Synthesis and Properties of Gold Nanoparticle Arrays Self-Organized

Article pubs.acs.org/cm

Synthesis and Properties of Gold Nanoparticle Arrays Self-Organized on Surface-Deposited Lysozyme Amyloid Scaffolds Olivier Deschaume, Bert De Roo, Margriet J. Van Bael, Jean-Pierre Locquet, Chris Van Haesendonck, and Carmen Bartic* Laboratory of Solid State Physics and Magnetism, KU Leuven, Celestijnenlaan 200D, BE-3001 Leuven, Belgium S Supporting Information *

ABSTRACT: In this study, amyloid fibers prepared from hen egg white lysozyme (HEWL) are specifically mediating the assembly of citrate-capped gold nanoparticles, on glass and silicon substrates. The organization of nanoparticles is investigated for nanoparticle diameters of 5, 15, and 25 nm, using variable deposition times, and under a range of pH, salt, citric acid and nanoparticle concentrations. The observed periodic self-organization of nanoparticles is mainly influenced by the interparticle interactions rather than by the spacing of binding groups at the surface of the amyloid fiber template. For a fixed ionic strength of 2.3 mM and particle concentration, the interparticle distance increases with the nanoparticle diameter in agreement with the values predicted by the Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory. UV−visible spectroscopy measurements show a red shift of the 520 nm plasmon absorption peak associated with spherical gold nanoparticles up to 650 nm upon aggregation or decrease in the interparticle distance. Such protein templates deposited on technologically relevant surfaces allow the self-assembly of inorganic nanoparticle arrays with functional optoelectronic properties.

■

INTRODUCTION Bioinspired and bioinorganic materials chemistry offers unique opportunities to produce and manipulate structures at the nanometer scale, under mild conditions and using minimal amounts of precursors.1 In particular, the soft and reversible interactions characteristic of natural biomineral systems allow a wide flexibility, but also a surprising specificity, in designing hybrid architectures made from building blocks with precisely defined grain sizes, shapes and crystalline orientations.2 Because of all these advantages, bioinspired materials chemistry is a perfect complement, and a potential alternative to traditional top-down nanofabrication techniques. The rapid development of plasmonics and metamaterials has further increased the interest in controlled arrangements of nanoparticles on biological templates.3−5 In such systems, the plasmonic properties of the material can be tuned by modifying the periodicity and orientation of nanoparticle assemblies, or by using particles having different shapes and sizes.6,7 The resulting properties can be predicted with excellent accuracy for the development of devices such as biosensors or waveguides. The toolbox of biological templates and self-assembly techniques available to produce novel materials targeting specific applications is growing each day, incorporating scaffolds based on DNA,4,8 proteins9 or virus capsids.10,11 Among potential biological templates, amyloid fibers are a particularly attractive class of protein or peptide assemblies, with excellent © 2014 American Chemical Society

mechanical and chemical resistance allied to dimensions at the nanometer scale.12 These natural templates have therefore been extensively investigated for the preparation of metal nanowires,13 among other applications.14 Amyloid fibers are characterized by a periodic arrangement of monomeric protein units. This periodic structure allows for the formation of precise arrays of functional groups suitable for selective particle binding in solution. Lysozyme is an amyloid-forming protein with high solubility and isoelectric point of 11.35, reflecting the large abundance of basic amino acid residues in its structure.15,16 Lysozyme fibers have been investigated as templates for silver nanowire synthesis,17 and have also been combined with quantum dots with the aim of studying fibrilisation.18 In this work, we demonstrate the ability of lysozyme amyloid fibers to specifically mediate the assembly of citrate-capped gold nanoparticles, directly onto a range of silicon oxide-based surfaces. Regular arrays of gold nanoparticles of different sizes have been assembled using amyloid fibers of a few nanometers diameter. The lysozyme fibers are generating self-assembled nanodomains that are electrostatically contrasting with the background substrate. The electrostatic nature of the assembly mechanisms offers a wide flexibility in tuning, predictable Received: July 18, 2014 Revised: September 2, 2014 Published: September 2, 2014 5383

dx.doi.org/10.1021/cm502652t | Chem. Mater. 2014, 26, 5383−5393

Chemistry of Materials

Article

168−192 h of heat treatment showed the best potential (longest fibers, low aggregation and bundling) for nanoparticle deposition experiments. Fiber batches selected for further experiments were dialyzed in pH 2 HCl for 2 days using 50 kDa dialysis membranes to enable the removal of monomeric protein units, and diluted twice in the pH 2 HCl solution to obtain the desired surface coverage. Five to ten microliters of the resulting solution was deposited on silicon and glass substrates, respectively, before being blown dry with a nitrogen flow after 30 min of incubation. To avoid drying during the incubation, we placed the samples in a Petri dish with a small amount of ultrapure water. Metal Particles Synthesis. Citrate-capped gold nanoparticles with mean diameters of 4.7, 14.8, and 24.8 nm were synthesized using variants of the Turkevich method.24−28 The nanoparticle sizes are respectively rounded to 5, 15, and 25 nm in the rest of the text for the sake of clarity. Briefly, 25 nm particles were obtained by pouring the metal precursor solution (5 mg of HAuCl4.3H2O in 9.3 mL of ultrapure water) in boiling sodium citrate solution (72 mg of Na3C6H5O7.2H2O in 50 mL of ultrapure water), and heating the mixture for 30 min before cooling down to room temperature. Fifteen nanometer particles were obtained by pouring the citrate salt solution (50 mg in 5 mL of water) into boiling metal precursor solution (10 mg in 100 mL of water), then maintaining heat for 30 min before cooling down gradually to room temperature. Five nanometer particles were obtained without heating, using citrate as a capping ligand, and sodium borohydride for precursor reduction. 0.6 mL of 0.1 M ice-cold sodium borohydride solution was mixed under efficient stirring into 20 mL of the metal precursor/citrate solution (containing 1.47 mg of Na3C6H5O7.2H2O and 1.96 mg of HAuCl4.3H2O). The resulting particles were measured by means of transmission electron microscopy (TEM), and images analyzed using ImageJ29 to obtain size distributions (see Figure S1 in the Supporting Information). Circular Dichroism Spectroscopy (CD). For CD characterization of lysozyme during fiber formation experiments, 3 μL of sample taken from the heated bath at regular intervals of time was diluted in the sodium azide solution used for protein solution preparation and CD baseline acquisition.30 Spectra were acquired using a Jasco 800 CD spectrometer between 250 and 185 nm, using a 20 nm/s scan rate, 1 nm resolution and continuous scan. The resulting spectra were deconvoluted for secondary structure determination using the dichroweb service31,32 with basis set 4 and Contin fitting algorithm.33 Ultraviolet−Visible Spectroscopy (UV−Vis). Single and multiple wavelength measurements were obtained on a GE healthcare Ultrospec 2100 Pro spectrophotometer, using a 1 nm scan step and a scan rate of 250 nm/min. Atomic Force Microscopy. An Agilent 5500 AFM system with MSNL-F cantilevers (f = 110−120 kHz, k = 0.6 N/m) with average tip radius of 2−12 nm was used for the morphological imaging. The height of gold nanoparticles measured by AFM was comparable with the one obtained from TEM characterization (see Figure S1 in the Supporting Information). The lateral dimensions of the particles, together with the dimensions of the bare fibers were used to check for tip convolution effects. The AFM topography images were leveled, line-corrected and measured (height profiles and histograms) using Gwyddion, a free and open-source SPM (scanning probe microscopy) data visualization and analysis program.34 2D radial distribution functions (RDF) were obtained using VMD35 and ImageJ software in order to determine the separation and periodicity of particles arrangement using 2.5 and 5 μm size images. For comparison, RDFs were also obtained from simulated images designed using Gwyddion, in which particles were distributed randomly or with perfect periodicity along fibers. Metal Particle Binding Conditions. For the first set of experiments, nanoparticle suspensions were diluted twice using ultrapure water, 10 mM hydrochloric acid, 1 mM citrate buffers with pH between 3 and 6, or 100 mM MES buffer with pH 6.5 to reach the desired deposition conditions. For the second set of experiments, the 15 nm gold suspension was purified by dialysis with 1 mM citrate buffer with pH 3.5, 4, 5, or 6, and with 0.1 mM citrate buffer for the deposition experiment at low citrate concentration. Salt concentrations

interparticle distances, and a potential extension to a large range of nanomaterials and biological templates. This electrostatically driven self-assembly mechanism differs from more specific approaches, based on the design of biomolecular templates from DNA and additional building blocks to generate nanoparticle assemblies with predetermined interparticle distances.19−21 Using a single biological template, we take advantage of an assembly mechanism able to control the formation of tunable nanostructures with properties depending mainly on the nanomaterial and processing conditions used. This concept is demonstrated by tuning materials using different deposition parameters (time, pH, ionic strength, nanoparticle concentration) and nanoparticle surface charge and size. We have investigated the morphology of the gold-lysozyme assemblies and their optical properties by atomic force microscopy (AFM) and UV−visible spectroscopy, respectively. The results illustrate that the assembly process is driven mainly by the interparticle interactions as also predicted by calculations based on the Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory.22,23

■

EXPERIMENTAL SECTION

Reagents and Surface Cleaning. Hen egg white lysozyme (HEWL, >90%, 40000 units/mg) and sodium azide (99.5%) were purchased from Sigma. Citric acid (>99.5%) was from Appli Chem, and trisodium citrate (99%), together with sodium borohydride (99%), were from Acros. Gold chloride trihydrate (>99.9%) was purchased from Aldrich. 2-(N-morpholino)ethanesulfonic acid (MES), 1 N hydrochloric acid and concentrated hydrochloric acid (37%) were obtained from Fisher chemicals. Sulfuric acid (97%), hydrogen peroxide (30%) and nitric acid (65%) were from Chem Lab. Sodium hydroxide (99%) and sodium chloride (99.7%) were from VWR, together with the 2.5 cm diameter glass coverslips (0.13 mm thickness) used as substrates. Ultrapure (UP) water was produced with a Sartorius Stedim Arium Pro VF system. One side-polished, 1 cm square doped silicon (100) substrates were cleaned for 30 min in piranha solution (H2SO4 (98%) H2O2 (30%) 2/1 v/v) (Warning! Piranha solution is a powerf ul oxidant, and should only be handled with adapted equipment and training), rinsed extensively in ultrapure water and dried under a nitrogen flow before use. As described in the discussion section, the glass coverslips were also initially subjected to the same cleaning protocol. For later experiments we first cleaned the coverslips for 30 min in aqua regia (HCl (37%) HNO3 (65%) 3/1 v/ v) to remove inorganic contaminants, followed by a rinsing in ultrapure water and the piranha cleaning protocol. Amyloid Fiber Preparation and Deposition. Lysozyme powder was dissolved in a 200 ppm aqueous sodium azide solution adjusted to pH 4 with HCl 1 N to reach an approximate concentration of 25 mg/ mL. The same sodium azide solution was also used for dialysis and concentration adjustments. HEWL solutions were purified by dialysis in 7 kDa tubing (Membra-Cel) with frequent changes of the pH 4 sodium azide dialysis solution. The concentration of the freshly purified HEWL solution was determined using UV−vis absorbance measurements at 280 nm and assuming an extinction coefficient of 2.65 L g−1 cm−1. The concentration was adjusted to 10 mg/mL by dilution with sodium azide solution, whereas the pH was set to 2 using small additions of 1 N HCl. Lysozyme fibers were obtained by heating portions of 10 mg/mL solution in eppendorf tubes placed in a water bath at 60 °C for up to 2 weeks, the fibril formation process being monitored using atomic force microscopy (AFM) in air and circular dichroism spectroscopy (CD) in solution. To monitor fiber growth with AFM, we took samples every 24 h from the heated batch and deposited and incubated 5 μL aliquots for 1 h on clean silicon substrates, before blowing dry with a flow of nitrogen, rinsing with ultrapure water, and drying again with nitrogen so as to avoid AFM tip contamination with loose protein monomers. Fibers obtained after 5384



Article

Figure 1. Fifteen nanometer diameter gold nanoparticle arrangements onto lysozyme-based amyloid fibers attached to silicon substrates, using nanoparticle suspensions diluted in (a) HCl pH 2, (b) ultrapure water, and (c) MES buffer 0.1 M pH 6.5.

water, HCl pH 2 (same conditions as for fiber deposition), or 100 mM MES buffer pH 6.5, which is commonly used for biochemical reactions and can be used for nanoparticle functionalization.37 Figure 1 shows the result of the binding of nanoparticles to lysozyme fibers using these initial conditions. On silicon, the particles bind to fibers with low selectivity when compared to the substrate background and low density along the fibers when deposited from HCl and 100 mM MES buffer. In HCl (pH 2), particles were already visibly destabilized after a day of storage, mostly because of the deprotonation of the citrate capping ligands. In the presence of MES buffer, the observed low selectivity/coverage observed arises from the high ionic strength of the solution, which screens the repulsive electrostatic potential between the silicon oxide and the citratecapped nanoparticle surfaces, together with the attractive electrostatic potential between the protein fibers and nanoparticle surfaces. The results obtained using ultrapure water dilution were far more promising, with an excellent binding selectivity toward the fibers, and a dense, periodic arrangement of particles along the biological templates. Initial tests on other substrates revealed lower selectivity. We therefore hypothesize that binding selectivity is related to the contrasting surface charges of the substrate and fiber, with a negative charge on the silicon oxide, and an array of regularly spaced, positively charged sites along the lysozyme templates. As observed and reviewed by different authors, the charge properties of silicon oxide surfaces are highly dependent on preparation and cleaning parameters.38 Hence the surface charge of native silicon oxide on silicon can differ substantially from that of thermally grown oxide or glass, notwithstanding inclusions of other compounds in glass. This difference becomes most evident when the charge versus pH dependence is considered. Therefore, we further evaluated the influence of variables including nanoparticle concentration and diameter, binding time, pH, ionic strength, and citrate concentration, on the self-assembly process and its reproducibility. Effect of pH and Incubation Time. In our initial experiments, gold nanoparticles with a diameter of 15 nm were adjusted to pH between 5 and 6 by dilution in 1 mM citrate buffers without further purification. The nanoparticles were deposited on glass substrates that were cleaned with a

were adjusted using dilute NaCl addition. For 5 nm gold nanoparticles, the colloidal stability was insufficient to achieve purification without aggregation; the suspension was therefore diluted in citrate buffers to reach the desired nanoparticle concentration and pH. For 25 nm gold, the deposition conditions and nanoparticle concentration were adjusted using three steps of centrifugation and redispersion of the pellet in the deposition buffer. With the exception of variable incubation time experiments, the resulting nanoparticle suspensions were deposited on lysozyme fiber-modified surfaces (5 μL for silicon substrates, 10 μL for glass substrates), and the surfaces were rinsed in a flow of ultrapure water after 1 min incubation, before being dried in a slow nitrogen flow.

■

RESULTS AND DISCUSSION Fiber Selection. The fibrilization process monitored by means of AFM and CD was in agreement with previous studies.16 AFM demonstrated the onset of fiber formation after 48 h of incubation at 60 °C, followed by a slow growth of fibers until 192 h, after which fibers tended to aggregate and break down. The structural reorganization characteristics of amyloid fiber formation was verified by CD, with a decrease in the relative amount of α-helical structures, correlated with an increase in the proportion of β-sheet structures during the first 200 h of heat treatment. Protein fiber samples obtained between 168 and 192 h of heat treatment at 60 °C were selected for nanoparticle binding experiments, as they contained a large density of non aggregated fibers with lengths exceeding 1 μm, and diameters of 3 nm and more rarely 6 nm (bundled fibers). More homogeneous distributions of fiber lengths have been obtained by other groups, for example using seeded growth starting from ultrasonicated amyloid fibers (e.g., lysozyme and insulin) and adding fresh protein monomers to study, for example, the kinetics of fiber formation.36 However, the use of a narrow length distribution of biological fibers is not the main focus of this study. Moreover, we will see that some degree of variation in fiber morphology enables one to observe different cases of particle arrangement along the template within a single sample, which can be useful in guiding the future design of specific nanostructures. Nanoparticle Arrangement on Surface-Deposited Lysozyme Fibers. The binding of nanoparticles with different sizes to protein fibers on silicon substrates with a native oxide layer and on glass coverslips was verified by AFM imaging. The nanoparticle suspensions were diluted to a 1:1 ratio in ultrapure 5385



Article

Figure 2. AFM topography images of 15 nm gold nanoparticles assembled on lysozyme fibers attached to glass substrates at pH between 5 and 6.

single piranha solution treatment and coated with lysozyme fibers. The relatively low citrate concentration was chosen so as to maximize electrostatic interactions (surface−fiber, surface− particle, fiber−particle, etc.) and therefore favor the selfassembly process while minimizing random particle aggregation. Moreover, 1 mM citrate is close to the citrate concentration obtained during the first experiments by diluting the gold particles suspension in ultrapure water (∼0.81 mM). As can be seen from Figure 2 (see Figure S5 in the Supporting Information for a wider field of view), nanoparticles decorate similarly the biological scaffolds for pHs between 5 and 6. The deposition on glass also enabled us to easily measure the global UV−vis absorption spectrum of the metalized fibers (see Figure S2 in the Supporting Information), which reveals an absorption peak centered around 520 nm, characteristic of gold nanoparticles that are sufficiently separated to experience little or no influence from their nearest neighbors. A few nanoparticles appeared to bind nonspecifically to the substrate, but we will see that this effect may also be due to the cleaning protocol used for this experiment or to the presence of some oligomeric lysozyme species underneath the particles. Following the information provided by the AFM topography images we investigated more quantitatively the interparticle spacing and periodicity by computing the radial distribution function (RDF) of the particle centers using 5 × 5 μm2 images (1024 × 1024 pixels). For more clarity, the experimental RDFs are compared with RDFs obtained from computer generated arrangements of particles (Figure 3). For a random distribution the probability to find a neighboring particle at a specific distance is relatively low, and quickly drops as the distance is increased. On the other hand, for a periodic distribution of objects along an axis, the probability density is high and shows a periodic function. In the experimental data a peak was observed between 25 and 30 nm for nanoparticles deposited on glass-bound lysozyme fibers between pH 5 and 6. This value corresponds to the average distance between particle centers observed along fibers. Secondary peaks were visible for some samples, e.g. for a deposition at pH 6 (Figure 3) and other samples presented in the Supporting Information (Figure S3).

Figure 3. Radial distribution functions of particle centers along lysozyme fibers, with examples of real (left inset picture) and simulated (right inset picture) particle arrangements.

However, small variations in interparticle distance broadened and merged secondary peaks into a decaying curve in many other cases. Differences in particle arrangement periodicities as a function of small variations in fiber bundling/charging, and the existence of closely associated fibers, can also blur the periodic variation when compared to the ideal case. A closer inspection of 5 × 5 μm2 AFM images indeed shows that particles are following two main patterns with respect to the periodicity of their arrangement on the biological scaffolds. Typical cases of dense and lower particle coverages, together with the bare fiber height profiles taken in regions of the fibers free of nanoparticles are presented in Figure 4. The most densely decorated constructs correspond to significantly thicker fibers (3 to 6 nm versus 2 to 3 nm for fibers with low particle coverage), probably produced by the bundling of single 3 nm diameter fibers. The higher particle density on bundled fibers can be explained in terms of two complementary mechanisms. The bundling leads to the formation of denser arrays of positively charged groups to which particles can bind. In addition, the repulsive force exerted by the surface on the particles is screened to a greater extent, which enables more particles to approach the scaffold and bind to it. 5386



Article

Figure 4. Effect of fiber bundling on nanoparticle binding to lysozyme scaffolds: (a) Average height profiles (over 7 measurements for different pHs) taken on bare regions of fibers carrying a dense or sparse nanoparticle coverage, and (b) corresponding AFM image insets from samples prepared at three different pHs. The scale bar is 100 nm long, the color scale is the same as for Figure 2

In a second series of experiments, the pH of the 15 nm particle suspensions was adjusted over a wider range, and the suspensions were purified by dialysis in 1 mM citrate buffers at pH 3.5, 4, 5, and 6. At pH 3.5, the main form of the ligand present in solution is citric acid, whereas the fully deprotonated form should prevail at pH 6. Above pH 6, the stability of lysozyme fibers on surfaces might be compromised by the deprotonation of both acidic and basic amino acids which ensure their adhesion to the substrates. Hybrid bioinorganic fibers were prepared in parallel onto glass and silicon (with native oxide layer) substrates, the glass surfaces being subjected to an additional cleaning step with aqua regia to remove possible inorganic contamination before piranha cleaning. In addition to pH, the effects of other parameters such as nanoparticle concentration and ionic strength were studied using the same experimental approach/surface preparation. In the following sections, the discussion will be illustrated with AFM images acquired in parallel on silicon and glassdeposited samples. In addition, UV−visible spectra were acquired using glass-deposited samples so as to obtain a more global picture of the nanoparticle coverage, and pinpoint eventual effects of interparticle coupling on the plasmon absorption characteristics of the hybrid constructs. AFM images acquired for a deposition pH of 3.5 are not presented, because of the extensive nanoparticle aggregation observed on surfaces, leading to imaging artifacts and tip contamination. The extensive particle agglomeration at this pH value could be expected due to the low charging of capping ligands on surfaces and in solution, and was also evidenced by a shift of the 520 nm plasmon absorption peak characteristic of isolated nanoparticles up to 550 nm. At pH 4 the binding selectivity toward the fibrilar scaffold was excellent, but the nanoparticle arrays were disorganized due to weak repulsive interactions between particles (Figure 5). The close proximity of nanoparticles along the template gave again rise to a broadening/

Figure 5. (a) Effect of pH on surface topography/scaffold coverage by 15 nm particles after 1 min of deposition from solution; UV−vis absorbance spectra of the hybrid material obtained using (b) 15 and (c) 25 nm gold nanoparticles. The color scale of AFM images is the same as for Figure 2

shifting of the plasmon absorption peak as compared to results obtained at higher deposition pHs (Figure 5b). A pH of 5 stands out as the optimal pH value for the formation of regular nanoparticles arrays on both silicon and glass, and led to the best overall coverage as seen from UV− visible measurements, with no shift of the plasmon absorption peak position from 520 nm. Nanoparticle deposition at pH 6 gave similar results as for pH 5, however the absorption peak at 520 nm decreased slightly and the particles appeared to bind preferentially to areas with high fiber densities. This is expected because repulsive interactions between the surface and the particles are the strongest at this pH, and binding is facilitated in areas where the lysozyme is screening the substrate charge in the most efficient manner. 5387



Article

substrate, the fiber coverage by nanoparticles was already high for contact times as short as 10 s, and the maximum coverage was obtained after 1 min. On the other hand, when glass was used, only few particles were observed on each fiber after 10 and 30 s, and arrays of nanoparticles with regular spacings could be obtained only after 30 min of incubation. These substrate-dependent effects can be explained by the more negative surface charge of glass when compared to native oxide formed on silicon substrates at a given pH, as reported by others.38 After reaching their maximum observed coverage by nanoparticles, fibers partially or fully detach from the substrates, possibly as a result of the increase in repulsive interactions between the bound nanoparticles and the substrate. This detachment enables the nanoparticles to reach fiber regions previously bound to the substrate, leading to denser arrays on both substrates after longer incubation times. The increase in the fiber-bound particle density, to the point of contact between neighboring nanoparticles, is reflected by the broadening of the plasmon absorption peaks for incubation times longer than 1 min. Effect of Salt, Citrate and Nanoparticle Concentration. In order to tune the positioning of nanoparticles along templates and modify the interparticle distance, changes in the electrostatic forces driving self-assembly can be induced by varying the citrate and salt concentrations of the nanoparticle suspensions. Considering nanoparticles of 15 nm diameter at a concentration of 1.16 × 10−9 M, and a surface occupancy of 0.3 nm2 per adsorbed citrate,39 the concentration of citrate ions should lie between 36 and 3600 times the concentration required to reach a complete particle coverage, for concentrations between 0.1 and 10 mM citrate. Therefore, the ligand depletion effects on the surface of the nanoparticles should be low under the conditions tested, and the effect of citrate concentration should be similar to that of salt concentration. We first kept the citrate concentration fixed at 1 mM, and varied the salt concentration independently up to 100 mM. In the case of 100 mM NaCl, all repulsive interactions are screened as predicted by the DLVO theory22,23 and the particles aggregate rapidly in solution. This leads to a visible

The effect of pH on particle binding was also clear for 25 nm particles adjusted by centrifugation to similar concentration as for the 15 nm particle suspension (Figure 5c for UV−vis, Figure S4 in the Supporting Information for AFM). For pH 3.5 and 4, nanoparticle aggregation and an additional plasmon absorption peak above 650 nm were observed, whereas a periodic distribution along scaffolds and a single, 520 nm absorption peak were obtained at pH 5 and 6. For 25 nm particles, the particle coverage appears higher at pH 6 from UV−vis measurements. This may be due to some degree of variability in particle binding, or because of a broadening of the absorption peak as a result of interparticle interaction/ aggregation effects at lower pHs. From the AFM characterization, it also appears that the nanoparticle coverage of fibrilar scaffolds was lower when glass was used as a substrate (Figure 5a). This effect was also significant in samples prepared with variable nanoparticle deposition time (Figures 6 and 7). When silicon was the

Figure 6. Effect of deposition time on UV−vis absorbance of the assemblies formed between lysozyme fibers and 15 nm gold nanoparticles.

Figure 7. Effect of deposition time on surface topography/biological scaffold coverage by 15 nm particles after 10 and 30 s and 10, 30, and 60 min deposition from solution. The color scale of AFM images is the same as for Figure 2 5388



Article

color change and a shift of the plasmon absorption peak to values above 600 nm (Figure 8b). At 10 mM NaCl, the

Figure 9. Effect of citrate concentration on the final morphology of scaffolds and UV−vis absorption spectrum of lysozyme fiber−gold nanoparticle assemblies. The color scale of AFM images is the same as for Figure 2

Figure 8. Effect of salt concentration on the final morphology of scaffolds and UV−vis absorption spectrum of lysozyme fiber-gold nanoparticle assemblies. The color scale of AFM images is the same as for Figure 2

silicon-deposited samples for 10C. For 0.1C, a much lower fiber coverage and little detachment of the fibers were observed (Figure 10b). Once more, the AFM images showed good agreement with UV−vis spectra, with an increase in absorbance at 520 nm correlated with the nanoparticle concentration used (Figure 10b). A small broadening of the plasmon absorption peak was observed for a concentration of 10C, as in the case of long incubation times leading to a similar particle coverage. Effect of Nanoparticle Diameter − Experiments and Modeling. The optimum deposition conditions determined for 15 nm gold nanoparticles were extrapolated to different gold nanoparticle sizes. The nanoparticle concentrations were adjusted to values similar to the ones used for 15 nm particles, by dilution in citrate buffer in the case of 5 nm particles, and by centrifugation/concentration in the case of 25 nm particles. A selective binding of the three different particle sizes on the fibers was obtained, still with a low substrate contamination (Figure 11b−d). From a visual inspection of the images, the interparticle spacing on a fiber appears to be directly proportional to the diameter of the nanoparticles. RDFs obtained with a resolution of 1 nm were fitted with exponential pulse functions (using OriginLab Origin 6.1 nonlinear curve fit tool) to define the position of the first

nanoparticle aggregation was still visible to some extent, and interparticle distances varied relatively broadly along the biological scaffolds (Figure 8a). At 1 mM NaCl no aggregation occurs in solution. On glass, an increased coverage of the fibers by nanoparticles was in particular observed, which can be attributed to the partial screening of the silicon oxide surface charge. The effect of citrate concentration was similar to that triggered by changes in salt concentration (Figure 9a, b), i.e., the formation of aggregates together with a lower fiber coverage and lower binding specificity for a citrate concentration of 10 mM. For citrate concentrations of 0.1 mM, particles were mostly accumulating around areas densely covered by fibers, where the repulsion between the surface and the particles is minimized. Nanoparticle concentrations of 0.1 and 10 times the initially tested value (0.1C, 10C, with C the initially tested concentration) were prepared by dilution and centrifugation, respectively, of a suspension of 15 nm particles in 1 mM citrate buffer at pH 5. As expected, higher surface coverages, but also a larger extent of fiber detachment was observed on glass and 5389



Article

Figure 11. Effect of nanoparticle diameter on the self-assembly with biological templates. (a) Pulse fits of radial distribution functions of particle centers calculated from AFM images of the biological scaffolds decorated with 5, 15, and 25 nm gold nanoparticles. Corresponding AFM images obtained after binding (b) 5, (c) 15, and (d) 25 nm gold nanoparticles to lysozyme fibers deposited on silicon substrates. The scale bars are 100 nm long. The color scale of AFM images is the same as for Figure 2.

to that of protein fibers used in this work.43−46 However, to the best of our knowledge, a single particle size was investigated in a vast majority of cases, and the main parameter used to vary the interparticle distance was ionic strength. The regularity of the particle arrangements obtained on the smallest surface features produced by e-beam lithography appears lower than in the current study, possibly because nanoparticles are experiencing a lower electrostatic repulsion from the surrounding substrate when binding sites on the fibers are situated 3 nm from the surface, than when they are directly bound to this surface, for example if silane or thiol layers are used to generate a chemical contrast. Moreover, these binding sites or charge domains are presented in a three-dimensionnal fashion along the biological template. Our final aim was to correlate experimental results with a theoretical model which would ultimately enable the prediction of interparticle spacing as a function of nanoparticles properties and deposition parameters. In most studies of particle adsorption on homogeneous surfaces or lithographically defined patterns of silanes or thiol layers, variations in interparticle distances are successfully interpreted with the random sequential adsorption model (RSA),47 together with DLVO theory.42,48 In the RSA model, hard spheres with a radius aeff corresponding to half of the minimum center to center distance between two particles are defined, and the pairwise particle interaction potential Upp(r) corresponding to two of such hard spheres in contact with each other is defined as

Figure 10. Effect of nanoparticle concentration on the final morphology of scaffolds and UV−vis absorption spectrum of lysozyme fiber−gold nanoparticle assemblies. The color scale of AFM images is the same as for Figure 2

probability peak (Figure 11a). (RDFs with 1 nm resolution are presented in Figure S6 in the Supporting Information before and after 5-point adjacent averaging smoothing for comparison with the fits). As hypothesized, the average distance to the nearest neighboring particle clearly and almost linearly increases with particle diameter (Figure 12a), with an interparticle spacing of 10 nm, 20 to 25 nm and 40 to 45 nm, respectively for 5, 15, and 25 nm diameter nanoparticles. This observation demonstrates that in the range of particle diameters tested, the interparticle distance is mostly determined by the interparticle interactions rather than by the particle−fiber interactions, and can therefore be modified by varying the deposition conditions. In the case of 5 nm particles, however, the interparticle distance of 10 nm probably lies close to the periodicity of monomeric proteins assembly within lysozyme fiber structure, and a visual inspection of AFM images indeed suggests a more complex binding pattern, reminding helical arrangements obtained when particles below 5 nm diameter are grown on fibers by reduction of bound precursors.40 Interparticle distances varying as a function of deposition parameters have been observed in the past with charged particles adsorbed on a range of substrate materials,41,42 and in particular on surfaces comprising nanostructures obtained by electron-beam or optical lithography, down to sizes equivalent

Upp(2aeff )/kT = 1/λ

(1)

Where kT is the thermal energy (defined by the Boltzmann constant k and the temperature T), and λ is a constant. The repulsive (Urep) and attractive (Uattr) potentials between two 5390



Article

Figure 12. (a) Interparticle distances determined by fitting experimental RDFs with pulse functions for the three particle sizes investigated (filled and hollow circles) and interparticle distances predicted by the DLVO theory for different calculation parameters (dashed and solid lines). (b) Theoretical interparticle potential (versus center to center distance) curves determined using the DLVO theory for a particle potential of −30 mV, together with different nanoparticle diameters, citrate, and salt concentrations.

particles of equal radius a, and surface potential ψ0, with centers situated at a distance r from each other, can be approximated as follows ⎛ kT ⎞2 1 Urep = 4πεa 2Y 2⎜ ⎟ e−κ(r − 2a) ⎝ e ⎠ r Uattr = −

as the particle size is decreased. Indeed, considering the interaction potential curves calculated at ψ0 = −30 mV, and λ = e as a limit for colloidal stability/aggregation, all the particles are stable in suspension for a pH of 5 and a citrate concentration of 0.1 and 1 mM. Five nanometer particles are, however, very close to this aggregation threshold, which explains the difficulty with purifying them and their fast aggregation when pH is slightly decreased below 5. For 10 and 100 mM NaCl and for a citrate concentration of 10 mM, the 15 nm suspension falls below the stability threshold, and the interval of ionic strength into which the interparticle distance will be tunable without drastic aggregation can therefore be predicted to lie between 0 and 12 mM. Effect of Sample Drying on the Assembly Process. In addition to electrostatic interactions, other processes can drive the self-assembly of nanoparticles at interfaces. In particular, capillary flows (due to solvent evaporation and temperature/ concentration gradients) can drive the migration of particles in a drop of colloidal suspension drying on a substrate. This phenomenon underlies the well-known coffee ring effect49,50 and can also be used to control the morphology of nanoparticle coatings by additionally tuning processing parameters or the properties of the substrate.51−53 Such drying process can thus drive alignments of nanoparticles parallel or perpendicular to the edge of the drop54 or pinning to surface boundaries and defects.55 Also phase transformations as a result of local changes in particle concentration may influence the assembly. Such effects are observed when drying anisotropic particle suspensions.56 In this work, however, we focused on the use of electrostatic interactions, as opposed to drying as driving force for the organization of nanoparticles on biological templates attached to surfaces. For this purpose, we minimized the effect of drying by removing unbound nanoparticles by carefully rinsing the substrates without drying of the nanoparticle suspension. The short incubation times and the rinsing steps ensure that no drying of the suspended nanoparticles occurs. As a control experiment, we also carried out the AFM characterization for a particular condition in liquid (see the Supporting Information for further experimental details), by progressively replacing the nanoparticle suspension in citrate buffer by nanoparticle-free buffer (for identical concentration, pH, and temperature to minimize the disturbance of the

(2)

AH ⎛ 2a 2 2a 2 r 2 − 4a 2 ⎞ + 2 + ln ⎟ ⎜ 2 2 6 ⎝ r − 4a r r2 ⎠

(3)

With ⎛ eψ ⎞ Y = 8tanh⎜ 0 ⎟ ⎝ 4kT ⎠

1 1+

1−

eψ 2κa + 1 tanh2 4kT0 (κa + 1)2

( )

Where e is the charge of an electron, AH the Hamaker constant, κ the inverse Debye length, and ε, the dielectric constant of the medium. The interparticle interaction potential, defined as the sum of the repulsive and attractive potentials, was calculated for the 15 nm diameter particles at variable ionic strengths (variable salt and citrate concentrations), and for the other two particle sizes (5 and 25 nm diameter), in the case of 1 mM citrate with no salt addition (Figure 12b). All calculations were done using the ionic strengths calculated at pH 5, and a Hamaker constant of 2.5 × 10−19 J was chosen, corresponding to the average value observed experimentally for gold.48 To obtain the range of interparticle distances under the different experimental conditions, the surface potential of gold was set at −45 (see Figure S7 in the Supporting Information) or −30 mV (Figure 12b) in calculations, which delimits the range observed for citratecapped gold nanoparticles in literature and for our own measurements. Moreover the value of λ used in the RSA model was varied in the range 1 < λ < e. The experimental interparticle distances are in good agreement with values predicted from the DLVO theory (Figure 12a), supporting our hypothesis of dominant interparticle−rather than template-particle−interactions determining the interparticle spacing. The same interpretation can be used to explain the breaking of periodicity of the particle chains, aggregation and tip contamination observed at increasing ionic strength, which becomes more pronounced 5391



Article

Notes

system). As predicted, the binding selectivity of nanoparticles for the biological template was similar to the results obtained by AFM imaging in air (see Figure S8 in the Supporting Information). However, further work should include the development of a quantitative model that accounts also for the wetting mechanisms.

The authors declare no competing financial interest.

■

ABBREVIATIONS HEWL, hen egg white lysozyme; DLVO, Derjaguin, Landau, Verwey and Overbeek; AFM, atomic force microscopy; MES, 2-(N-morpholino)ethanesulfonic acid; CD, circular dichroism spectroscopy; TEM, transmission electron microscopy; RDF, radial distribution function; VMD, visual molecular dynamics; RSA, random sequential adsorption

■

CONCLUSIONS We demonstrated the specific self-assembly of citrate-capped gold nanoparticles of various sizes onto lysozyme based nanofiber scaffolds deposited on glass and silicon substrates. Nanoparticles form chains along the templates with constant interparticle spacing in a pH range between 5 and 6, with a citrate concentration of 1, and without salt addition. In particular, periodic arrangements could be obtained with 5, 15, and 25 nm gold nanoparticles, with the nanoparticle density along the template being proportional to the fiber diameter. For lower pHs, and increased salt or citrate concentrations, the particle arrangement along the fibers became more random, and ultimately the fiber specificity was lost due to the reduction of electrostatic repulsion between the substrate and particles. The interparticle distances observed and calculated using radial distribution functions are determined by the electrostatic interaction between particles, and can be predicted with good accuracy using the DLVO theory. The theory was also used to predict a range of ionic strengths for which the interparticle distance could be tuned without causing aggregation. The repulsive interaction between the substrate and the citratecapped gold surface seems to be responsible for the fiber specificity of the binding, but can also lead to fiber breakdown, in particular when large particles are used. In order to optimize the production of hybrid fibers, this effect should, however, be minimized by using surface-immobilized scaffolds rather than thicker biological fibers, in order to produce chains of particles with a well-defined size.

■

■

(1) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. (2) Davis, S. A.; Breulmann, M.; Rhodes, K. H.; Zhang, B.; Mann, S. Chem. Mater. 2001, 13, 3218−3226. (3) Lalander, C.; Zheng, Y.; Dhuey, S.; Cabrini, S.; Bach, U. ACS Nano 2010, 4, 6153−6161. (4) Matczyszyn, K.; Joanna, O.-B. J. Nanophotonics 2012, 6, 64501− 64505. (5) Kitching, H.; Shiers, M. J.; Kenyon, A. J.; Parkin, I. P. J. Mater. Chem. A 2013, 1, 6985−6999. (6) Halas, N.; Lal, S.; Chang, W.-S.; Link, S.; Nordlander, P. Chem. Rev. 2011, 111, 3913−3961. (7) Noguez, C. J. Phys. Chem. C 2007, 111, 3806−3819. (8) Zheng, J.; Constantinou, P. E.; Micheel, C.; Alivisatos, A. P.; Kiehl, R. A.; Seeman, N. C. Nano Lett. 2006, 6, 1502−1504. (9) Shira, L.-S.; Noa, C.-H.; Hila, M.-D.; Wine, Y.; Freeman, A. Curr. Opin. Biotechnol. 2006, 17, 569−573. (10) Lee, Y. J.; Yi, H.; Kim, W.-J.; Kang, K.; Yun, D. S.; Strano, M. S.; Ceder, G.; Belcher, A. M. Science 2009, 324, 1051−1055. (11) Shenton, W.; Douglas, T.; Young, M.; Stubbs, G.; Mann, S. Adv. Mater. 1999, 11, 253−256. (12) Hamley, I. Angew. Chem., Int. Ed. 2007, 46, 8128−8147. (13) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X.-M.; Jaeger, H.; Lindquist, S. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4527−4532. (14) Knowles, T.; Buehler, M. Nat. Nanotechnol. 2011, 6, 469−479. (15) Krebs, M. R.; Wilkins, D. K.; Chung, E. W.; Pitkeathly, M. C.; Chamberlain, A. K.; Zurdo, J.; Robinson, C. V.; Dobson, C. M. J. Mol. Biol. 2000, 300, 541−549. (16) Arnaudov, L. N.; de Vries, R. Biophys. J. 2005, 88, 515−526. (17) Malisauskas, M.; Meskys, R.; Ludmilla, M.-R. Biotechnol. Prog. 2008, 24, 1166−1170. (18) Vannoy, C. H.; Xu, J.; Leblanc, R. M. J. Phys. Chem. C 2010, 114, 766−773. (19) Lo, P. K.; Karam, P.; Aldaye, F. A.; McLaughlin, C. K.; Hamblin, G. D.; Cosa, G.; Sleiman, H. F. Nat. Chem. 2010, 2, 319−328. (20) Geerts, N.; Schreck, C. F.; Beales, P. A.; Shigematsu, H.; O’Hern, C. S.; Vanderlick, T. K. Langmuir 2013, 29, 13089−13094. (21) Wen, Y.; McLaughlin, C. K.; Lo, P. K.; Yang, H.; Sleiman, H. F. Bioconjugate Chem. 2010, 21, 1413−1416. (22) Derjaguin, B.; Landau, L. Acta Physicochim. URSS 1941, 14, 633−662. (23) Verwey, E.; Overbeek, J.; Van Nes, K. Theory of the Stability of Lyophobic Colloids: The Interaction of Sol Particles Having an Electric Double Layer; Elsevier: Amsterdam, 1948. (24) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55−75. (25) Frens, G. Nature 1973, 241, 20−22. (26) Ojea-Jiménez, I.; Bastús, N. G.; Puntes, V. J. Phys. Chem. C 2011, 115, 15752−15757. (27) Sivaraman, S. K.; Kumar, S.; Santhanam, V. J. Colloid Interface Sci. 2011, 361, 543−547. (28) Jana, N. R.; Gearheart, L.; Murphy, C. J. Langmuir 2001, 17, 6782−6786. (29) Schneider, C. A.; Rasband, W. S.; Eliceiri, K. W. Nat. Methods 2012, 9, 671−675.

ASSOCIATED CONTENT

S Supporting Information *

Nanoparticle characterization results (TEM images and corresponding size distributions), additional AFM images for 25 nm particles organization and wide field view of templates decorated with 15 nm particles, UV−visible measurements, detail of fits for Figure 11, interparticle potential curves for a surface potential of −45 mV and protocol/results of AFM imaging in liquid. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. Funding

Part of this research was supported by a KU Leuven START grant awarded to C.B; B.D.R. and J.-P.L. acknowledge financial support from the Agency for Innovation by Science and Technology in Flanders (IWT) and from the European Union (FP7 project Snow Control); C.B. and C.V.H. acknowledge financial support from Hercules Foundation (Project HER/09/ 021). 5392



Article

(30) Kelly, S. M.; Price, N. C. Biochim. Biophys. Acta 1997, 1338, 161−185. (31) Whitmore, L.; Wallace, B. A. Nucleic Acids Res. 2004, 32, W668−73. (32) Whitmore, L.; Wallace, B. A. Biopolymers 2008, 89, 392−400. (33) Provencher, S. W.; Glöckner, J. Biochemistry 1981, 20, 33−37. (34) Nečas, D.; Klapetek, P. Cent. Eur. J. Phys. 2011, 10, 181−188. (35) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graph. 1996, 14 (33−38), 27−28. (36) Buell, A. K.; White, D. A.; Meier, C.; Welland, M. E.; Knowles, T. P. J.; Dobson, C. M. J. Phys. Chem. B 2010, 114, 10925−10938. (37) Hermanson, G. T. Bioconjugate Techniques; Academic Press: New York, 2013. (38) Raghavan, S.; Keswani, M.; Venkataraman, N. In Handbook for Cleaning for Semiconductor Manufacturing: Fundamentals and Applications; Reinhardt, K. A.; Reidy, R. F., Eds.; Wiley: New York, 2011; pp 3−37. (39) Wagener, P.; Schwenke, A.; Barcikowski, S. Langmuir 2012, 28, 6132−6140. (40) Leroux, F.; Gysemans, M.; Bals, S.; Batenburg, K. J.; Snauwaert, J.; Verbiest, T.; Van Haesendonck, C.; Van Tendeloo, G. Adv. Mater. 2010, 22, 2193−2197. (41) Adamczyk, Z.; Zembala, M.; Siwek, B.; Warszyński, P. J. Colloid Interface Sci. 1990, 140, 123−137. (42) Lundgren, A.; Björefors, F.; Olofsson, L.; Elwing, H. Nano Lett. 2008, 8, 3989−3992. (43) Le, J. D.; Pinto, Y.; Seeman, N. C.; Musier-Forsyth, K.; Taton, T. A.; Kiehl, R. A. Nano Lett. 2004, 4, 2343−2347. (44) Khatri, O. P.; Han, J.; Ichii, T.; Murase, K.; Sugimura, H. J. Phys. Chem. C 2008, 112, 16182−16185. (45) Gilles, S.; Kaulen, C.; Pabst, M.; Simon, U.; Offenhäusser, A.; Mayer, D. Nanotechnology 2011, 22, 295301. (46) Yang, J.; Ichii, T.; Murase, K.; Sugimura, H. Langmuir 2012, 28, 7579−7584. (47) Feder, J. J. Theor. Biol. 1980, 87, 237−254. (48) Kim, T.; Lee, K.; Gong, M.; Joo, S.-W. Langmuir 2005, 21, 9524−9528. (49) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827−829. (50) Deegan, R.; Bakajin, O.; Dupont, T.; Huber, G.; Nagel, S.; Witten, T. Phys. Rev. E 2000, 62, 756−765. (51) Park, J.; Moon, J. Langmuir 2006, 22, 3506−3513. (52) Belgardt, C.; Blaudeck, T.; von Borczyskowski, C.; Graaf, H. Adv. Eng. Mater. 2014, DOI: 10.1002/adem.201400245. (53) Cai, Y.; Zhang Newby, B. J. Am. Chem. Soc. 2008, 130, 6076− 6077. (54) Watanabe, S.; Mino, Y.; Ichikawa, Y.; Miyahara, M. T. Langmuir 2012, 28, 12982−12988. (55) Lallet, F.; Olivi-Tran, N. Phys. Rev. E 2006, 74, 061401. (56) Sharma, V.; Park, K.; Srinivasarao, M. Mater. Sci. Eng. R 2009, 65, 1−38.

5393


Synthesis and Properties of Gold Nanoparticle Arrays Self-Organized

Recommend Documents