Synthesis and Internal Structure of Finite-Size DNAâGold Nanoparticle

Article pubs.acs.org/JPCC

Synthesis and Internal Structure of Finite-Size DNA−Gold Nanoparticle Assemblies Anne Buchkremer,† Malte J. Linn,‡ Jan U. Timper,§ Thomas Eckert,∥ Joachim Mayer,§ Walter Richtering,∥ Gero von Plessen,‡ and Ulrich Simon*,† †

Institute of Inorganic Chemistry, RWTH Aachen University and JARA-Fundamentals of Future Information Technology, Landoltweg 1, 52074 Aachen, Germany ‡ Institute of Physics (IA), RWTH Aachen University, Sommerfeldstraße 14, Turm 28, 52074 Aachen, Germany § Central Facility for Electron Microscopy, RWTH Aachen University, Ahornstraße 55, 52074 Aachen, Germany ∥ Institute of Physical Chemistry, RWTH Aachen University, Landoltweg 2, 52074 Aachen, Germany S Supporting Information *

ABSTRACT: Spatially defined networks of 15 nm-sized DNA-functionalized gold nanoparticles (DNA−AuNPs) were studied using dynamic light scattering (DLS), small-angle Xray scattering (SAXS), as well as optical extinction spectroscopy (OES). We use a combination of these techniques with Monte Carlo simulations of pair-distance distribution function (PDDF) curves and generalized Mie theory simulations as well as in situ-transmission electron microscopy (in situ-TEM) to analyze the internal structure of the finite-size assemblies. The DLS data show that monodisperse, spherical networks with hydrodynamic radii of ca. 30 nm are found for reaction mixtures of complementarily functionalized DNA−AuNPs between 1:15 and 1:20. Different interparticle distances within these assemblies are identified and quantified. By controlling the network morphology through selection of the reaction mixture, center-shell geometries are obtained. The number of shell-AuNPs surrounding each center-AuNP is determined from the SAXS data and Monte Carlo simulations. This number is quantified to be ca. 10, with the exact number depending on the linking DNA double strand. The optical spectra of the networks are found to be consistent with the structural properties. The structural information gained here enables a quantitative description of optical and other physical properties, which is expected to prove useful for the construction and application of such systems, for example, in drug release, gene regulation, or external-stimuli-responsive materials.

1. INTRODUCTION The synthesis and precise construction of metal nanoparticle− biomolecule hybrid systems have gained immense interest due to their unique combination of optical properties and biomolecular recognition capabilities. In particular, conjugates of AuNPs and DNA have become an important area of focus in nanoscience research.1−14 The ability to form such hybrid systems with a high degree of control allows fabricating micrometer-sized periodically ordered superstructures, which are assembled via DNA hybridization and thus offer a high level of predictability.6−8 These superstructures consist of AuNPs connected by uniform DNA spacers of a few nanometers in length and thus are heterogeneous on the nanometer scale. The uniformity of the DNA spacers results in the existence of a distinct DNA-melting temperature (Tm) based on a cooperative mechanism.1 Upon heating beyond Tm, these structures disassemble and reassemble upon cooling to form thermodynamic products. These structures are of fundamental interest, since, e.g., their electrical or optical properties may be expected to be different from both the individual particles and the bulk solid. © 2014 American Chemical Society

Besides the periodically ordered superstructures, tailored finite-sized model systems such as dimers,15,16 trimers,17,18 or satellite structures3,19,20 have been introduced as well. Finitesize assemblies can be linked to form hierarchical structures that may exhibit new structural and functional properties. Recently, we reported the synthesis of superaggregates consisting of DNA-linked 14 nm AuNPs that exhibit a hierarchical sequence of structural sizes from the scale of the individual nanoparticles, to that of finite-size networks (approximately 0.3 μm), to that of the superaggregates themselves (approximately 1.3 μm). We demonstrated that thermal and photothermal dissociations occur in distinct steps and that the photogenerated temperature rise inside the laser-irradiated networks increases with aggregate size.21 The dissociations showed that the macroscopic physical properties of such a system can be switched by temperature and light as external stimuli.22 Received: December 16, 2013 Revised: March 1, 2014 Published: March 4, 2014 7174

dx.doi.org/10.1021/jp412283q | J. Phys. Chem. C 2014, 118, 7174−7184

The Journal of Physical Chemistry C

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Scheme 1. Two-Dimensional Simplified Representation of the Three-Dimensional Network Assemblya

a

The networks consist of two types of 15 nm-sized AuNPs that are functionalized with two pairs of complementary DNA base sequences (DNA-1 and -2 or DNA-3 and -4, respectively), and are assembled at temperatures below the DNA-melting temperature Tm. Molar ratios, which are denoted as 1:1, 1:2, 1:5, 1:10, 1:12, 1:15, 1:18, 1:20, and 1:50, were varied. A priori various network geometries are conceivable. We will show in section 3 that core−shell geometries are realized when one compound is added in huge excess. The reason is that each of the minority particles will be completely surrounded by majority particles, so that further network growth is inhibited. Using SAXS-derived information such as the AuNP core radius (R(SAXS) AuNP ), the center-AuNP to shell-AuNP, DNA-tethered, interparticle distance (dcenter−shell) and the shell−shell, structure-induced AuNP interparticle distance (dshell−shell) will be modeled in section 3.3 for networks in which one compound is added in huge excess. The hydrodynamic radius for a single shell of DNA−AuNPs surrounding a central core DNA−AuNP can be calculated on the basis of data obtained by SAXS (AuNP (DLS) (DLS) radius R(SAXS) AuNP and interparticle distance dcenter−shell; cf. section 3.3) and DLS (AuNP hydrodynamic radius RH,AuNP), which results in a RH,core−shell value of ca. 30 nm. Note that, for clarity, DNA and AuNP dimensions are not drawn to scale.

Here we report the analysis of the internal structure of DNA−AuNP networks and determine two interparticle distances within finite-size networks that have sizes in between very small structures such as dimers and trimers and long-range ordered extended structures. For this purpose, we use a combination of dynamic light scattering (DLS), SAXS with concomitant Monte Carlo simulations, optical extinction spectroscopy (OES), and in situ-transmission electron microscopy (in situ-TEM). The networks consist of two types of 15 nm-sized AuNPs that are functionalized with complementary DNA base sequences. The networks were assembled at temperatures below the DNA-melting temperature Tm, as shown in Scheme 1. The network size can be adjusted by the molar building-block ratios,5,21,25−28 which were chosen to be 1:1, 1:2, 1:5, 1:10, 1:12, 1:15, 1:18, 1:20, and 1:50 (as listed in Scheme 1). Core−shell geometries are expected when one compound is added in huge excess, since then each of the minority particles will be completely surrounded by majority particles, so that further network growth is inhibited. In this scenario, which is illustrated in Scheme 1, the shell particles are bound to the center particle via Watson−Crick base pairing, while the packing within the shell is determined by the space filling of equicharged particles with noncomplementary base sequence. In the present paper, this

Compared to extended superstructures with long-range order, which can be characterized using X-ray diffraction, finite size structures require elaborate techniques to verify the internal structure. Different interparticle distances can occur due to different coupling modes, such as tethering the particles by direct hybridization of surface-bound DNA single strands, cross-linking through linker DNA or unspecific, structureinduced vicinity. First steps toward characterizing the internal structure have been reported by Mirkin and co-workers and Gang and co-workers.5,23 In 2007, Maye et al. applied smallangle X-ray scattering (SAXS) to determine the average distance between DNA-tethered AuNPs within networks of different sizes by analyzing the structure factor.5 Using the same approach accompanied by a detailed theoretical description, they as well as Mastroianni et al. were able to fully describe the internal structure of AuNP dimers, which are the most basic unit of AuNPs linked by DNA.16,24 In order to fully describe the internal structure of finite-size AuNP networks, it is vital to quantify and distinguish the different interparticle distances present in these heterogeneous networks, e.g., to discriminate DNA-tethered from unconnected neighboring particles. This would, e.g., enable the development of a quantitative model to describe the optical and (photo)thermal properties of these networks. 7175



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charge-coupled device (CCD) chip (Andor Technologies) was used. In situ liquid cell TEM experiments were performed on a Zeiss Libra 200FE TEM equipped with an in-column energy filter and a Köhler illumination system. A Hummingbird Liquid Flow Holder was utilized in combination with two Hummingbird silicon blank chips with 200 μm by 50 μm windows, sealed by 50 nm thick Si3N4 membranes. In order to reduce contamination artifacts and improve the wettability of the otherwise hydrophobic membranes, an air-plasma was applied to the membranes for 10 s prior to sample preparation. Samples were then prepared for observation under static conditions by pipetting 0.5 μL of the sample solution between the two silicon chips. Images were acquired with energy filtering and image generation from an energy window of about 100 eV around the most probable loss (MPL) energy. To prove the presence of a liquid film after insertion into the high vacuum, the presence of a water-plasmon in the EELS spectrum was repeatedly checked during imaging. Furthermore, occasional diffusion of particles into or out of the focus plane was taken as proof for the presence of the solvent. The beam current was carefully adjusted for the shortest possible exposure times without removing particles from the illuminated region due to charging effects at high beam intensities. Standard AuNP-network solutions were diluted 1:9 with ultrapure water.

scenario is confirmed using the combination of techniques listed above. The interparticle distances between the centerAuNP and a shell-AuNP (dcenter−shell in Scheme 1) within the networks as well as the interparticle distance between the respective shell-AuNPs (dshell−shell in Scheme 1) are investigated by SAXS and modeled using Monte Carlo simulations of the pair distance distribution function (PDDF). We deduce the number of shell-AuNPs surrounding a center-AuNP in core− shell model structures that exhibit hydrodynamic radii in the range of ca. 30 nm. Also, networks in the range from 18 to 160 nm were synthesized and characterized. Furthermore, simulations of optical spectra based on generalized Mie theory are employed to evaluate the OES data, as also recently shown by Urban et al. for a similar system comprising polymer-linked nanoparticles.29 In situ-TEM images support the proposed network size. To investigate the influence of the interconnecting DNA double strands on the network structure, we interconnected AuNPs with two different recognition sequences (DNA-1/2 and DNA-3/4) and compared the results.

2. EXPERIMENTAL METHODS Thiol-functionalized and carboxyfluorescein (FAM)-labeled single stranded (ss)-oligonucleotides were purchased from Thermo Fisher Scientific GmbH (purified by high-performance liquid chromatography). Water was purified using a Purelab Plus system and subsequently filtered through a 0.2 μm nylon syringe filter. Tetrachloroauric acid and dithiothreitol (DTT) were purchased from Sigma Aldrich, and trisodium citrate dihydrate was purchased from Merck. 47 nm gold nanoparticles were purchased from BBI/Plano and used without further purification. Citrate-stabilized AuNPs were synthesized according to Turkevich et al.,31 and ssDNA−AuNP composites were synthesized following a scheme derived from those of Mirkin and co-workers and Brust and co-workers. 1,30 AuNP dispersions (600 μL, 8 nM) were incubated with thiol-ssDNA-1, -2, -3, and -4 (see Scheme 1 for sequences; each 50 μL, 0.1 mM) at 50 °C. Fluorescein-labeled ss-DNA-2F and -3F (F5′-TTA TTG TTA A10-3′-(CH2)-SH; F-5′-GCG ATT CAG GAT A10-3′-(CH2)-SH) were used for the DTT assay.37,38 AuNP concentrations were determined using atom-absorption spectroscopy (AAS). The respective networks were assembled in buffer solution (0.1 M NaCl, 10 mM phosphate (pH 7)). Scanning electron microscopy in transmission mode (SEMT) was performed using 10-fold-diluted solutions of various samples on a FE-SEM, LEO/Zeiss Supra 35 VP in bright field SEM-T mode on carbon-coated copper grids (Plano). DLS measurements were performed with a standard light scattering device from ALV GmbH Langen using a He−Ne laser (JDS Uniphase, λ = 633 nm) with a two-avalanchephotodiode pseudocross detection system. Measurements were performed at scattering angles of θ = 30, 50, 70, 90, 110, and 130° and lasted 300 s each at 20 °C. Hydrodynamic radii were calculated applying the Stokes−Einstein equation to the diffusion coefficients D obtained from cumulant analysis. SAXS measurements were performed with an S-Max3000 system with a MicroMax-002 + X-ray microfocus generator by Rigaku in a q range from 0.004 to 0.16 Å−1 at 20 °C. OES measurements were performed by irradiating the sample solution inside a quartz cuvette with white light. For detection, a grating spectrometer (Andor Technologies, Shamrock SR-303i-B) in combination with a Peltier-cooled

3. RESULTS AND DISCUSSION 3.1. Synthesis. As a first step, 15 nm-sized citrate-stabilized AuNPs were synthesized according to the Turkevich method.31 They exhibit a plasmon peak position of λmax = 520 nm and a particle size determined via scanning electron microscopy in transition mode of R(SEM‑T) = 7.6 ± 2.0 nm (standard deviation; AuNP s.d.). This combination of plasmon peak position in optical spectra and size is in accordance with the literature.31,32 SAXS measurements were performed, which led to a sphere radius of R(SAXS) AuNP = 8.2 ± 0.5 nm (s.d.) of the AuNPs as a mean of the PDDF maximum and the first and second minimum of the form factor. The slight difference in AuNP radii obtained from SEM-T and SAXS is due to the different measuring setup and evaluation for both techniques. While SAXS measurements are ensemble measurements and evaluated using mathematical functions, SEM-T images are graphically evaluated by encircling more than 200 individual particles based on the contrast of the respective image, which leads to a larger uncertainty and a less accurate value for the AuNP radius. Nevertheless, the values obtained from both methods agree within their standard deviation. DNA functionalization was performed using the following four base sequences. The sequences used were 5′TAA CAA TAA A10-3′-(CH2)3-SH; 5′-TTA TTG TTA A10-3′(CH2)3-SH; 5′-CGC ATT CAG GAT A10-3′-(CH2)3-SH; and 5′-ATC CTG AAT GCG A10-3′-(CH2)3-SH. In the following, they will be denoted as DNA-1, -2, -3, and -4, respectively. DNA-1 is complementary to DNA-2, while DNA-3 is complementary to DNA-4. DNA-3 and DNA-4 were chosen so as to be longer than DNA-1 and DNA-2. Theoretically, a mismatched DNA hybridization between the TAA group at the 3′ end of DNA-1 and the ATT group at the 5′ end of DNA-2 may occur, which would result in a double strand dehybridization temperature which is below room temperature and it would not yield the thermodynamically most stable conformation. No such mismatched hybridization could be experimentally observed in DLS- and OES-melting experiments in earlier works using the same DNA sequences.21 The citrate 7176



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the observed single-stage sigmoidal decline of the correlation functions in Figures SI2 and SI3 (Supporting Information), Figure SI4 (Supporting Information) leads to the conclusion that these networks are spherical within the error range of the experimental evaluation. A single-stage sigmoidal decay is only seen for spherical structures.39 Thus, the hydrodynamic radii are valid without further modeling. The diffusion coefficients D were derived using the second order cumulant coefficients, and subsequently, the hydrodynamic radius RH was determined using the Stokes−Einstein relation for each q,36 as seen in Figure 1. The system presented

shell is replaced in a ligand exchange reaction with the thiolmodified single stranded DNA (ssDNA), which does not significantly affect the metal core size of the nanoparticle. Hence, similar values as for citrate−AuNPs were obtained for DNA−AuNPs with R(SEM−T) DNA−AuNP = 7.2 ± 1.1 nm (s.d.) and = 8.0 ± 0.5 nm (s.d.), respectively. A slight shift in R(SAXS) DNA−AuNP λmax from 520 to 522 nm was observed, which is attributed to a change in dielectric environment and the binding of the DNA to the AuNP surface.32 Furthermore, the hydrodynamic size of the AuNPs increased from R(DLS) H,AuNP = 9.9 ± 0.2 nm (s.d.) to = 12.2 ± 0.3 nm (s.d.), giving additional evidence R(DLS) H,DNA−AuNP for a successful replacement of the citrate shell by ssDNA.33−35 The hydrodynamic radii were calculated via the Stokes− Einstein relation using the diffusion coefficient derived from the second order cumulant analysis.36 In contrast to the citratestabilized AuNPs in water, the DNA-functionalized AuNPs are kept in PBS-buffer solutions. The AuNP average surface coverage was adjusted to 4.9 × 1012 ssDNA/cm2, which corresponds to 38 strands per particle, determined using fluorescein-labeled ssDNA and a DTT assay.37,38 The number of Au atoms per particle was determined by atomic absorption spectroscopy (AAS) and SAXS. Subsequently, the molar extinction coefficients of the AuNPs were determined by means of OES, and then ratios of complementarily functionalized DNA−AuNPs were calculated on the basis of the DNA−AuNP’s quantitative optical extinction. The particles were mixed and were left overnight to form networks. Number ratios of 1:1 and 1:2 of complementarily functionalized DNA−AuNPs (cf. Scheme 1) led to the formation of networks that sedimented due to their large size. Molar excesses higher than 1:2 were chosen to yield finite sized networks that were stable in solution over several hours, as shown in the Supporting Information, Figure SI1. This stability was preserved also over weeks. The ratios of 1:5, 1:10, and 1:20 had proven advantageous to generate stable networks in earlier works, while networks from a reaction ratio of 1:50 are expected to be significantly less numerous than single, unhybridized AuNPs, which are also present in the solution.21 For extended DNA−AuNP superstructures, fcc- and bccpatterns have been observed in the literature.6−10 In the superstructures, number ratios between 1:8 and 1:12 of complementarily functionalized components were found. In the present work, also networks from number ratios of 1:12, 1:15, and 1:18 were prepared; they comprise the nine-base-pair recognition sequences DNA-1/2 and the 12-base-pair recognition sequences DNA-3/4 (Scheme 1), to further investigate the influence of the interconnecting DNA double strand on the network structure. 3.2. DLS. The correlation functions of DLS measurements on both DNA−AuNP network species at scattering vectors of q = 0.0068, 0.0111, 0.0152, 0.0187, 0.0217, and 0.024 nm−1 (q = 4πn0/λ sin(θ/2), with n0 as the refractive index, λ as the wavelength of the incident laser light and θ, as the scattering angle), corresponding to scattering angles of θ = 30, 50, 70, 90, 110, and 130°, respectively, are shown in Figures SI2 and SI3 in the Supporting Information. No rotational diffusion is observed, as seen in small Y-axis intercepts of the plot of the decay rate of the second order fit versus q2 for all investigated samples in Figure SI4 (Supporting Information). In this plot, rotational diffusion, if present, would be observable in Y-axis intercepts that diviate substantially from 0. The deviation from 0 of the Y-axis intercepts is less than 5% of the maximum value of the decay rate of the second order fit. In combination with

Figure 1. The hydrodynamic radii (RH) of DNA-1/2−AuNP networks synthesized from different reaction mixtures. The RH, in dependence of q, were derived using the cumulant coefficients of second order. Polydisperse network solutions display strongly varying values for RH. Samples exhibiting high excesses of one component are less polydisperse, as seen in RH values that are independent of q. The corresponding plot for DNA-3/4−AuNP networks can be found in Figure SI5 in the Supporting Information.

here does not guarantee a rigid interparticle distance due to the adenine10-spacer between the AuNP surface and the DNArecognition base sequence, and thus, the network size is an average value. In Figure 1, the hydrodynamic radii are plotted versus q for DNA-1/2−AuNP networks. While a reaction mixture of 1:5 exhibits a mean value over all q of RH = 67 nm, hydrodynamic radii of 35 nm for a reaction mixture of 1:10, 46 nm for 1:12, 28 nm for 1:15, 27 nm for 1:18, and 25 nm for 1:20, respectively, were determined. A variation of RH of less than 2 nm (less than 6%) between all scattering angles and thus practically monodisperse compounds are observed for ratios of 1:10, 1:15, 1:18, 1:20, and 1:50. For a ratio of 1:50, almost the size of single DNA−AuNPs is obtained with a network radius of RH = 15 nm. One needs to take into account the measuring method involved in DLS, which generates an average value for all species in solution. Thus, also single DNA−AuNPs contribute to the average size, in addition to the networks, which probably are not predominant within the 1:50 reaction mixture.40,41 A more detailed analysis of the samples with larger networks from reaction mixtures 1:5 and 1:12 was not possible due to their polydispersity. For higher excesses of one component than 1:12, the differences in size between networks and single particles were too small to allow distinguishing between the two species in solution. The same experiments were performed with DNA-3/4− AuNP networks, whose hydrodynamic radii are presented in Figure SI5 in the Supporting Information. Since the AuNPlinking DNA-3/4 double strands consist of 12 recognition base pairs instead of the 9 in the DNA-1/2 double strands, larger networks are expected and observed. Mean values of RH over all 7177



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Scheme 2. Conceivable Internal Structures of Networks Composed of Complementarily Functionalized DNA−AuNP That All Exhibit a Radius of ca. 30 nma

a

Since one DNA−AuNP species (DNA-2- or DNA-4−AuNP, respectively; red) is added in a molar excess of 1:15 and higher as compared to the other species (DNA-1- or DNA-3−AuNP, respectively; blue), scenario 1 is most likely. Note that for clarity DNA and AuNP dimensions are not drawn to scale.

Figure 2. SAXS curves of networks consisting of DNA-1- and DNA-2−AuNPs (a) and DNA-3- and DNA-4−AuNPs (b), synthesized using ratios of 1:5, 1:10, 1:20, and 1:50, respectively, next to the respective scattering patterns (descending with increasing excess of one component). The curves are shifted along the ordinate for better comparison. The minima of the form factor are visible for all network sizes, as well as the forward scattering at small q values that indicates the presence of networks. SAXS-PDDFs of networks consisting of DNA-1- and DNA-2−AuNPs (c) and DNA-3- and DNA-4−AuNPs (d), synthesized using ratios of 1:5, 1:10, 1:20, and 1:50, respectively, are presented. The peak denoted as i describes the distances within the AuNP itself, while peaks ii and iii at higher r values can be attributed to scattering pairs inside two spatially close AuNPs and can be associated with the interparticle distances dcenter−shell and dshell−shell; cf. Scheme 1. With increasing excess of one component, the height of peak i relative to that of peak ii is increased and the network size is decreased for both network species (as seen in decreasing dmax values, with dmax as the r value at which p(r) = 0).

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3.3. SAXS and Monte Carlo simulations. Although DLS delivers information on particle and network sizes, SAXS measurements are needed to obtain information on the internal structure of the networks.5,7,8,21,23 With SAXS, it is possible to gain information on the spatial distribution of the particles inside the networks, and a conclusion on the length of the interconnecting DNA strands can be drawn. Increasing numbers of AuNP-linking DNA bases imply increasing interparticle distances, as confirmed by the following experimental results. The SAXS curves of networks consisting of DNA-1- and DNA-2-functionalized AuNPs (a) and DNA-3and DNA-4-functionalized AuNPs (b) in reaction mixtures of 1:5, 1:10, 1:20, and 1:50, respectively, are plotted in Figure 2, next to the respective scattering patterns. The SAXS curves are shifted along the ordinate for better comparison, descending with increasing excess of one component. The minima of the form factors at q = 0.05 Å−1 are clearly visible for all reaction mixtures. The characteristic forward scattering at small q values indicates the presence of large networks for networks from a reaction mixture of 1:5. This forward scattering is reduced for networks from reaction mixtures with a higher excess of one component, which also show smaller hydrodynamic radii in DLS. This trend is observed for both network species. Inside the monodisperse networks synthesized in a 1:20 DNA-1/2− AuNP reaction mixture, a surface-to-surface distance of ca. 7 nm, which corresponds to a center-to-center distance of ca. 22 nm, to the closest scattering neighbor was derived from the structure factor peak at the smallest q value around 0.01 Å−1. The structure factor is calculated as the quotient of networkscattering curve and single-particle curve.21,23 For 1:20 DNA-3/ 4−AuNP networks, a surface-to-surface distance of ca. 8.5 nm, corresponding to a center-to-center distance of 23.5 nm, was derived. Fourier transformation of the presented scattering curves leads to the pair distance distribution function (PDDF, denoted as p(r)). Like the structure factor, the PDDF reflects the accumulation of scattering centers and their spatial relation to each other.43,44 For better comparison, the PDDFs are normalized to exhibit an intensity of 1 at the peak associated with the single DNA−AuNPs (peak “i”). The maxima indicate the most frequent distances between scattering centers within each network. Several peaks are observed in the PDDFs of all samples in Figure 2c for DNA-1/2−AuNP networks and in Figure 2d for DNA-3/4−AuNP networks. The peak denoted as i represents the distance distribution for scattering-center pairs inside individual AuNPs, while peaks at higher r values can be attributed to pairs of scattering centers inside two different AuNPs. The peak denoted as ii is therefore a measure of the distance of neighboring particles. Peak iii and the tail for larger r values originate from scattering centers which are further apart from each other. The adenine10-spacer between the AuNP and the recognition sequence can be partially physisorbed to the particle surface; this can lead to a variation of particle distance and consequently the peaks ii and iii are broadened. The size determined by DLS of networks synthesized in reaction mixtures of 1:10, 1:20, and 1:50 correlates well with their maximum dimensions that can be derived from SAXS from the r value at which p(r) = 0 in Figure 2c and d; we denote this r value as dmax. For example, for the monodisperse networks synthesized in a 1:20 reaction mixture, values of ca. 60 nm in diameter are obtained, which is in accordance with the DLS

scattering angles are 177 nm for a 1:5 reaction mixture, 70 nm for 1:10, 51 nm for 1:12, 44 nm for 1:15, 25 nm for 1:18, 33 nm for 1:20, and 19 nm for 1:50. As also seen for the DNA-1/ 2−AuNP networks, a trend of decreasing RH with increasing excess of one component as well as almost monodisperse networks for reaction mixtures between 1:12 and 1:50 are observed. In addition, the size distributions of all investigated DNA-1/2− and DNA-3/4−AuNP networks, calculated using CONTIN fits for DLS measurements performed at various angles, are presented in the Supporting Information, Figures SI6 and SI7. Generally, a decrease of the mean values of RH over all scattering angles with increasing excess of one component is observed. The only observed exceptions to this trend are the larger RH for the reaction mixture of 1:12, which are larger than that for 1:10 (DNA-1/2−AuNP networks), and for the reaction mixture of 1:18, which are larger than for 1:20 (DNA-3/4− AuNP networks). No sound explanation has been found for these repeatedly observed exceptions yet. For the 1:5 system, RH is large for small q due to the presence of large, polydisperse networks that show significant forward scattering at small q and thus at small scattering angles θ. This effect vanishes for smaller networks, which can therefore be considered as less polydisperse. Networks with a hydrodynamic radius of ca. 30 nm, which is the value RH,core−shell expected for spherical networks from adding all relevant dimensions in Scheme 1 and taking into account the measured data from section 3.3, are observed for ratios between 1:15 and 1:20. DLS probes the collective diffusion of particles and solvent. The fact that the solvent flows through the networks might affect the diffusion coefficient. To exclude such an influence, we compared DNA-1/2−AuNP networks of the composition 1:20 and single, large (47 nm in diameter as measured in SEM-T) citrate-stabilized AuNPs in SAXS and DLS measurements. The maximum size of the networks determined by SAXS (as = 29 nm. The described in section 3.3) was R(PDDF;SAXS) max maximum size of the citrate−AuNPs was found to be = 28 nm. A hydrodynamic radius of RH = 26 nm R(PDDF;SAXS) max was found for both systems, indicating that the networks are already in the so-called nondraining limit,42 which means that diffusion of solvent molecules through the networks during DLS measurements can be neglected. Taking the assembly behavior of complementarily functionalized DNA−AuNPs into account (sketched in Scheme 2 as red and blue particles, respectively), different possible internal structures could be conceived, if the mixing ratios were arbitrary, as sketched in Scheme 2. The DNA−AuNP networks on which we will focus in the following are formed from molar ratios of the respective complementarily functionalized components of 1:15 and higher. These networks exhibit hydrodynamic radii of ca. 30 nm, and the respective solutions are stable over weeks; i.e., no further DNA hybridization takes place. In scenarios 2 and 3 of Scheme 2, unbound complementary valences of both particle species would be simultaneously available for further hybridization at the network periphery, leading to further growth. Furthermore, scenario 4 is least likely due to the large excess of DNA-2 and -4 AuNPs (red particles in Scheme 2). Thus, the probability of appearance of the structures proposed in Scheme 2 decreases from left to right, making the core−shell structure proposed in scenario 1 most likely. 7179



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Figure 3. Calculated PDDFs based on Monte Carlo simulations (red) in comparison with experimental data (cyan) are seen for (a) DNA-1/2− AuNP networks and (b) DNA-3/4−AuNP networks, synthesized in a 1:20 reaction mixture. A core−shell interparticle distance of 7 nm for part a and 8.5 nm for part b fitted best to the experimental data, which is in accordance with the structure factor peak determined using SAXS. The experimental data agree best with simulations of eight equidistantly placed shell particles for DNA-1/2−AuNP networks and with nine particles for DNA-3/4−AuNP networks. The greater height of the particle peak at small r values in the experimental data as compared to the simulations can be explained with excess single particles in the experiments, which are not included in the simulations.

tively) have been found to agree best with the experimental PDDFs. These values also agree with those determined from the experimental structure factor peak. A core−shell model with equidistantly placed shell particles is assumed in the simulations.45 In Figure 3, the simulated PDDFs are shown together with the experimental data for DNA-1/2−AuNP networks (a) and DNA-3/4−AuNP networks (b). During the simulations, the number of shell-AuNPs comprised within networks exhibiting dcenter−shell = 7.0 and 8.5 nm, respectively, was varied, which revealed differences in the shape of the PDDF. The simulated data giving the best agreement with the experimental data was found for networks comprising eight shell-AuNPs for DNA-1/2−AuNP networks and nine shell-AuNPs for DNA-3/4−AuNP networks. The excessive single particles present in a 1:20 reaction mixture are not included in the simulations, which leads to the reduced intensity of the particle peak at low r values in the simulated PDDFs in Figure 3. To support the assumption that the shell is spatially saturated with this number of particles, Monte Carlo simulations for the PDDFs of 1:50 reaction mixtures for both network species have been performed and can be seen in the Supporting Information, Figure SI8. Since the same numbers of shell-AuNPs per core−shell network are obtained from the best fits for the 1:50 reaction mixtures, these simulations underline the fact that already at a reaction mixture of 1:20 the network shell seems saturated. Looking at these model networks (eight shell particles for DNA-1/2−AuNP networks and nine shell particles for DNA-3/ 4−AuNP networks), a mutual surface-to-surface interparticle distance between neighboring shell particles of dshell−shell = 11.9 and 12.2 nm, respectively, is determined taking into account the network circumference. The noncomplementarily functionalized DNA−AuNPs involved in dshell−shell experience mostly repulsive interactions due to their polyanionic charge and steric hindrance due to the wider dimension. The charge is overcompensated by attractive hydrogen bonds in Watson− Crick base pairing, which is involved in dcenter−shell.5 Thus, it indeed seems plausible that dshell−shell is larger than dcenter−shell = 7.0 and 8.5 nm, respectively. Since dshell−shell is almost equal for the DNA-1/2 and DNA-3/4 core−shell networks, the effectively higher charge of DNA-3 and DNA-4 single strands seems to be compensated by a larger overall network volume induced by a larger dcenter−shell providing enough space for nine rather than eight particles.

data (cf. section 3.2). For the networks synthesized in a 1:5 reaction mixture, dmax does not coincide with the RH, which is due to the limited q range accessible with our experimental setup. Therefore, the complete internal structure of these largest networks and their maximum dimension cannot be investigated in detail in here. For the reaction mixtures 1:10, 1:20, and 1:50, SAXS is able to quantify the interparticle distances and dmax is found to decrease for networks exhibiting a higher excess of one component. While single DNA− and citrate−AuNPs only show peak i in their PDDFs (not shown here), networks additionally exhibit peaks ii and iii with relative heights that depend on the overall network size. For the 1:5 reaction mixture of DNA-3− and DNA-4−AuNPs, peaks ii and iii exhibit higher values than peak i. This can be attributed to the higher contribution to the structure factor from the largest networks. From 1:5 to 1:10 to 1:20 to the 1:50 networks, the height of peak ii decreases, and peaks at higher r values become less pronounced. This leads to the conclusion that smaller networks are formed in reaction mixtures with a higher excess of one component. This conclusion is in accordance with the DLS results, where smaller hydrodynamic radii are observed for these samples. To determine the number of shell-AuNPs present in a core− shell network, Monte Carlo simulations of PDDFs have been performed for networks synthesized in a 1:20 reaction mixture. This reaction mixture was chosen due to its monodispersity displayed in DLS measurements and the internal structure being completely accessible to SAXS. To calculate the PDDF of a model network, every three-dimensional coordinate in the calculation domain was, in a first step, associated with a value of ρ = 1 when lying inside a gold nanoparticle, or with a value of ρ = 0 when lying outside a nanoparticle.43 These values represent the normalized electron density at a specific point in space, assuming that X-ray scattering is much stronger for gold atoms than for water and organic materials. Therefore, points outside the gold nanoparticles were neglected as scattering centers. The next step was to randomly select two different points in space, points i and j, with normalized electron densities ρi and ρj, to calculate their mutual distance dij and the product of the associated normalized density values ρdij = ρi·ρj and to repeat this step for all other point pairs. By summing up ρdij for a specific distance interval, a pair distance distribution function was determined. As a result of the Monte Carlo simulations, core−shell interparticle surface-to-surface distances of 7 and 8.5 nm (for DNA-1/2− and DNA-3/4−AuNP networks, respec7180



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3.4. OES and Mie Calculations. Another ensemble technique to investigate DNA−AuNP networks is OES, which yields the plasmon peak position λmax. λmax depends on the number and mutual distance of AuNPs within each nanoparticle network. Therefore, λmax is a measure of the aggregation state of the AuNPs in solution. By comparing the first derivatives of the optical extinction spectra shown in Figure 4 of a single-nanoparticle solution and a solution containing

An analogous model for networks synthesized from DNA-3and -4-functionalized AuNPs is presented in the Supporting Information (Figure SI10). Due to the longer double-stranded DNA (ds-DNA) bonds, these networks can be modeled with approximately nine shell particles. Again, the calculated extinction spectra agree well with the experimental OES data. The terminated network growth, monitored by the development and saturation of the red-shift of λmax with progressing time after combining the complementarily functionalized components, is plotted in Figure SI1 in the Supporting Information for the hybridization of DNA-1− and DNA-2− AuNPs in different ratios of the components. As expected, a more pronounced red-shift of λmax for low excesses than for high excesses is observed when the network formation is completed. With regard to the reaction mixture of 1:50, it seems likely that many unbound DNA−AuNPs are present in the solution, since λmax is close to the value λmax = 522 nm for single DNA−AuNPs. The λmax values of the 1:5, 1:10, and 1:20 ratios, on the other hand, indicate defined networks which seem to have terminated their growth after approximately 600 min. Here, the plasmon peak positions are λmax = 532 nm for a ratio of 1:5, λmax = 525 nm for 1:10, and λmax = 524 nm for 1:20. Values of λmax = 534 nm for a ratio of 1:5, λmax = 527 nm for 1:10, λmax = 525 nm for 1:20, and λmax = 523 nm for 1:50 are obtained for networks consisting of DNA-3− and DNA-4− AuNPs one day after synthesis. 3.5. Summary of the Discussion. The combination of different experimental techniques enables us to carry out a comprehensive analysis of the structure of finite-sized DNA− AuNP networks. DLS is sensitive to the network size, shape, and dispersity, especially when performed at low scattering angles. The DLS data discussed above show that spherical, almost monodisperse networks with RH values of ca. 30 nm are found for reaction mixtures between 1:15 and 1:20. Thus, a core−shell formation is likely for these reaction mixtures. However, DLS does not provide information on the internal structure of the networks. Therefore, SAXS in combination with Monte Carlo simulations was employed, which allowed determining the interparticle distances present in networks with core−shell architecture. The internal structure of the core− shell networks is based on a center-AuNP surrounded by ca. 10 DNA-tethered shell-AuNPs, with the exact number depending on the interconnecting DNA double strand. Experimental OES data and spectra computed with generalized Mie theory are consistent with these results. 3.6. In Situ Liquid-Cell Energy Filtered TEM Imaging. To image the proposed structure of the core−shell AuNP networks, we applied in situ liquid-cell TEM. By imaging the particle networks in liquid, uncertainties in the interpretation of TEM images caused by drying artifacts can be avoided. However, one has to keep in mind that the image resolution is strongly dependent on the sample thickness, although energy filtering can, to some extent, increase image quality. A sample with a reaction mixture of 1:20 was chosen for in situ-TEM analysis to visualize samples exhibiting the model structure proposed in section 3.5. The high contrast of gold in electron microscopy makes it possible to image small particles up to relatively large sample thicknesses. The local sample thickness of the probed cell volume can be estimated from the most probable loss (MPL) energy in the plasmon peak,48 which was in the range of 600 eV and corresponds to several micrometers between the membranes of the cell. Clusters of aggregates are seen. The micrographs (a, b) and the size distribution plotted

Figure 4. (a) Comparison of experimentally determined extinction spectra of single particles (cyan curve) and a 1:20 reaction mixture of DNA-1- and DNA-2−AuNPs (blue curve) with simulated spectra based on generalized Mie theory (gray curve for single AuNP, black curve for networks). The spectral positions of the experimental extinction maxima were determined from the first derivative of the spectra, yielding λmax values of 522 nm for the single particles and 524 nm for the networks. (b) Model of the nanoparticle positions in DNA1/2−AuNP networks according to experimentally obtained interparticle distances from SAXS for an idealized network comprising eight shell particles surrounding a center-AuNP.

AuNP networks (1:20 reaction mixture, DNA-1/2 networks), a small difference in the position of the plasmon resonance peaks (cyan and blue curves in Figure 4a) is observed. Due to mutual electromagnetic coupling of the nanoparticles in the networks, the plasmon peak position λmax is slightly shifted to larger wavelengths and the line width is increased with respect to the plasmon peak of the single-nanoparticle solution. Here, the redshift is ca. 2 nm, from λmax = 522 nm to λmax = 524 nm. Using generalized Mie theory, it is possible to calculate the extinction cross sections Cext of nanoparticle networks consisting of ideal spheres.46,47 Figure 4b shows the above suggested core−shell network model consisting of one core particle and eight shell particles. The calculated extinction spectra for this model, whose core−shell interparticle distance matches the experimentally determined dcenter−shell, and for a single particle are shown in Figure 4a. The calculated spectra qualitatively reproduce the measured extinction spectra. Since the generalized Mie calculations consider the particles as perfectly spherical and neglect inhomogeneous broadening, the calculated plasmon peaks appear sharper. The calculations show a shift of 4 nm in λmax from λmax = 522 nm to λmax = 526 nm as compared to the single particles. In the experimental spectra, this red-shift is reduced to a value of 2 nm. This is due to the fact that the measured spectrum is an average over the signals from the networks and single particles, which are also present in the solution. In Figure SI9 (Supporting Information), the calculated λmax are plotted in dependency of the number of particles nAuNP in a DNA-1/2−AuNP network (center particle + shell particles). Due to enhanced plasmonic coupling of shell-AuNPs with decreasing interparticle distance, higher values of λmax are observed with increasing nAuNP per network. The λmax obtained for a network comprising nine AuNPs (526 nm) agrees well with the experimental data (524 nm), taking into account that also single particles are present in the solution. 7181



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Figure 5. In situ liquid-cell TEM images of DNA-1/2−AuNP networks from a 1:20 reaction mixture with diameters in the range of 60 nm: micrographs (a, b) and histogram (c). At this resolution, no single particles could be imaged.

Statistical analysis of the network diameters, represented by the size histogram in Figure 6b, shows good agreement with the network sizes evaluated from lower resolution images (such as those in Figure 5) as well as with data obtained by DLS and SAXS. A detailed analysis of the different interparticle distances as was carried out for the SAXS data is not straightforward for 2D projections of 3D networks, such as the one imaged here, and also not possible with high accuracy with the current image quality. Due to organic residues in the buffer solution that hinder high sample resolutions, all images were acquired from 10-folddiluted samples in DI-water. Under these conditions, the DNAhybridization efficiency and thus the melting temperature of the networks are expected to be lower than in the otherwise used buffer solution due to decreased ionic strength. Nevertheless, under irradiation, no change in structure during the course of measurements was observed. Thus, we consider in situ-TEM to be a nondestructive method to investigate our samples in their native state in solution.

in a histogram (c) in Figure 5 strongly suggest the presence of particle networks. The diameter is about 60 nm, which corresponds to 3 times the AuNP diameter including the dsDNA. Conventional, high-resolution TEM in the dried state showed no single particles larger than 30 nm. Therefore, within the accuracy of the measurement, the mean network size agrees well with the assumed core−shell network model. However, for the given thickness of the aqueous layer in the sample, no individual AuNPs could be distinguished and no single AuNPs could be imaged. Thus, the structures imaged in Figure 5 correspond to clusters of AuNP networks, in which individual networks have an approximately spherical appearance. All structures observed here were adsorbed on the Si3N4 membrane. Freely diffusing single particles, though sometimes observed in search mode, show translational speeds which would require image acquisition times that are far from practical with moderate beam currents. High beam currents, however, induce charging of the sample, quickly leading to a removal of the particle networks from the observed volume. Individual AuNPs could be resolved in the networks for relatively thin samples with a MPL energy of about 200 eV, which can be achieved at the very edge of exactly aligned Si3N4 windows. The particles and particle networks are in the expected diameter range of ca. 15 nm for particles and 60 nm for networks. A tendency of network aggregation can be observed, as is already seen in Figure 5 in clusters of ca. 60 nmsized aggregates. Figure 6a shows an enlarged section from an

4. CONCLUSIONS In conclusion, we have demonstrated the synthesis of spatially defined DNA−AuNP networks and their characterization by means of DLS, SAXS in combination with Monte Carlo simulations, OES in conjunction with Mie calculations, and in situ-TEM. By varying the molar concentration ratio of two sets of complementarily functionalized DNA−AuNPs, hydrodynamic radii ranging from 160 to 18 nm were measured with DLS. Using SAXS in combination with Monte Carlo simulations, two characteristic interparticle distances in small networks have been identified. Their values depend on the precise composition of the networks and are controlled by the linking double-stranded DNA. By controlling the network morphology, the model structures that give the best fits to the SAXS data have core−shell geometries in which the interparticle distance dcenter−shell was found to be significantly smaller than dshell−shell, supposedly due to electrostatic repulsion and steric hindrance of the polyanionic ssDNA. The number of shell AuNPs is quantified to be ca. 10. According to the experimental data, a larger number of particles is needed during network synthesis than the number of shell particles which fit around a center particle. For instance, to obtain a center-AuNP surrounded by ca. 10 shell-AuNPs, a reaction mixture of 1:20 is needed. Having investigated the internal and external structure of finite-size DNA−AuNP assemblies in the present study, a purification of the networks from excessive single DNA−AuNPs and an investigation of the kinetic stability of isolated networks are of interest for future work. One way to achieve this goal could be purification via

Figure 6. In part a, an enlarged image section from the TEM image series presented in the Supporting Information (Figure SI11) is shown. Individual particles can clearly be distinguished. Part b shows a statistical evaluation of the network diameters observed at an MPL energy of about 200 eV.

image series (Figure S11, Video S1 (jp412283q_si_002.mpg), Supporting Information) of a small NP network, which adsorbed and desorbed multiple times to and from the membrane during observation, thus diffusing in and out of focus and assuming different orientations. Careful analysis of the images reveals a network of about seven nanoparticles. 7182



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flow-field flow fractionation (F-FFF), which is based on the potentially different mobilities of single particles and networks under exertion of a force perpendicular to the direction of the original flow. Another method could be the use of an analytical ultracentrifuge, which enables separation of components via their differences in sedimentation coefficient.49,50 The knowledge on the spatial distribution of AuNPs within networks will help to improve DNA−AuNP networks as nanoplasmonic functional materials in future applications.

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ASSOCIATED CONTENT

S Supporting Information *

Further description of the material: Network growth monitored by OES, detailed DLS measurements of all materials, Monte Carlo and Mie simulations, in situ-TEM image series, and a video. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +49 (0)241 80 99 003. Phone: +49 (0)241 80 94 644. Author Contributions

The manuscript was written with contributions from all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank D. Carmele for helpful discussions of the SAXS data. Further, the authors would like to thank the German Research Foundation (DFG; Deutsche Forschungsgemeinschaft) Graduate School “Biointerface” (Nr. 1035), the Excellence Initiative of the German federal and state governments and the Collaborative Research Center “Functional Microgels and Microgel Systems” (SFB 985) for financial support.

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