不同大小AuNP合成4

Published: July 05, 2011 r 2011 American Chemical Society 11098 dx.doi.org/10.1021/la201938u | Langmuir 2011, 27, 11098–11105 ARTICLE pubs.acs.org/Langmuir Kinetically Controlled Seeded Growth Synthesis of Citrate-Stabilized Gold Nanoparticles of up to 200 nm: Size Focusing versus Ostwald Ripening Neus G. Bast�us,*,† Joan Comenge,†,‡,§ and Víctor Puntes*,†,§,|| †Institut Catal�a de Nanotecnologia (ICN), Campus UAB, 08193 Bellaterra, Barcelona, Spain ‡International Iberian Nanotechnology Laboratory (INL), Av. Central 100, 4710-229 Braga, Portugal §Universitat Aut�onoma de Barcelona (UAB), Campus UAB, 08193 Bellaterra, Barcelona, Spain )Institut Catal�a de Recerca i Estudis Avanc) ats (ICREA), 08010 Barcelona, Spain bS Supporting Information ’ INTRODUCTION Size and shape control of metal nanoparticles (NPs) has been the focus of continuous efforts during the past decades. In particular, Au NPs have been intensively studied because of their size- and shape-dependent physicochemical properties,1 which are of enormous interest for applications in photonics, catalysis, electronics, and biomedicine.1,2 The synthesis of citrate-stabilized Au NPs based on the single-phase aqueous reduction of tetra- choloauric acid (HAuCl4) by sodium citrate, initially developed by Turkevich et al.3 in 1951 and further refined by Frens et al.,4 still remains the most commonly employed aqueous method. Following this strategy, it is possible to control the size of AuNPs from 5 to 150 nm by simply varying the reaction conditions (sodium citrate to gold salt ratio,4 solution pH,5 and solvent6). However, the quality (size and size distribution) of the particles is quite poor, and the shapes obtained are nonuniform and irregular, such as quasi-spheres, ellipsoids, and triangles.7 Nowadays, seeding-mediated synthesis strategies based on the temporal separation of nucleation and growth processes are considered to be very efficient methods to control the Au NP size and shape precisely.8,9 Natan and co-workers pioneered the seeded growth of Au NPs using mild reducing agents such as sodium citrate10 or hydroxylamine11,12 to obtain Au NPs of up to 100 nm in diameter. Although the resultant NPs showed improved physical properties compared to those obtained by the citrate reduction of Au3+ (Frens method), its synthesis was accompanied by a second population of rod-shaped particles. This work was further improved by Murphy13 and Liz-Marzan14 in reporting the synthesis of monodisperse Au NPs of up to ∼180 nm in diameter using ascorbic acid as a reducing agent and cetyl-trimethylammonium bromide (CTAB) as a cationic sur- factant. Although CTAB-based methods allow the control of Au NP morphology, the use of molecules that strongly bind to the Au surface restricts the possibility of further functionalization because their replacement/displacement by thiols is difficult to achieve.15 This condition is especially important in biomedicine, where the ability to render a biological functionality to inorganic nanostructures is one of the cornerstones of this emerging field. In this context, citrate-stabilized Au NPs are unique candidates because the loosely bound capping layer provided by the sodium citrate can easily be exchanged by thiolated molecules that pseudocovalently bind (∼45 kcal/mol) to the gold surface.16 In this way, Au NPs derived with proteins, peptides, antibodies, and thiolated DNA17�19 have been used in promising applica- tions in the fields of diagnosis, therapy, delivering, and sensing, among others.20 Therefore, the development of alternative routes leading to both high monodispersity and versatile surface chemistry still Received: May 24, 2011 Revised: June 30, 2011 ABSTRACT: Monodisperse citrate-stabilized gold nanoparti- cles with a uniform quasi-spherical shape of up to∼200 nm and a narrow size distribution were synthesized following a kineti- cally controlled seeded growth strategy via the reduction of HAuCl4 by sodium citrate. The inhibition of any secondary nucleation during homogeneous growth was controlled by adjusting the reaction conditions: temperature, gold precursor to seed particle concentration, and pH. This method presents improved results regarding the traditional Frens method in several aspects: (i) it produces particles of higher monodispersity; (ii) it allows better control of the gold nanoparticle size and size distribution; and (iii) it leads to higher concentrations. Gold nanoparticles synthesized following this method can be further functionalized with a wide variety of molecules, hence this method appears to be a promising candidate for application in the fields of biomedicine, photonics, and electronics, among others. 11099 dx.doi.org/10.1021/la201938u |Langmuir 2011, 27, 11098–11105 Langmuir ARTICLE remains a challenging task for Au NPs larger than 40 nm. In this context, several seed-mediated synthesis strategies, based on the use of 2-mercaptosuccinic acid21 or ascorbic acid22 as reducing agents in aqueous solvents, have been recently reported. In the first case, as when CTAB is used,13,14 the growth is controlled by the strongly binding surfactant molecules, which compromises further functionalization of the NPs. In the second case, Au NPs over a large range of sizes were grown by the incorporation of small gold clusters on the surfaces of seed particles. Further heat- mediated recrystallization led to quasi-spherical Au NPs via an interparticle ripening process. In this work, we study the condi- tions by which it is possible to obtain large, citrate-stabilized Au NPs via a seeded growth method whereas the new nucleation of particles is suppressed. This method allows better control of the final Au NP size and provides extremely stable and readily functionalized particles. Herein, a seeded growth strategy for the synthesis of size- and shape-controlled large citrate-stabilized Au NPs based on the classical Turkevich/Frens reaction system is described. It focuses on the inhibition of secondary nucleation during the homoge- neous growth process, allowing the enlargement of presynthe- sized Au NPs via the surface-catalyzed reduction of Au3+ by sodium citrate. The success of the method relies on the kinetic control of growth process by adjusting the reaction conditions, in detail, temperature, pH level, and seed concentration. It is noteworthy in several respects: (i) it produces particles of high monodispersity; (ii) it allows smaller particles to be grown into larger particles of a predetermined size; and (iii) it leads to higher Au NP concentrations relative to the Frens method (from 3 � 1012 NPs/mL, 8.5 nm to 5 � 109 NPs/mL, 180.5 nm). Addi- tionally, it provides key aspects, identifying and explaining the important synthetic variables that must be controlled in order to control the Au NP morphology. Results show the reproducible preparation of citrate-stabilized Au NPs with a uniform quasi- spherical shape from 10 to ∼180 nm, a narrow size distribution, and controlled concentration. These results allow the expansion of the well-known applicability of these NPs in biological and biomedical applications. Thus, large Au NPs arise as ideal candidates for biolabeling because of their higher electronic contrast.20 Similarly, the intense visible absorption of larger Au NPs (which is translated into greatly enhanced local electro- magnetic fields) promotes its use as surface-enhanced Raman spectroscopy (SERS) agents with superior activity.23 The differ- ent accumulation/penetration behavior of Au NPs in tumors makes accurate control of the Au NP size necessary.24 Moreover, the increase in the Au NP size (which entails a decrease in the radii of curvature) increases the number density of molecules that can be delivered. Additionally, the size-dependent biodistribu- tion of Au NPs together with the elucidation of their potential toxicity,25 usually related to the NPmorphology, composition, or surface coating,26 requires a wide catalog of NPs with controlled size and morphology and identical structure, composition, and surface chemistry. This method can be potentially adapted to continuous processes as in flow reactors, where the production of large numbers of particles of the highest quality is a limitation of Au NP technology implementation. ’EXPERIMENTAL SECTION Materials. HAuCl4 3 3H2O (99%) and trisodium citrate (99%) were purchased from Sigma-Aldrich. Milli-Q water was used in all experiments. All glassware was cleaned with acetone, rinsed with deionized water, and stored at 150 �C before use. Synthesis of Gold Nanoparticles. Synthesis of Au Seeds. A solution of 2.2 mM sodium citrate inMilli-Q water (150mL) was heated with a heating mantle in a 250 mL three-necked round-bottomed flask for 15 min under vigorous stirring. A condenser was utilized to prevent the evaporation of the solvent. After boiling had commenced, 1 mL of HAuCl4 (25 mM) was injected. The color of the solution changed from yellow to bluish gray and then to soft pink in 10 min. The resulting particles (∼10 nm, ∼3 � 1012 NPs/mL) are coated with negatively charged citrate ions and hence are well suspended in H2O. Seeded Growth of Au NPs of Up to 30 nm in Diameter. Immediately after the synthesis of the Au seeds and in the same vessel, the reaction was cooled until the temperature of the solution reached 90 �C. Then, 1 mL of sodium citrate (60 mM) and 1 mL of a HAuCl4 solution (25 mM) were sequentially injected (time delay∼2 min). After 30 min, aliquots of 2 mL were extracted for further characterization by transmis- sion electron microscopy (TEM) and UV�vis spectroscopy. By repeat- ing this process (sequential addition of 1 mL of 60 mM sodium citrate and 1 mL of 25 mM HAuCl4), up to 14 generations of gold particles of progressively larger sizes were grown (further details in Scheme S1). The concentration of each generation of NPs was approximately the same as the original seed particles (∼3 � 1012 NPs/mL). SeededGrowth of AuNPs of Up to 180 nm inDiameter. Immediately after the synthesis of the Au seeds and in the same reaction vessel, the reaction was cooled until the temperature of the solution reached 90 �C. Then, 1 mL of a HAuCl4 solution (25 mM) was injected. After 30 min, the reaction was finished. This process was repeated twice. After that, the sample was diluted by extracting 55 mL of sample and adding 53 mL of MQwater and 2 mL of 60 mM sodium citrate (further details in Scheme S2). This solution was then used as a seed solution, and the process was repeated again. By changing the volume extracted in each growth step, it is possible to tune the seed particle concentration. Characterization Techniques. UV�Vis Spectroscopy. UV� visible spectra were acquired with a Shimadzu UV-2400 spectrophot- ometer. A Au NP solution (1 mL) was placed in a cell, and spectral analysis was performed in the 300 to 800 nm range at room temperature. In the case of time-dependent measures, aliquots of the solution were taken out and samples were cooled in ice water to quench the reaction. Transmission Electron Microscopy. Gold nanoparticles were visua- lized using 80 keV TEM (JEOL 1010, Japan). Ten microliter droplets of the sample were drop casted onto a piece of ultrathin Formvar-coated 200-mesh copper grid (Ted-pella, Inc.) and left to dry in air. TEM images of the prepared colloidal Au NPs were used for the size distribution measurements. For each sample, the size of at least 100 particles wasmeasured and the average size and the standard distribution were obtained. Mie Calculations. Calculations of the scattering coefficient of colloi- dal Au NPs of different diameters were obtained usingMie plot software considering Au spheres embedded in water at 25 �C. In all cases, mean size and standard distribution measured by TEM were used for the calculations. ’RESULTS Seeded Growth Synthesis of Au NPs of Up to 30 nm.Gold seeds (∼10 nm,∼3� 1012 NPs/mL) were prepared by injecting an aqueous HAuCl4 precursor solution into a boiling solution of sodium citrate (SC), and the reaction was run until it reached a red-wine color. After that, the temperature of the solution was decreased to 90 �C and more sodium citrate and HAuCl4 precursor were injected. The decrease in the temperature to 90 �C allows both the inhibition of the new nucleation of AuNPs and the slowing down of the reaction, which helps in the ljx 线条 ljx 线条 11100 dx.doi.org/10.1021/la201938u |Langmuir 2011, 27, 11098–11105 Langmuir ARTICLE monitoring of reaction kinetics and the delay of the defocusing period (vide infra). Figure 1A shows the morphological char- acterization of Au NPs obtained after different growth steps. As can be seen, the size of the Au NPs increased from 13.5( 2.1 to 30.5 ( 3.9 nm after 13 consecutive growth steps to obtain Au NPs with diameters of 19.2 ( 2.6 and 24.8 ( 3.4 nm after three and six growth steps, respectively (Figure S1). The optical properties of Au colloidal solutions measured by UV�vis spectroscopy are depicted in Figure 1B. Spectra were normalized to 400 nm, where the absorbance is mainly due to interband transitions, in order to facilitate a comparison.14 In all cases, the spectra show a rather symmetric surface plasmon absorption band, which red shifts (inset of Figure 1B) and increases in intensity as a consequence of particle growth. Interestingly, when comparing the size of the Au NP size measured from TEM images after different growth steps, it can be seen that the growth of the Au NPs is not homogeneous and decreases as the particle size increases. This is simply due to the fact that the rate of growth is determined by the relation between the diameter of the growing particle to the amount of gold added (considering a constant NP concentration). Three different aspects are of crucial importance to obtaining Au NPs of large size and high stability: (i) the temperature of the solution, (ii) control of the pH of the solution, and (iii) the number of Au atoms injected in each growth step. First, because the kinetics of the gold reduction significantly depends on the temperature (Figure S2), we would expect that at low tempera- tures the nucleation rate decreases, leading to more favorable conditions for particle growth. Thus, the temperature was decreased to 90 �C to prevent any secondary nucleation during the growth step. This assumption was experimentally confirmed by repeating the above-described experiment at 100 �C.Whereas particles synthesized at 90 �Cpresented excellentmorphology and narrow size distributions, those obtained at 100 �C (Figure S3) showed two different size populations, one of them smaller than the initial seed Au NPs. This indicates that secondary nucleation was not prevented. Another important aspect is the role of sodium citrate as a pH buffer in the reaction. Because each growth step involves the addition of a Au3+ precursor that acidifies the solution,5 the pH of the solution progressively decreases as the growth process is taking place. This was experimentally proven by repeating the reaction without the addition of sodium citrate and measuring the pH of the solution after each growth step (Figure S4). We found a decrease of 0.3 pH unit after each injection, achieving a value of 3.14 after 14 consecutive injections. At this point, the solution of Au NPs precipitated. This can be explained by considering the pKa values of sodium citrate (6.4, 4.8, and 3.2). Because at pH 3.14 the protonation of all of the carboxylic groups occurred, the electro- static interactions cannot impart stability to particles in solution. Thus, the amount of sodium citrate added in each growth step was the experimentally determined to maintain a constant pH of ∼7 during the whole growth process. The last point to address is the amount of Au precursor added in each growth step. As previously discussed, the growth of the Au NPs is determined by the ratio between the gold atoms added and those present at the seed surface, which leads to a growth rate that drastically decreases as the particles become larger. This problem cannot be solved by injecting larger amounts of gold salt. The injection of larger amounts (5�) broadens the size distribution, leading to nonspherical (oval) shapes probably because of the cementing of different seeds during the growth process,27 whereas the injec- tion of larger amounts (10�) induces the nucleation of new Au NPs (Figure S5). In this context, we studied the possibility to decrease the seed particle concentration as NPs grew. Seeded Growth Synthesis of Au NPs of Up to 180 nm. To get larger particles with a controlled size, morphology, and concentration, we studied the possibility to grow a successive generation of spherical particles with larger sizes by adjusting both the seed particle concentration and the total number of gold atoms injected into the solution. Thus, after two growth steps, Au seeds were diluted and more gold precursor and sodium citrate were added. By repeating this process, we synthesized Au NPs with increasing size and decreasing concentration. The morphol- ogy of the resultant Au NPs was determined by TEM micro- scopy. Figure 2 shows how the final size of the particles increases as the number of growth steps increases, and it is possible to synthesize spherical and monodisperse Au NPs of up to ∼200 nm in diameter (Table 1). Consequently, the concentra- tion of the colloidal gold solutions depends on the particle size. Thus, initial seed particles are present in a concentration of∼3� 1012 NPs/mL but at∼5 � 109 NPs/mL for the largest Au NPs. Moreover, although the particles are rather faceted and thus not Figure 1. Seeded growth without the dilution of the seed solution. (A) Transmission electronmicroscopy images of Au seed particles and those obtained after different growth steps. The particle size increases from 13.5( 2.1 to 19.2( 2.6, 24.8( 3.4, and 30.5( 3.9 nm after 3, 6, and 13 growth steps, respectively. (B) Absorption spectra of gold colloids obtained after different growth steps and evolution of the SPR wave- length as a function of the growth step. All spectra are normalized at 400 nm to facilitate comparison. 11101 dx.doi.org/10.1021/la201938u |Langmuir 2011, 27, 11098–11105 Langmuir ARTICLE perfectly spherical, the growth is very uniform up to ∼180 nm without the formation of elongated particles or a second popula- tion of smaller particles (S6 and S7). The synthesis was finished when we achieved a particle size of ∼180 nm as the colloidal transparency limit, but there is no reason against carrying out subsequent growth steps to achieve larger Au NPs. Monitoring the Growth Process of the Gold Nanoparti- cles. To determine whether the growth of the Au NPs was via atom addition or the aggregation of smaller units forming compact aggregates,22,28 we monitored a single growth step by extracting different aliquots of the reaction after the initialization event (addition of Au3+ precursor to the seed NP solution). Several aliquots were taken at different reaction times and were immediately quenched by sample immersion in an ice/water mixture for further TEM and UV�vis analysis. Evidence of Au NP growth can be seen from the UV�vis data (Figure 3), and two different behaviors can be observed. At short times (from 15 s to 6 min), a great red shift (from 524.5 to 532.5 nm) in the surface plasmon resonance (SPR) band position can be observed whereas absorbance values increased only slightly (from ∼0.31 to ∼0.48). At longer times (from 6 min on), a great increase in the absorbance (from ∼0.48 to ∼1.21) and a slight blue shift (from 532.5 to 529.5 nm)were observed. The same aliquots were analyzed by TEM. Initially, seed particles were 29.8( 3.3 nm in diameter, and they grew to 30.5( 3.5 nm (15 s), 32.2( 3.3 nm (30 s), 32.8( 3.3 nm (45 s) and 34.6( 3.8 nm (1 m). At these initial times, Au NPs were accompanied by aggregates (15 s) or small particles, which were not taken into account to calculate