectors, qj, the apparent translational collective diffusion coefficient (D) at finite concentration can be calculated from the relaxation rate, Γ, which is obtained from the first moment of the translational mode in the relaxation time distribution: D ) lim qf0

() Γ q2

(2)

where q is the magnitude of the scattering vector (q ) 4πn0sin(θ/ 2)/λ, where n0 is the refractive index of water, λ is the incident wavelength, and θ is the scattering angle) and Γ ) 1/τ. Here, D is obtained from the slope of Γ as a function of q2, where Γ has been measured at different scattering angles. The apparent hydrodynamic radius (Rapp H ) is related to the collective diffusion coefficient at finite concentration through the Stokes-Einstein relationship: Rapp H )

kT 6πη0D

(3)

where k is Boltzmann’s constant, T is the absolute temperature, and η0 is the viscosity of water. Cryogenic Transmission Electron Microscopy (Cryo-TEM). The samples were prepared in a controlled environment vitrification system. The climate chamber temperature was 25-28 °C, and the relative humidity was kept close to water vapor saturation to prevent evaporation from the sample during preparation. A 5 µL sample drop was placed on a carbon-coated holey film supported by a copper grid and gently blotted with filter paper to obtain a thin liquid film (20-400 nm) on the grid. The grid was then rapidly plunged into liquid ethane at -180 °C and transferred into liquid nitrogen (-196 °C). The vitrified specimens were stored in liquid nitrogen and transferred into a Philips CM120 BioTWIN microscope equipped with a post-column energy filter (Gatan GIF 100) using an Oxford CT 3500 cryo-holder and its workstation. The acceleration voltage was 120 kV, and the working temperature was kept below -182 °C. The images were recorded digitally with a CCD camera (Gatan MSC 791) under low-dose conditions with an underfocus of approximately 1 µm.

Results and Discussion In this study, the interactions of two different types of DNA macromolecules, a thiolated single-stranded DNA (thiol-ssDNA) of 46 bp and a longer double-stranded DNA (dsDNA) of 146 (24) Nicolai, T.; Brown, W.; Johnsen, R. M.; Stepanek, P.; Macromolecules 1990, 23, 1165-1174. (25) Stepanek, P. In Dynamic Light Scattering; Brown, W., Ed.; Clarendon Press: Oxford, 1993; p 177. (26) Johnsen, R. M.; Brown, W. In Laser Light Scattering in Biochemistry; Harding, S. E., Satelle, D. B., Bloomfield, V. A., Eds.; Royal Society of Chemistry: Cambridge, 1992; p 449. (27) Schille´n, K.; Brown, W.; Johnsen, R. M. Macromolecules 1994, 27, 48254832.

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Table 1. Measured Apparent Hydrodynamic Radii (in nm) of All Studied Samples samplea

Rapp H fast mode

NPs in water Step 1 thiol-ssDNA+NPs Step 1 dsDNA+NPs Step 2 thiol-ssDNA+NPs Step 2 dsDNA+NPs Step 3 dsDNA+NPs Step 4 thiol-ssDNA+NPs, after 2 h Step 4 dsDNA+NPs, after 2 h Step 4 thiol-ssDNA+NPs, after 72 h Step 4 dsDNA+NPs, after 72 h NPs in buffer thiol-ssDNA+NPs, after 2 h dsDNA+NPs, after 2 h thiol-ssDNA+NPs, after 72 h dsDNA+NPs, after 72 h

10 ( 1 11 ( 1 12 ( 1 14 ( 1 12 ( 1 74 ( 13 19 ( 1 79 ( 6 26 ( 1 115 ( 5 83 ( 1 14 ( 1 24 ( 1 14 ( 1 26 ( 1

Rapp H slow mode 267 ( 63 378 ( 150 374 ( 78 74 ( 13 42 ( 21 635 ( 48 640 ( 246

213 ( 7

a Step 1 indicates the mixture of DNA and NP in water. Step 2 refers to ionic strength adjustment to 0.31 M. Step 3 indicates solvent evaporation, and Step 4 indicates the sample after centrifugation, washing, and redispersion of the pellet.

bp, which is not thiolated, with gold nanoparticles were investigated. Both DNAs are short with a contour length that is less than or equal to the persistence length of DNA at this ionic strength. To reveal the effect of each of the preparation steps for making the thiol-ssDNA-functionalized gold nanoparticles (ssDNA-thiol-NPs) according to Kanaras et al.,1 an aliquot of the dispersion was taken after each step of analysis using dynamic light scattering as the tool of investigation. For comparison, the same procedure was applied using nonthiolated dsDNA. Further information regarding the morphology of the aggregates formed was obtained by cryo-TEM imaging of both samples after the last preparation step. In addition, apparent hydrodynamic radii of all investigated samples in this study are summarized in Table 1. The Thiol-ssDNA/Gold Particle System. Dynamic light scattering experiments were first performed on the thiol-ssDNA (46 bp)-functionalized gold nanoparticle system. Figure 1 shows the relaxation time distributions obtained from the RILT analysis of the corresponding intensity correlation functions measured at the scattering angle θ ) 70° at different steps of the preparation procedure. The data for gold nanoparticles dispersed in water and in 10 mM phosphate buffer pH 7 containing 0.3 M NaCl are also included in the figure as a reference. For the particles dispersed in water, the relaxation time distribution exhibits a large, narrow mode positioned at a relaxation time of 102 µs, which represents the major fraction of the particle population, and a low amplitude mode at around 103.1 µs. Both relaxation modes are due to translational motion (i.e., the corresponding relaxation rates are q2 dependent), the faster corresponds to a diffusing particle with a hydrodynamic radius (Rapp H ) of 10 nm while the slower is attributed to larger aggregates of nanoparticles. Note that the relaxation time distributions are intensity distributions, and therefore, the larger aggregates are fewer for the same intensity. If instead the particles are dispersed in buffer, only one large translational mode is observed that is broader and displaced to a longer relaxation time (around 103 µs), which gives Rapp H ) 83 nm (see also Table 1). This growth in particle size when adding salt is a typical and known example of decrease in colloidal stability of charged particles: higher ionic strength leads to screening of the negative charges on the surface of the gold particles and thus induces particle aggregation. Addition of thiol-ssDNA to the nanoparticle dispersion in water induces considerable particle aggregation with increased

Figure 1. Relaxation time distributions at θ ) 70° of gold nanoparticles dispersed in water, phosphate buffer (10 mM pH 7 and 0.3 M NaCl), and after various steps of the procedure to produce thiol-ssDNA-modified gold nanoparticles. Step 1 indicates the mixture of DNA and NP in water. Step 2 refers to ionic strength adjustment to 0.31 M, and Step 4 indicates the sample after centrifugation, washing, and redispersion of the pellet.

Figure 2. Schematic representation of the ssDNA-thiol-NP showing the adsorbed flat conformation (a) and extended conformation of the oligo chain (b). The pentagons represent the hydrophobic nitrogenous bases. Note that the drawing is not to scale.

polydispersity of the size distribution, as evidenced by the development of translational modes at longer relaxation times (Step 1). It should be stressed that the DLS analysis using RILT analysis of the DLS correlograms can sometimes give a multimode appearance if the relaxation time distribution is very wide, especially at longer relaxation times. This has been observed in investigations of semidilute polymer systems, where the correlograms also were fitted to a sum of two distributions; see, for example, ref 28 and the references therein. In this work, we are only interested in the fact that aggregation occurs, and we do not interpret each individual peak at the slower side of the distribution. The fastest mode corresponds to the particles with a hydrodynamic radius of 11 nm. Given that the ionic strength is the same as that for bare nanoparticles in water, this small increase in radius (1 nm) may indicate that the thiol-ssDNA interacts with particles but lies flat on the surface (as schematized in Figure 2a). This was also observed by Parak et al. who found, using gel electrophoresis, that for low coverage thiol-ssDNA wraps nonspecifically around the Au nanoparticle.29 The loss in configurational entropy may be balanced by the decreased exposure of the hydrophobic base to water. This conformation could be easily understood taking into account that the persistence (28) Nicolai, T.; Brown, W.; Hvidt, S.; Heller, K. Macromolecules 1990, 23, 5088-5096. (29) Parak, W. J.; Pellegrino, T.; Micheel, C. M.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. Nano Lett. 2003, 3, 33-36.

Thiol Interactions between DNA and Au NPs

length of ssDNA is somewhat lower than that of dsDNA.30,31 The slower modes of the relaxation time distribution correspond to sizes of Rapp H ) 266 nm and larger, and must thus be due to bridging between different particles through nonspecific interactions between the hydrophobic moieties of ssDNA and the gold nanoparticle as also have been observed on polystyrene particles17,18 or even through limited base-to-base interactions between two or more ssDNA chains on different NPs.1 If now the ionic strength of the dispersion is increased to 5 mM phosphate buffer pH 7 and 0.1 M NaCl (Step 2), the translational mode for the ssDNA-thiol-NP coated by a single oligo layer increases to 14 nm, while there is a considerable reduction in the population of aggregated particles (Rapp H ) 373 nm and larger) as evidenced by the decrease in the amplitude of the slower modes. The ssDNA-thiol-NP dispersion was then concentrated (Step 3) by vacuum centrifugation, increasing the ionic strength of the solution, which helps to suppress nonspecific ssDNA binding to the surface. Following the concentration step, the pellet can be redispersed in 10 mM phosphate buffer pH 7 and 0.3 mM NaCl (Step 4), where the “excess thiol-ssDNA” and other “looser aggregates” with low number density are removed by repeated centrifugation and redispersion of the pellet. Correspondingly, the aggregates have almost completely disappeared in the dispersion 2 h after of resuspending the pellet. The fast mode gives a hydrodynamic radius of 19 nm. The contour length of the ssDNA macromolecules used here is about 14 nm. Thus, the increase in hydrodynamic radius from 10 nm (bare NP) to 19 nm (ssDNA-thiol-NP) indicates that the thiol-ssDNA molecules are now adsorbed in an extended conformation, as depicted in Figure 2b. This extended conformation does not necessarily imply that the ssDNA chain behaves as a rod. On the contrary, one would expect that the molecule is still quite flexible16 and thus be able to bend and partially hide its hydrophobic nitrogenous bases. However, the particles do aggregate with time as the relaxation time distribution becomes bimodal again after 72 h of resuspending the pellet. At the same time, the hydrodynamic radius of the fastest mode further increases to 26 nm. Cryo-TEM images of the redispersed sample after 72 h also show the appearance of nonordered gold nanoparticle aggregates of various sizes (Figure 3c,d). Although some aggregation is observed as a function of time in the thiol-ssDNA-gold particle system, we can still conclude that the presence of thiol-ssDNA does stabilize the gold nanoparticle dispersion. This is evident when comparing the relaxation time distribution of the bare nanoparticles in 10 mM phosphate buffer pH 7 and 0.3 M NaCl with that of the freshly prepared ssDNA-thiol-NP dispersion with the same ionic strength (Figure 1). Without ssDNA, the monomodal distribution is centered at a Rapp H ) 83 nm, whereas with ssDNA, it is centered at a Rapp ) 19 nm. This is indeed expected as the oligo chains H increase the negative charge of the bare NPs and therefore the stability of the colloidal particles. In addition, one would expect that formation of a layer of flexible chains on the particle surface would also provide some steric stabilization. However, possible nonspecific interactions between the hydrophobic bases of the ssDNA molecule and the surface of the gold nanoparticles could promote interparticle interactions, and this could cause aggregation within a rather short period of time. Indeed, recent studies support this idea, showing a strong binding affinity of de(30) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Macromolecules 1997, 30, 5763-5765. (31) Baumann, C. G.; Smith, S. B.; Bloomfield, V. A.; Bustamante, C. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6185-6190.

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Figure 3. Cryo-TEM images of (a,b) gold nanoparticles dispersed in water; (c,d) after Step 4 (72 h) of the procedure to produce thiolssDNA-modified gold nanoparticles; and (e,f) after Step 4 (72 h) of the procedure to produce dsDNA-modified gold nanoparticles.

oxynucleosides to gold nanoparticles9 and polycrystalline gold films.32 The dsDNA/Gold Particle System. For comparison, the interaction between nonthiolated double-stranded DNA and gold nanoparticles was also investigated using DLS. The contour length of this DNA macromolecule is about 50 nm, which is about the same as the persistence length of double-stranded DNA for solutions with ionic strength larger than 10 mM.31 Therefore, the dsDNA molecule used in the present study may be considered as a rigid rod. The relaxation time distributions obtained from RILT analysis of the intensity correlation functions for gold nanoparticles mixed with 146 bp dsDNA according to the standard procedure are presented in Figure 4. Also shown in the figure are the data for gold nanoparticles simply dispersed in water and in 10 mM phosphate buffer (pH 7) containing 0.3 M NaCl. Addition of dsDNA to the gold nanoparticle dispersion in water (Step 1) causes a slight shift of the fast relaxation mode from Rapp H ) 10 to 12 nm and appearance of the slow modes. When the ionic strength is increased (Step 2), the fast mode remains invariable (Rapp H ) 12 nm), whereas the slower mode of the distribution indicates an increased aggregation. Once the dispersion is concentrated by means of vacuum centrifugation (Step 3), the relaxation time distribution changes considerably (Figure 4). A very broad size distribution is observed, where the peak has been significantly shifted to longer relaxation times and radii (>50 nm). After centrifugation and redispersion of the pellet in (32) O ¨ stblom, M.; Liedberg, B.; Demers, L. M.; Mirkin, C. A. J. Phys. Chem. B 2005, 109, 15150-15160.

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Figure 5. Schematic representation of the dsDNA-NP showing the proposed conformation at Steps 1, 2 (a) and 3, 4 (b) of the preparation procedure. The pentagons represent the hydrophobic nitrogenous bases. Note that the drawing is not to scale.

Figure 4. Relaxation time distributions at θ ) 70° of gold nanoparticles dispersed in water, phosphate buffer (10 mM pH 7 and 0.3 M NaCl), and after various steps of the procedure to produce dsDNA-modified gold nanoparticles. Step 1 indicates the mixture of DNA and NP in water. Step 2 refers to ionic strength adjustment to 0.31 M. Step 3 shows sample after solvent evaporation, and Step 4 indicates the sample after centrifugation, washing, and redispersion of the pellet.

10 mM phosphate buffer pH 7 and 0.3 M NaCl (Step 4), the distribution is somewhat narrower but still bimodal. This, however, may also be an effect from the RILT procedure, where a broad distribution sometimes can be split into two modes. The dispersion was remeasured 72 h after redispersion of the pellet to reveal an even broader distribution centered at about Rapp H ) 115 nm. The dispersion at this time consists mainly of nanoparticle aggregates, as shown by the cryo-TEM images in Figure 3. It is noteworthy that the color of the dispersion completely changed from pink (Steps 1 and 2) to blue (Step 3) and finally to be uncolored (Step 4). Another interesting observation is that redispersion was only possible once in buffer since the pellet could not be easily separated from the supernatant. The increase in hydrodynamic radius from 10 to 12 nm in Steps 1 and 2 is a clear indication that dsDNA adsorbs to the surface of the gold nanoparticles, even though there is no thiol group present to drive the adsorption process. This might not be what one would expect when considering that these gold nanoparticles bear a slightly negative charge. Previously, studies which describe the interaction of gold nanoparticles and dsDNA have required cationic nanoparticles with positively charged ligand shells to facilitate an electrostatic DNA-nanoparticle interaction.33,34 On the other hand, previously, we have shown that dsDNA adsorbs to latex particles even if they also are negatively charged.17,18 In those studies, we concluded that the adsorption process was driven by hydrophobic interactions between the nitrogenous bases of the DNA and the hydrophobic tails present on the surface of the particles. Moreover, as shown in these studies, there is an entropic gain when releasing the water molecules confined at the hydrophobic surfaces. Interestingly, Sandstro¨m et al.15 reported in an electrophoresis study that (33) Sastry, M.; Kumar, A.; Datar, S.; Dharmadhikari, C. V.; Ganesh, K. N. Appl. Phys. Lett. 2001, 78, 2943-2945. (34) Warner, M. G.; Hutchison, J. E. Nat. Mater. 2003, 2, 272-277.

high amounts of strong, nonthiol-mediated binding to gold nanoparticles occurs for both single- and double-stranded DNA. The authors suggested that the mechanism for the interaction between double-stranded DNA and the gold nanoparticles is ion-induced dipole dispersive interactions, where the highly negative charge of dsDNA induces dipoles in the highly polarizable gold nanoparticle. Adsorption of dsDNA to the gold nanoparticles increases the negative charge of the particles and thus the colloidal stability of the particle dispersion. Indeed, the dsDNA-NP dispersion prior to vacuum concentration is more stable than the ssDNAthiol-NP one, as observed in the relaxation time distributions shown in Figures 1 and 4 (Steps 1 and 2). However, whereas concentrating the ssDNA-NP dispersion in Step 3 increases the colloidal stability, aggregation is induced in the dsDNA-NP dispersion as the particle concentration is increased in the same manner. This could be due to bridging as the short and stiff dsDNA macromolecule may adsorb to more than one particle simultaneously. Perhaps the binding occurs by the more hydrophobic ends of the dsDNA molecule, as depicted in Figure 5. Interestingly, the removal of the “excess dsDNA” does not improve the stability of the colloidal dispersion (comparison of Step 4 in Figures 1 and 4). This is a clear indication that the mechanism of adsorption for dsDNA and thiol-ssDNA to gold particles is different. In the case of dsDNA, centrifugation of the dispersion leads to irreversible aggregation, while for thiolssDNA, it leads to the proper removal of some loose aggregates and the excess oligonucleotides leaving a relatively stable colloidal dispersion with a single layer of oligonucleotides adsorbed onto the gold particles. To further explore the effects of the preparation procedure on the aggregation of the DNA-coated gold particles, additional experiments were performed. In these experiments, the concentration of the added DNA stock solution was decreased, and 1 volume unit of dsDNA or thiol-ssDNA in buffer was mixed with 1 volume unit of gold NPs in water to have the same final concentration of DNA, NPs, and buffer as in the previous experiment. The corresponding relaxation time distribution measured after 2 and 72 h of equilibration time is shown in Figure 6. Addition of dsDNA to the gold NP dispersion shifts the translational mode of the particles toward longer relaxation times, which indicates particle growth or the appearance of aggregates. The hydrodynamic radius of the particles increases from 10 nm in water to 24 nm when measured 2 h after mixing the sample. When the sample is reexamined 72 h later, the translational mode of the particles slightly moves to longer ) 26 nm) and a new slower mode relaxation times (Rapp H develops that indicates even broader size distribution of aggregates. On the other hand, the aggregation is more pronounced when thiol-ssDNA is used and the shift in the translational mode of the particles is considerably more modest (Rapp H ) 14

Thiol Interactions between DNA and Au NPs

Figure 6. Relaxation time distributions at θ ) 70° for gold nanoparticles dispersed in water, phosphate buffer (10 mM pH 7 and 0.3 M NaCl), and 1:1 volume mixtures of dsDNA or thiolssDNA in buffer and gold NP in water.

regardless of the equilibration time elapsed). These results support the idea that thiol-ssDNA adsorbs in a flatter conformation to the gold particles than dsDNA does, as indicated in Figures 2a and 5a. It also confirms that the vacuum centrifugation step (Step 3) markedly improves the stability of the resulting ssDNANP dispersions.

Conclusions The focus of this study has been to unravel the influence of two types of DNA macromolecules, a thiol-modified singlestranded DNA and a nonthiolated double-stranded DNA, on the stability of gold nanoparticle dispersions. For this purpose, we utilized dynamic light scattering and cryo-TEM to gain knowledge of the size distributions and aggregation behavior of these systems. The DLS results showed that thiol-ssDNA initially adsorbs to the gold nanoparticles in a flat conformation with a single oligonucleotide layer, which was expressed by a small but significant increase in the hydrodynamic radius of the particles. It is expected that the thiol-ssDNA molecules bind to the gold surface through thiol group interactions. However, aggregates

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composed of several ssDNA-coated nanoparticles were observed. This indicates that nonspecific interactions between the hydrophobic bases of the ssDNA molecule and the gold surface are also active. These aggregates could easily be removed by vacuum concentration giving a more stable colloidal dispersion than without ssDNA present. Importantly, this demonstrates that nonspecific binding of ssDNA to gold nanoparticles is strongly dependent on the ionic strength of the medium and can be suppressed by using sufficiently high electrolyte concentrations. Both the cryo-TEM and DLS results reveal that dsDNA adsorbs to gold nanoparticles even though it does not contain thiol groups and although the particle surface is slightly negative. In this case, the adsorption is driven by the nonspecific hydrophobic interaction between the nitrogenous bases of the DNA and the hydrophobic tails that are present on the gold nanoparticle surface. There may also be another mechanism present, for example, the ion-dipole dispersive interaction. The dsDNA binding to the nanoparticles through the more hydrophobic end groups gives a more extended conformation of the layer, compared to the ssDNA case. This in turn leads to bridging to other gold particles and irreversible aggregation during the course of the preparation procedure. The fact that dsDNA readily adsorbs to gold surfaces must thus be taken into account when examining the adsorption of complementary strands to prefunctionalized ssDNA gold substrates. In other words, ssDNA strands could adsorb to a prefunctionalized ssDNA gold substrate not only by finding its complementary strand but also through nonspecific interactions to the bare gold surface. We believe that our findings will be useful when designing methods for bio-recognition and for the development of diagnostic devices. The occurrence of nonspecific binding to and cross-linking of gold nanoparticles by DNA has been underestimated in past studies and does in fact explain why it is in practice often difficult to control the programmed assembly of DNA-gold nanostructures. In particular, our cryo-TEM data demonstrate unequivocally that significant aggregation can occur even in the absence of complementarity. This study emphasizes the importance of suppressing nonspecific interactions when using DNA as a building material for nanostructures, and it outlines the conditions under which this can be achieved. Acknowledgment. This work was supported by EU-STREP FP6 Project BIOSCOPE (Contract No. NMP4-CT-2003-505211). LA0530438