Turkevich Method for Gold Nanoparticles

Research will be limited to:
- Material: Gold Nanoparticles
- Method: Turkevich
- Reagent: Citrate
- Size: 15-50 nm

Strategy: The objective is to establish a precise protocol for synthesizing gold nanoparticles (AuNPs) within the 15-50 nm range using the Turkevich method. I will focus on the quantitative relationship between the citrate-to-gold molar ratio, reaction temperature, and stirring speed as they relate to final particle size and monodispersity. Specifically, I need to find empirical data or kinetic models that map these parameters to the 15-50 nm target range.
Queries:
- Turkevich method gold nanoparticles citrate ratio size control 15-50 nm
- effect of stirring speed on gold nanoparticle size Turkevich method
- temperature dependence of gold nanoparticle nucleation Turkevich method 15nm 50nm
- citrate to gold molar ratio correlation particle diameter 15-50nm
- optimized Turkevich protocol monodisperse gold nanoparticles 15-50 nm range

Executing 5 concurrent searches:
- Turkevich method gold nanoparticles citrate ratio size control 15-50 nm
- effect of stirring speed on gold nanoparticle size Turkevich method
- temperature dependence of gold nanoparticle nucleation Turkevich method 15nm 50nm
- citrate to gold molar ratio correlation particle diameter 15-50nm
- optimized Turkevich protocol monodisperse gold nanoparticles 15-50 nm range

Found 8 papers. Key excerpts:

Gold Nanoparticles: A Didactic Step-by-Step of the Synthesis Using the Turkevich Method, Mechanisms, and Characterizations — Ana Elisa F. Oliveira et al., 2023

1. IntroductionDuring the 1850s, Faraday fabricated gold slides and examined them using shining light. For this purpose, he used sodium chloroaurate (NaAuCl4) with a reducing suspension, such as phosphorus in carbon disulfide, where the color changed from bright yellow to deep ruby. The resulting liquid is known as Faraday gold [1,2,3]. While shining a beam of light through the liquid, Faraday observed that a portion of the light was scattered, leading to the divergence of the light beam. This was explained by the presence of fine particles of gold dispersed in the liquid “in a state of extreme division”, which was not visible in any microscopy. This behavior is known as the Faraday–Tyndall effect. Faraday’s studies recognized the emergence of nanoscience and nanotechnology [1,2,3].Gold nanoparticles (AuNPs) of various sizes and shapes have been fabricated using different techniques and routes. AuNPs have attractive physical properties, including surface plasmon resonance (SPR), the ability to quench fluorescence, surface-enhanced Raman scattering (SERS), and redox activity. Over the last decade, these properties have been used in the fabrication of electronic devices, imaging, sensing, printable inks, photodynamic therapy, therapeutic agent delivery, sensors, catalysis, probes, and others [4,5,6,7,8,9,10,11].In addition, AuNPs exhibit excellent biocompatibility owing to their high binding affinities to biomolecules. Both covalent and noncovalent approaches have been designed to conjugate AuNPs. The most common covalent conjugation is the direct attachment of the thiolate molecule to AuNPs. Non-covalent interactions are usually due to electrostatic interactions, hydrophobic interactions, and specific binding affinities [4,12].The synthesis of nanoparticles, in general, is usually classified by two methods: top-down and bottom-up approaches. In the top-down method, bulk metals decompose into smaller particles, generating the required nanostructures. The bottom-up method assembles atoms or molecules into larger nanostructures to generate nano-sized materials [11,12,13]. These synthetic approaches have been performed using physical, chemical, and biological methods. Each

fabrication method has its own advantages and disadvantages.The most common approach for synthesizing AuNPs is chemical methods. It usually employs three components: a metal precursor, reducing agent, and stabilizing/capping agents. The metal precursor is a metallic salt, such as gold. Chemical reduction can be performed using various chemical reductants such as sodium citrate (Na3C6H5O7), hydrazine (N2H4), ascorbic acid (HC6H7O6), and sodium borohydride (NaBH4) [14]. In addition, the stabilizing agent was absorbed onto the AuNPs surface to prevent agglomeration. The most common are phosphorus ligands, trisodium citrate dihydrate (C6H9Na3O9), cetyltrimethylammonium bromide (CTAB), chitosan, surfactants, and other polymers [12,13,14,15].Although there are many articles discussing the synthesis of gold nanoparticles, to the best of our knowledge, we did not find an article that explained didactic step-by-step synthesis, the role of each reagent, or showed pictures of the entire process, including a well-explained mechanism and characterization study. Therefore, in this study, gold nanoparticles were synthesized using the well-known Turkevich method, and these aspects are considered in the discussions. This classical method was presented by Turkevich in 1951 using trisodium citrate as a reducing agent. Since then, several articles have proposed syntheses using a modified version of the Turkevich method [16,17,18,19]. 2. Materials and Methods 2.1. ReagentsSodium citrate (Na3C6H5O7) was purchased from Synth (Diadema, São Paulo, Brazil). Sodium tetrachloroaurate (III) dihydrate (NaAuCl4) was purchased from Sigma-Aldrich (Barueri, São Paulo, Brazil). All reagents were of analytical purity and all solutions were prepared with purified water using a Millipore Milli-Q system with a resistivity of 18.2 MΩ cm (at 25 °C). 2.2. Synthesis of Gold NanoparticleThis paper will explain the didactic step-by-step process of the synthesis

, showing pictures of the entire process, including a well-explained mechanism and characterization study. The synthetic process is illustrated in Figure 1. The gold nanoparticles were fabricated using classical citrate synthesis, also known as the Turkevich method.Stock solutions of sodium citrate (10 mg mL−1) and sodium tetrachloroaurate (NaAuCl4) (0.125 mol L−1) were prepared. Subsequently, 420 µL of NaAuCl4 was added to 94.6 mL of deionized water, and the solution was agitated and heated to 90 °C. When the required temperature was achieved, 5 mL of sodium citrate was quickly added. Agitation and heating at 90 °C were performed for 20 min. The color of the suspension changed from light yellow to grey and then to red, indicating the formation of AuNPs. The AuNPs suspension was then slowly cooled to room temperature and maintained at 4 °C. 2.3. Characterization TechniquesNaAuCl4 and AuNPs samples were characterized using different techniques. UV-Vis Spectrometry was performed using a spectrophotometer UV-2550 (Shimadzu, Kyoto, Japan) at 300–800 nm. Both samples were diluted in deionized water using a quartz cuvette.Scanning electron microscopy (SEM) images were recorded on a JEOL JSM 300-LV instrument (Tokyo, Japan). The measurements were performed by Prof. Dr. Lucas Franco Ferreira of the Federal University of the Jequitinhonha and Mucuri Valleys (UFVJM). X-ray diffraction analysis (XRD) was performed using a Shimadzu model XRD 6000 (30 kV, 30 mA) and Cu-Kα (λ = 1.54 Å) in the 30–90° range. In both techniques (SEM and XRD), the NaAuCl4 and AuNPs samples were placed on an acetate slide and dried at 70 °C.Atomic force microscopy (AFM) was performed using a Bruker Multimode 8 (MM8) microscope. A scan size of 5.00 µm and amplitude of 5000 mV were used. AFM was conducted only for AuNPs. The sample was dried on a graphite substrate and placed on a circular metallic AFM holder using silver tape.

How does the size of gold nanoparticles depend on citrate to gold ratio in Turkevich synthesis? Final answer to a debated question — Li Shi et al., 2017

Introduction

Gold nanoparticles (AuNPs) are the most widely used metallic nanoparticles and drive a variety of applications in nanomedicine, sensing, optoelectronics and catalysis. The fine control of AuNPs individual characteristics is a prerequisite to exploit the macroscopic properties, that emerge from collective effects. Although the syntheses in non-aqueous solvents were preferred so far for the synthesis of high quality building blocks, great efforts have been done in the last decade to optimize synthesis pathways directly in water. The prominent member of this group of aqueous synthesis is the 'Turkevich' protocol introduced in 1951. This method enables to obtain monodisperse citrate-stabilized AuNPs by simply changing the relative concentration of citrate molecules that are quickly injected in a boiling HAuCl 4 aqueous solution.
In spite of its advantages, this approach suffers from lack of reproducibility and of predictability due to a poor theoretical understanding of the mechanism of formation. This is due to the fact that citrate and gold ions exist both in different chemical forms, which can be reversibly converted into each other. This is also due to the multiplicity of roles played by citrate molecules (i.e. reductive agent, stabilizing agent and pH mediator), which results in multiple intricate steps that are hard to probe separately. However, one can attempt the following description. First, Au III is slowly reduced to Au I thanks to citrate decarboxylation into dicarboxy acetone (DCA) which leads to electron transfer. Differential functional theory simulations have shown that the most favorable reaction path is obtained at low pH where [AuCl 4 ] -is the dominant structure of aureate complex due to the presence of highly labile Cl -ions around Au III . [10,22] We wish to underline that pH is controlled by citrate to gold molar ratio (X) at fixed gold concentration. As formed Au I atoms can be assumed to form multi-molecular complexes with DCA but experimental proofs are lacking. [11,32] Au 0 atoms could be formed in bulk when the concentration of Au I species increases locally to a 'high enough' level ([AuCl 2 ] - [?] 10 nM ) to trigger homogeneous disproportionation. Further disproportionation may lead to larger aggregates of

Au 0 atoms and when the aggregate size reaches a critical radius of order 1.5 nm, a stable nucleus of gold atoms should be formed. [13,15] Disproportionation can also occur at the particle surface, leading to nucleus growth and also to the regeneration of some Au III . The reactant molar ratio, X, is the eldest and probably the most frequent lever for controlling the size and the polydispersity of AuNPs. Forty years ago, Frens mentioned a steep decrease of AuNPs' size by a factor 6 when X was varied from 0.8 to 2 at [Au] = 0.25 mM. This result has been confirmed by several studies [8, together with a concomitant decrease of polydispersity. It is also recognized that this evolution of size is restricted to low values of X. Indeed, above a certain value, noted X in figure 1.c, which vary with the absolute gold concentration, the different results are conflicting. This is shown in figure 1.a which summarizes the results obtained for the most popular Conditions of synthesis conditions of synthesis: [Au] = 0.25 0.05 mM and T = 98 2 degC. One can discern two families of behaviors: (i) On one hand, several studies [14,16,27] showed a discontinuous evolution of the size with a sharp minimum at X [?] 3.5. [14,27] Ji et al. observed that this minimum corresponds to a pH at which the dominant structures of aureate complexes and citrate ions modify (Figure 1.b). These authors propose that the observed discontinuity may be related to a modification of the mechanism of formation (Figure 1.c). A two-steps process involving nucleation followed by a slow diffusion-controlled growth should dominate above X*, while a three-steps process involving fast nucleation and random binding, followed by an intra-particle ripening, should dominate below X. This last mechanism may involve transient "nanowire-shaped" clusters that have been detected by TEM, [20,26] but never by SAXS or in situ AFM, [15,21] suggesting that they result from the reduction of Au I and Au III species during drying. Fig. 1. Evolution of the averaged radius of citrated AuNPs as a function of (a) the molar

ratio X and (b) the pH for similar conditions of synthesis given in Inset of figure 1.a and summarized in table 1 of S.I. All radii are number averaged values obtained by TEM except for which corresponds to intensity averaged hydrodynamic radii. The line corresponds to Kumar' model prediction. The different colors of patterns and the numbers in figure 1.b characterize the regions of the speciation diagrams. (c) Scheme representing the different results and the proposed reaction pathways.
(ii) On the other hand, several studies [26,30] showed that the size decreases continuously with X on the whole range of X, with lowest radius ranging between 2.5 nm and 10 nm at X = 20. A so-called four-steps "seed-mediated" growth mechanism, which is supposed to be congruent with a monotonic decrease of the size, was proposed on the basis of coupled in situ XANES and SAXS during AuNPs formation, for X > X. Such proposal is based on the observation that gold equilibrium is shifted from [AuCl 4 ] -to less reactive [AuCl 3-
-during the fast seed particle formation ( 30 s after mixing), so that for X >= X, the kinetics of seed formation and pH neutralisation should be approximately the same irrespective of the molar excess thus explaining the size independence. For X < X, the molar excess might still be good enough to reduce Au III species but not to shift the gold complex equilibrium.
Hence, homogeneous and heterogeneous disproportionation can also occur unselectively during the entire synthesis, increasing the polydispersity. At very low X, seed particles should grow up to a size that could be stabilized by the available citrate molecules. Interestingly, the continuous evolution of size with X has been theoretically predicted by Kumar et al. until R [?] 7.5 nm at X = 20, but with a model neglecting the dependence of the aureate complexes structure and reactivity with the pH. In summary, we note that there is no consensus on the evolution of the size with X. We believe that this dispersion of results is due (i) to the variation of certain parameters that are often not mentioned in the protocols, and (ii) to the use of a single characterization technique (TEM or DLS). In this article, we want

Citrate-Capped AuNP Fabrication, Characterization and Comparison with Commercially Produced Nanoparticles — Abdul Ghaffar Memon et al., 2022

1. IntroductionVarious metallic nanoparticles are being synthesized using green methods; such nanoparticles include gold [1], silver [2], copper [3], zirconium oxide, platinum [4], zinc oxide [5], titanium oxide & Silica along with nanoclay [5,6,7], and many more. Among these metallic nanoparticles, gold nanoparticles (Au-NPs) are the material of choice for researchers due to their remarkable properties, such as chemical stability, broad spectra, antimicrobial activity, surface-enhanced Raman scattering, and nonlinear optical behavior [8]. A broader spectrum of the fungicidal and bactericidal activity of Au-NPs has made them an extremely popular component in the fields of medicine, agriculture, food preservation, biosensing, and consumer products [9]. Nanoparticle (NP) fabrication has become a new paradigm in the field of material science, as NPs have found a variety of applications, including environmental, pharmaceutical, medicinal, textile, and many more. The application of AuNPs in the development of sensors that can be used for heavy-metal detection, medicine, and drug delivery along with renewable energy applications [10,11,12,13,14] are of particular interest. AuNPs are also used as catalysts in a number of reactions. Some researchers have also shown effective results of AuNPs in fuel-cell technology. Therefore, in order to maintain the constant supply of AuNPs, several cost-effective and high-yielding techniques have been developed [15,16]. Recently, the synthesis of AuNPs from green sources, using cost-effective methods, has evolved and is the widely used technique for particle production. Due to its cost effectiveness and simple steps, Turkevich is viewed as the most frequently used method [3]. Since the first reported fabrication of AuNPs in 1973 by Michael Faraday [16], the citrate reduction method developed by Turkevich et al. [17] received a great deal of attention. This green method offers a number of advantages over other conventional methods, such as cost savings, less energy consumption, and ambient-condition processing, thus eliminating the strict requirements for inert atmosphere maintenance [4,5,15]. Different sizes and shapes, such as spheres, cubes [18], rods [19], shells [20], stars [21], and prisms [22], have been synthesized via this method and applied in

various applications, including biosensing. This is considered one of the most commonly used techniques for formulating spherical AuNPs since AuNPs prepared using this method can have sizes down to 1–2 nm. The basic principle of this technique involves the reduction of gold ions (Au3+) to produce gold atoms (Au) by using reducing agents, such as amino acids, ascorbic acid, UV light, or citrate. The stabilization of AuNPs is achieved by using different capping/stabilizing agents. At the beginning, the applications of the Turkevich method were finite because of the limited range of AuNPs that could be synthesized using this technique. Later, after several advancements to the basic method, researchers were able to extend the size range of particles synthesized using this method as it was initially established by varying the ratio of reducing, as well as stabilizing, agents. Therefore, with the help of this advancement, AuNPs within the range of 16 to 147 nm can be produced, and that size of NPs can play a significant role as well. Also, these Turkevich methods give great control over the size and shape of the particles [23,24]. Due to the stable structure and strong electrostatic properties of localized surface-plasmon resonance (LSPR) [25,26], spherical-shaped colloidal gold nanoparticles have been widely applied in the development of labels [27], label-free [28], colorimetric [29], and evanescent-wave optical biosensing strategies [30]. Apart from the Turkevich method, seed-mediated growth and digestive ripening have also been reported in the literature [17]. According to the literature, the formulation of AuNPs involves two main stages. In the first stage, the gold precursor, which is usually an aqueous gold salt solution, is reduced to gold nanoparticles using a specific reducing agent, such as citrate. In the second stage, the stabilization of gold nanoparticles is carried out by a specific capping agent. The capping agents hinder the agglomeration of metallic nanoparticles.In this research, the Turkevich method has been employed to synthesize gold nanoparticles, and prepared particles were fully characterized by various means, including SEM, TEM, UV–vis, EDS, XRD, zeta sizer and particle-size distribution, etc. In the literature, no research was found

to provide the full characterization of AuNPs, as per the best knowledge of the authors. Moreover, optimization of the synthesis process was also carried out by variations in salt concentration and relevant parameters. The synthesized particles also exhibited greater thermal stability even after months of storage. Thus, this study will be helpful for researchers to understand the synthesis and development of gold nanoparticles, as several characterizations and tests were performed. The developed gold nanoparticles are the potential materials for developing various types of calorimetric sensors for various purposes. 2. Experimental Work 2.1. Materials and MethodsGold chloroauric acid salt (HAuCl4) was purchased from the Sinopharm Chemical Reagent Company (Shanghai, China). The trisodium citrate (C6H5Na3O7.2H2O), HNO3, HCl, and other solutions of analytical reagent grade were bought from Beijing Chemical Reagent Company. All the solutions were prepared using sterile, molecular-grade DNase-, RNase-, and Protease-free water of USP standard. 2.2. Preparation of Gold NanoparticlesA fresh stock of the gold colloidal solution with a 13 nm diameter was synthesized through citrate reduction (Turkevich method) [17,31,32,33,34,35,36,37]. In brief, 4 mL of the gold chloric acid at 25 mM was added to 196 mL of molecular-grade water in a piranha solution (HCl: HNO3 3:1), pre-rinsed in a 250 mL round-bottom flask containing magnetic beads, and heated to above 100 °C in an oil bath. Later, 20 mL of the citrate solution at 38.8 mM was rapidly injected into the heating solution and continued to be vigorously stirred for 30 min. The color of the reaction changed from light, pale gold to colorless and then to a red-wine. The synthesized AuNPs were then stored at 4 °C for further use. A small volume was taken for concentration calculation and further characterization. 2.3. CharacterizationThe UV–Vis Spectrophotometer U-3900 of Hitachi, Japan was used for recording absorption spectra in the concentration calculation and measurement. Scanning electron microscope GeminiSEM 500 (Carl Zeiss Microscopy GmbH, Jena, Germany), high-resolution transmission electron microscopy (HR-

New Insight Into the Size Tuning of Monodispersed Colloidal Gold Obtained by Citrate Method — Li Shi et al., 2016

nanoparticles (AuNPs). This dependence is still a matter of debate for X 3 where the
polydispersity is yet minimized. Indeed, there is no consensus between experiments proposed so far for comparable experimental conditions. Nonetheless, the sole available theoretical prediction has never been validated experimentally in this range of X. We show unambiguously using 3 techniques (UV-Vis spectroscopy, dynamic light scattering and transmission electronic microscopy), 2 different synthetic approaches (Direct, Inverse) and 10 X values for each approach that AuNPs’ size decay as a monoexponential with X. This result is, for the first time, in agreement with the sole available theoretical prediction by Kumar et al. on the whole studied range of X.
INTRODUCTION. Gold nanoparticles (AuNPs) are probably the most widely used and studied metal nanoparticles and have driven a variety of applications in nanomedicine, sensing, optoelectronics and catalysis. [1-5] The control of AuNPs individual characteristics (i.e. size, shape, and size/shape distribution) is fundamental to exploit at a higher length scale their properties that are often related to collective effects. [6] Although, the syntheses in non-aqueous solvents were often preferred for the synthesis of high quality building blocks, great efforts have been done in the last decade to optimize green synthesis pathways directly in water. [7] The proeminent member of this group of aqueous synthesis is probably the ‘Turkevich’ protocol introduced in 1951 for the synthesis of citrate-stabilized AuNPs. [8] This synthesis enables to obtain quite monodisperse AuNPs in a wide size range by simply changing the relative concentrations of trisodium citrate molecules that are quickly injected in a boiling HAuCl4 aqueous solution. In contrast with the simplicity of the experimental protocol, the mechanism of AuNPs formation is still obscure on several aspects. [7] This is in part due to the multiple roles played by citrate molecules which result in multiple intricate steps that are hard to probe experimentally. However one can attempt the following basic description. At the beginning, Au III is slowly reduced to Au I thanks to citrate decarboxylation into dicarboxy acetone (DCA) which leads to electron transfer. Ojea-Jime

́nez and Campanera have shown by differential functional theory simulations that the most favorable reaction path can be decomposed in four steps: (i) substitution of a Cl - ions by a citrate ligand in the auric acid, (ii) deprotonation of the second most acid carboxylic group, (iii) conversion of the Au equatorial coordination from the initial carboxylate ligand to the hydroxyl group and (iv) formation of transition state. [10] They reveal that the major part of the total activation energy (G ffi ) corresponds to the two extreme steps and that G ffi decreases from 37.4
kcal/mol to 26.8 kcal/mol when the pH is decreased from neutral to 4. This pH dependence is mainly explained by the fact that Cl - ions are much more labile around Au III when pH decreases thus facilitating their substitution by citrate. [10]
As formed Au I atoms may form multimolecular complexes with DCA. [8,11] Au 0 atoms could be formed in bulk when the concentration of Au I species increases to a level ‘high enough’ ([AuCl2] − ≈ 10 nM [9] ) to trigger homogeneous disproportionation. [12] Further disproportionation may leads to formation of still larger aggregates of gold atoms. When the size of the aggregate reaches a critical diameter of about 2 nm, a nucleus of gold atoms may be formed. [13,14] A common feature with other NPs synthesis is that AuNPs with narrow size distribution could be obtained by increasing the speed of this nucleation step. This can be done by favouring the formation of Au I (i.e. by increasing [Citrate]t=0/[HAuCl4] t=0 = X, decreasing pH and/or increasing temperature) and also by increasing the concentration of DCA. [15-20] Once particles are formed, disproportionation can also occurs at particle surface leading to nucleus growth and also to the regeneration of some Au III species. In contrast with common homogeneous growth of monodisperse particles, several studies
performed at [Citrate]t=0 / [Au]t=0 = X 6.7 have shown that these growing AuNPs could assemble into more or less aniosotropic and crystalline aggregates of several tens

of nanometers. [13,15,21-23] The presence of these intermediate aggregates could be understood by considering that several nuclei could be generated by the same Au I /DCA complex. As the reaction proceeds the size of the constituent particles seems to increase until a certain stage at which the aggregates decompose into individual rather monodisperse particles with a typical average diameter of about 15 nm. This mechanism of disaggregation is not readily explained to the best of our knowledge. [9]
Other studies [14,15] show that monodisperse particles can be obtained without such intermediate
aggregates. Considering the pH dependence of the standard redox potential of the different Au III complexes and of the reducing agent pointed by Goia and Matijevic, [24] Ji et al. [15] proposed a pH-dependent mechanism of particle formation. It would proceed either in two steps without intermediate aggregates (pH > 6.5), or in three steps, with intermediate aggregates (pH < 6.5). The final AuNPs are almost spherical when X 3.5 in absence of metal contamination. [25] They
display an anionic surface charge due to a stabilizing shell composed of deprotonated citrates directly adsorbed on the NP‟ surface. The dependence of the shell‟ structure with pH is still matter of discussion. According to a recent study by Park et al. [26] the citrate molecules are coordinated by the central carboxylate group at pH = 3.2 which corresponds to X ≈ 1.
Interestingly, they show that this first layer weakly interacting with the gold surface (ECOO-/Au 2 kcal/mol) could be hydrogen bonded (E 7 kcal/mol for one hydrogen bond of carboxylic acid
dimer or 28 kcal/mol for the total citrate interaction) to a second layer of monodeprotonated citrate which give rise to the surface negative charge. The terminal carboxylate groups are progressively coordinated to the gold surface when the pH increases thus suppressing H bond sites and leading to the second layer disappearing. Following the work of Frens, [27] several recent studies have enable to identify and optimize the main levers (i.e. [Au III ], [27,28] X, [8,27-33] [DCA], [15,

Effect of different physical factors on the synthesis of spherical gold nanoparticles towards cost-effective biomedical applications. — Zahra Bahmanyar et al., 2023

Recently, gold nanoparticles (AuNPs) have attracted considerable biomedical interest in high biocompatibility, physicochemical properties, and characteristics tunability in synthesis [ 1 , 2 ]. Gold nanoparticles have been studied in a wide range of biomedicine applications from diagnosis to treatment, including biosensors [ 2 ], gene and drug delivery [ 3 , 4 ], phototherapy and hyperthermia [ 5 ], and antimicrobial applications [ 6 ] in different shapes of nanostars [ 7 ], nanorods [ 5 ], nanocages [ 8 ], and nanosphere [ 9 ]. It is widely accepted that physicochemical properties of nanoparticles (NPs), predominantly size and morphology, determine their action inherently in in vitro and in vivo applications [ 10 ]; previous findings indicated that the smaller sizes of AuNPs, between 10 and 30 nm inserted easier into cancerous tumour cells than larger sizes [ 11 ]. Surface‐coated AuNPs showed more cell uptake in smaller sizes, 20 nm, than 40 and 80 nm [ 12 ].
Furthermore, The NPs' size and shape play a fundamental role in long circulation, biodistribution, and releasing drugs in delivery systems, so the smaller size and spherical morphology is a good candidate in this regard. Smaller size NPs allow faster drug release due to the larger surface‐to‐volume ratio and the more potential for cellular uptake of spherical morphology [ 13 ]. As reported by previous studies, the shape of NPs is one of the significant determinative factors of desirable applications in biomedicine; since the spherical shapes of AuNPs revealed higher sensitivity and specificity in biosensors [ 14 ], while elongation and increase of sharpness of nanostructure made them more favoured in photothermal therapy and imaging due to more substantial near‐infrared absorbance [ 15 ].
Although the smaller sizes of AuNPs are typically preferred in various applications, this may present some potential drawbacks. The minimal size of these NPs, under 5 nm, is reported to have higher toxicity due to their chemical reactivity [ 16 ]. Moreover, previous findings reported that spherical AuNPs with a size of 1.4 nm could induce oxidative stress, mitochondrial damage, and necrosis in studied cell lines. In contrast, there was no evidence of cell damage for 15 nm spherical AuNPs with the same surface group [ 17 ]. Therefore, given that the toxicity of AuNPs is size‐dependent, the

AuNPs should be prepared in the optimal and appropriate size for each application type, along with fewer adverse effects. Accordingly, we aimed to design various synthesis experiments to optimise the fabrication of AuNPs with a desirable size of nanospheres and acceptable synthesis productivity towards biological applications. For this purpose, different experiments were designed using the Turkevich method by manipulating reaction conditions of this common synthesis approach, including initial temperature, initial PH of reaction, and a various range of Trisodium citrate/HAuCl 4 molar ratios. Turkevich method is a relatively convenient and reproducible technique to achieve the small size of gold nanospheres by using Trisodium citrate salt (Na 3 Cit) as a reducing and stabilising agent. In this synthesis method, the reduction of gold chloride salt in aqueous solution results in the synthesis of monodisperse AuNPs suspensions with tunable particle size. In this reaction, to achieve a particle size of less than 20 nm, 1 ml of 1% Na 3 Cit solution should be suddenly added to boiling HAuCl 4 solution with a concentration of 0.01 by weight. After 5 min, the complete colour change indicates the formation of AuNPs [ 18 ]. Moreover, given the significant impact of the additional orders of precursors on final particles' characteristics and synthesis efficiency [ 19 ], in the current study, all designed synthesis experiments were also carried out in two different addition orders of reagents, HAuCl 4 and Na 3 Cit salt.
Tetrachloroauric (III) acid trihydrate (HAuCl 4 .3H 2 O) and Trisodium citrate (Na 3 C 6 H 5 O 7 .2H 2 O) were respectively purchased from Shirazchem Co. and Kimia mavad Co, Iran. Sodium hydroxide (NaOH) and Hydrochloric acid (HCl) were purchased from Sigma Chemical Co., St. Louis, Mo.
Before each synthesis process, the round‐bottom flask was washed with freshly prepared aqua regia acid solution, a mixture of NaOH and HCl with a molar ratio of 1:3, to prevent contamination. Then, 10 ml of deionised water was added to 58 μL of 0.05 M HAuCl 4 . Formerly, the specific concentrations of Na 3 Cit solution were suddenly added to the mixture during vigorously stirring.

Constant air pressure is needed before the addition of the reducing agent. The molar ratio of 0.7, 1.4, 2.1, 2.8, 3.6, 4.3, 5, 5.7, 6.5, and 7.2 were considered for Na 3 Cit/HAuCl 4 in designed experiments. After about 5 min vigorously stirring at boiling temperature and complete colour change, the synthesis process was performed. Due to the size of the final synthesised NPs, this colour change can be in the range of orange‐red to violet. It was gradually cooled to room temperature and finally stored at 4°C. The Na 3 Cit/HAuCl 4 molar ratio: 0.7–7.2 and initial temperature: 25, 55, 65, 75, 85, 95 and initial pH value: 1–9. The synthesis experiments were performed in two methods: adding the specific concentrations of Na 3 Cit solution to boiling gold salt solution in the method I and adding HAuCl 4 solution to boiling Na 3 Cit solution in different concentrations, method II.
Since there is a necessity to develop cost‐benefit techniques of NPs synthesis and acceptable efficiency, in this study, the efficiency of different designed experiments was compared with the benefit of the Beer‐Lambert law. Given the Beer‐Lambert law, the final concentration of synthesised AuNPs directly correlated with surface plasmon resonance absorbance in the maximum wavelength [ 20 ]; thus, in this study, high absorbance was considered more effective for synthesis (Equation ( 1 )). (1) A = b C ϵ
A = absorbance b = length of light path C = concentration ϵ = molar absorptivity.
Gold nanoparticles synthesised with different experiments were characterised by Fourier transform infrared spectroscopy (FTIR Spectroscopy, Vertex 70, Bruker, Germany) to assess their chemical properties. Dynamic light scattering (DLS analyser, Microtrac) to calculate particle size distribution and UV/Visible spectroscopy in the 400–700 nm region (UV/Visible spectrometer, PG Instruments Ltd) to estimate surface plasmon resonance. In addition, the more detailed characterisation tests, including transmission electron microscopy (TEM, Zeiss‐EM10C‐100 kV, Zeiss co)

Effect of high gold salt concentrations on the size and polydispersity of gold nanoparticles prepared by an extended Turkevich–Frens method — Kara Zabetakis et al., 2012

The synthesis of colloidal gold via citrate reduction was first introduced by Turkevich et al. in 1951 [ Gold nanoparticles display a variety of properties [ Variations of the Turkevich–Frens method have been investigated in the past years. Ji et al. [ Herein, by expanding the conditions of the Turkevich–Frens synthesis, we report a systematic study regarding the evolution of both size and size distribution of the GNPs formed, when the concentration of initial gold salt solutions and their respective citrate/gold(III) ratios are increased. To examine these trends, GNP solutions have been made under different starting conditions: a series of seven gold salt concentrations (ranging from 0.3 to 2 mM) have been investigated, and Ct/Au molar ratios from 2:1 to 18:1 have been studied. The sizes and polydispersity indices (PDI) of resulting gold nanoparticles have been measured via dynamic light scattering (DLS) spectroscopy and analyzed to assess for trends in the particle sizes and size distributions. The concentration of the starting gold solution is found to have a significant effect on the size and PDI of the formed nanoparticles. Simulations have also been performed in order to better understand these observations.
Materials/chemicals Tetrachloroauric acid monohydrate (HAuCl Gold nanoparticle synthesis The GNP solutions were synthesized using a modified Frens method [ DLS measurements Samples from the obtained GNP solutions were analyzed by DLS spectroscopy to quantify the average hydrodynamic diameter (Zave) and PDI. DLS measurements were performed with a Malvern Zetasizer Nano ZS (Malvern, Southborough, MA) equipped with a 633-nm He–Ne laser and operating at an angle of 173°. The software used to collect and analyze the data was the Dispersion Technology Software version 5.02 from Malvern. The GNP solutions were stored securely in a hood. These stored solutions have been monitored by DLS over a period of up to 3 months, with an average of four DLS measurements for each stored solution. All the DLS measurements have been then averaged for each individual GNP solution, and the resulting Zave and PDI have been recorded. Model The model used for predictions of the GNPs sizes was the model of Kumar et al. [
To date, the previous studies on

the formation of GNPs based on the Turkevich–Frens method have mostly involved lower initial concentrations of gold salts (≤0.25 mM). The use of higher concentrations of gold salts has received little attention and their effect on the size of the formed GNPs has not been systematically investigated. Herein, we report the study on the formation of GNPs, starting with a series of gold salt concentrations ranging from 0.3 to 2 mM and using Ct/Au molar ratios ranging from 2:1 to 18:1 for each of these concentrations. DLS spectroscopy has been used to measure the hydrodynamic diameter (HD) and the PDI of each prepared GNP solution. Effect of the initial gold salt concentration on the GNP size distribution Figures  Effect of the initial gold salt concentration on the GNP diameter When varying the initial gold chloride concentration, we find that the size evolution of the formed GNPs follows different trends (as a function of the Ct/Au ratios) for gold salt concentrations in the range below 0.8 mM (Fig.  The GNPs synthesized from 0.3 and 0.6 mM gold salt solutions display large hydrodynamic diameters (HD was around 32 and 27 nm, respectively) when a Ct/Au ratio of 2:1 is used (Fig.  The size evolution found for GNPs prepared from 0.8 mM gold salt solutions is intermediate between the trends observed with 0.3 and 0.6 mM gold(III) solutions and the trends displayed with Au Figure  In order to assess the reproducibility of the size trends observed when varying Ct/Au ratios, we used initial gold salt solutions of 1.5 mM as a representative example. We performed three gold nanoparticles syntheses in triplicates, using Ct/Au ratios of 4:1, 6:1, and 10:1. The resulting sizes were plotted in Figure S To help visualize the difference in the GNP sizes obtained using same Ct/Au ratios but different gold(III) concentrations, Figs.  On the other hand, for the same Ct/Au ratio, different initial gold chloride concentrations give GNPs of different sizes (Figs.  Effect of pH The pH of the reacting mixture (gold salt + citrate) has an important role in the formation of the gold nanoparticles,

as discovered by Ji et al. [ Modeling The model of Kumar et al. [ As shown in Fig.  Figure  Relationship between the evolution of GNP sizes and their respective PDI Interestingly, for each initial concentration of gold chloride solution, the evolution of the GNP diameter as a function of Ct/Au ratio follows a similar trend as to the evolution of its PDI. Consequently, for gold(III) solutions below 0.8 mM, the largest monodisperse GNPs (PDI < 0.1) [ The sizes and size distributions of GNPs were also measured by TEM to verify the trends observed by DLS. Samples of GNPs with initial gold(III) concentrations of 0.3, 0.6, 1.2, and 2 mM were selected. For each of these concentrations, TEM images were taken for Ct/Au ratios of 4:1, 6:1, and 10:1. The mean sizes and size distributions were measured for each of the 12 samples (Fig. 
By extending the conditions of the Turkevich–Frens method, it has been found that two groups of gold chloride concentrations present two different behaviors as a function of the Ct/Au ratios. Gold salt solutions below 0.8 mM lead to the formation of highly monodisperse GNPs for Ct/Au ratio over 4:1. The size of the formed GNPs presents a minimum at Ct/Au ratios around 4:1–5:1 and saturates at high ratios (>10:1). However, gold salt solutions over 0.8 mM lead to the formation of monodisperse GNPs (PDI < 0.1) [
The authors are grateful to Dr. Tiberiu-Dan Onuta for his very helpful suggestions and comments on this work, as well as for the creation of the residual data in Figure S Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited.

Turkevich in New Robes: Key Questions Answered for the Most Common Gold Nanoparticle Synthesis — Maria Wuithschick et al., 2015

and Discussion

Question #1: What is the general growth mechanism of the Turkevich synthesis?

As mentioned in the introduction, the number of proposed growth mechanisms is almost the same as the number of publications on the topic itself. Therefore, the starting point for a comprehensive study on the Turkevich synthesis needs to be a discussion of the general growth mechanism.
Most of the deduced Turkevich mechanisms are derived from analytical methods which (i) might not reflect the properties of the colloidal solution (ex-situ methods, e.g. electron microscopy), (ii) cannot deliver decisive information on the particle concentration (e.g. UV-vis spectroscopy), or (iii) cannot be applied in a sufficient time-resolution (e.g. in-situ AFM 20 ). SAXS was shown to be a versatile tool for the investigation of nanoparticle growth mechanisms since it delivers in-situ information on the particle size distribution and the relative particle concentration. Combined with a free liquid jet, the method offers the benefits of a container-free measurement (avoiding contamination problems) and minimized inducing effects of the incident X-ray beam. If the setup is applied at a synchrotron light source, the time-resolution can be in the range of milliseconds. Coupled with XANES, simultaneous tracking of the chemical reduction becomes available. Therefore, time-resolved SAXS/XANES measurements deliver the decisive information necessary to deduce a comprehensive growth mechanism of the Turkevich synthesis.
A schematic, which can be found in Fig. 1, illustrates the mechanism deduced from SAXS/XANES measurements by Polte et al. 26,27 which was refined recently. 32 The mechanism comprises four steps.
The first step is a partially reduction of the gold precursor and the formation of small clusters from the Au monomers. In a second step, these cluster form seed particles with radii > 1.5 nm. The remaining gold ions are attracted and attached in the electronic double layer (EDL) of the seed particles as co-ions.
The third and fourth steps comprise the reduction of the ionic gold (first slowly, then fast) whereby the generated gold monomers grow exclusively on top of the seed particles' surfaces until the precursor is fully consumed. Therefore, no new particles are formed during the last two steps. It should be mentioned that as early as

1958, Takiyama also recognized that the number of particles remains constant after a short initial phase although the time resolution of his applied experimental procedure was very limited. 14 Although Takiyama deduced a crucial point of the growth mechanism, his work remained widely unrecognized. Figure 1. General growth mechanism of the Turkevich synthesis as deduced by Polte. 32 Herein, this growth mechanism is shown to be valid for a wide range of parameter variations.
The described mechanism can be referred to as seed-mediated growth mechanism. It has to be distinguished clearly from a so-called nucleation-growth mechanism. The latter is based on the formation of "nuclei", a term which refers to small clusters consisting of very few atoms. In contrast, the herein described growth mechanism is based on the formation of seed particles, which already have stable sizes and consist of some hundred atoms (for example, a AuNP with r = 1.5 nm consist of ~840 atoms).
As mentioned above, several publications claim the formation and subsequent fragmentation of large gold aggregates in the beginning of the Turkevich synthesis. 9, This assumption is based on the shift of the plasmon band maximum from 530 nm to approx. 520 nm during the early stage of the synthesis (corresponding to a color change from blue-purple to red), time-resolved DLS measurements and ex-situ TEM images which show large networks of particles. To disprove this hypothesis, ultra-small angle X-ray scattering (USAXS) measurements at different reaction times were made at the DORIS III synchrotron light source using a flow-through capillary. USAXS allows to detect AuNP aggregates since the determination of large objects (r > 25 nm) demands the measurement at very small angles (corresponds to low q values, with scattering vector magnitude defined as q = (4p/l)sinth). Fig. 2 shows selected scattering curves and corresponding fits for a Turkevich synthesis carried out under same conditions as described in 26 (T = 75degC, final concentrations [HAuCl 4 ] = 0.25 mM, [Na 3 Ct] = 2.5 mM, mixing of equal reactant volumes). Even at the minimal accessible angle, which corresponds to q = 0.04 nm -1 , no significant scattering signal is detected throughout the synthesis. This confirms that large structures are not formed at any

time of the Turkevich synthesis. The bluish color of the reaction solution at the early stages is most likely caused by the attachment of gold ions in the EDL of the seed particles and a change of their electronic properties. Charging effects are known to influence the optical properties of AuNP significantly. 15,33 Aggregates which were observed in TEM images of samples taken during early stages of the synthesis are most likely artefacts formed during the sample preparation process (drying) and do not represent the actual properties of the colloidal solution. A detailed discussion on the existence or non-existence of aggregates can be found in SI-2. [Na 3 Ct] = 2.5 mM, both mixing of equal reactant volumes). 25 To investigate the general validity of the deduced growth mechanism, another SAXS study with various sets of parameters including a variation of reactant concentrations, the way of mixing the reactants and temperature was carried out at the ESRF synchrotron light source. The results are shown in the supporting information (see SI-1). For all investigated parameter combinations, the different growth steps are observed. Only the duration of each particular step and the corresponding particle mean radii depend on the reaction conditions. The influence of reaction parameters on the growth process will be discussed in detail later in this contribution. From the parameter study it can be concluded that the deduced growth mechanism is valid for a wide range of reaction conditions-at least for sets of parameters commonly used in Turkevich synthesis protocols.

Summary #1:

The Turkevich synthesis is characterized by a seed-mediated growth mechanism.
Stable seed particles with sizes of r > 1.5 nm are formed in the beginning of the synthesis. Remaining gold ions are attached and reduced in the EDL of the seed particles and grow exclusively onto the existing particles. New particles are not formed. Therefore, the total number of particles at the end of the synthesis corresponds to the number of seed particles and is determined already in the beginning of the synthesis. The formation of large aggregates ("nanowires") during the course of the reaction can be excluded.

Question #2: When is the final particle size determined?

According to the growth mechanism of the Turkevich synthesis, the final number of particles corresponds to the number of seed particles formed in step 2. The number of particles determines the final size because it defines to how many particles the

Little adjustments significantly improve the Turkevich synthesis of gold nanoparticles. — Florian Schulz et al., 2014

Introduction

Gold nanoparticles (AuNPs) are among the most widely used and studied nanomaterials and have numerous applications in nanomedicine, biotechnology, microelectronics, optics and catalysis.
1-8 The Turkevich protocol 9 for the synthesis of citrate-stabilized AuNPs is considered most popular for several reasons. The procedure is very straightforward and reliably produces AuNPs with diameters from 5 to 150 nm which are nontoxic and well stabilized by citrate ions. 10 The citrate stabilizer can easily be exchanged by ligands with higher affinity to gold, especially thiols, allowing for (multi)functionalization of the AuNPs.
The mechanism of the Turkevich synthesis is much more complex than the simple procedure might suggest and until today studies aim at a better understanding of the mechanism to assist optimization of the protocol. Regarding such optimization, improvements of the size distribution, indicated by a low coefficient of variation (CV), uniformity, reproducibility and control of the AuNP-diameter were addressed by most studies, but scaling up has also gained interest. The state of the art in the synthesis of AuNPs was reviewed recently. 10 To understand the influence of the various parameters on the outcome of the synthesis it is helpful to consider separately the chemical reactions and processes involved on the one hand and the mechanisms of particle nucleation and growth on the other hand. These are presented in Figure 1.
It can be considered consensus that a fast nucleation in the Turkevich synthesis leads to AuNPs with narrow size distribution. 10,14,23,27 A fast nucleation can be achieved by increasing the reactivity of the precursor or by increasing the concentration of the intermediate acetondicarboxylate [ADC] ( Figure 1). 14,23,27 [ADC] can be increased easily by reversing the reagent addition of the original Turkevich protocol, i.e., injecting the Au precursor into a boiling aqueous solution of sodium citrate. This inverse addition of reagents promotes the thermal oxidation of sodium citrate (SC) and the subsequent formation of ADC, which results in a faster nucleation and an improved dispersity of the final AuNPs. 23 This method is referred to herein as inverse method.
The following key parameters for the synthesis can be identified based on available literature: the concentrations of precursor, [HAuCl

4 ], sodium citrate, [SC], and acetondicarboxylate, [ADC], the electrolyte concentration, the temperature and heating time and the pH. 9,18,23,27,28 Among these, the pH seems to be the essential parameter in the synthesis, but due to the complex interplay of parameters, its role can be conflicting. Especially interesting is the work of Peng's group who studied the pH-dependent reactivity of the Au precursor. 14 Similarly, Puntes' group reported the interplayed role of [ADC], solution pH and the AuNPs' CV. 23 In another study, Xia et al. reduced the buffer effect of the citrate (leading to an increase in pH) by fast mixing of the reagents and catalysis of ADC formation with silver(I)-ions to obtain uniform AuNPs with low CV. 9,14-16,27,29 a.) The Redox-reaction of citrate and the Au(III)-precursor yields Au(I)-ions and acetonedicarboxylate (ADC). b.) ADC organizes Au(I)-ions in polymolecular complexes to yield high local Au(I)-concentrations, which allow c.) disproportionation of Au(I) to Au(0) and Au(III) and d.) the nucleation and growth of Au(0) to AuNPs. The pH influences not only the protonation of citrate and therefore the electrostatic stabilization of the growing and final AuNPs but also the reactivity of the precursor and thus the rate and extent of nucleation. A fast nucleation and good stabilization promote a narrow size distribution of the AuNPs, whereas slow nucleation and low stabilization result in a broad size distribution of the AuNPs due to temporal overlap of nucleation and growth and aggregative growth.
Here, we show that in fact at high [SC]/ [HAuCl 4 ]-ratios AuNPs with very low CV can be synthesized by controlling the pH at a rather low value of ~5.5. At this low pH, the dispersity was further improved by optimizing the mixing conditions. Interestingly, the shape uniformity of the AuNPs was significantly improved by the addition of small amounts ethylenediaminetetraacetate (EDTA). With optimal conditions, 1000 ml of ~3.5 nM

quasispherical AuNPs with high uniformity and a CV as low as 5-6 % were synthesized in a highly reproducible manner. The relative standard deviation (SD rel ) of the mean diameter, d ~12 nm, of several batches was < 3 %. To our best knowledge, this is the lowest CV and the best reproducibility of citrate-stabilized AuNPs in this size regime demonstrated so far, which are also superior to all commercially available AuNPs. At the same time the protocol is very straightforward and robust and can be easily implemented by researchers to synthesize large quantities of high quality AuNPs. This is a great advantage for any research objective and application that benefits from high reproducibility and comparability of different batches and for improved quantification of surface-related parameters like ligand coverage and catalytic activity. Also, for the comparison of experiment and theory, e.g. in the field of plasmonics, for their use as a standard for calibration and for the selfassembly of highly ordered superstructures well-defined particles are highly desirable.

Methods

Materials. Tetrachloroauric(III) acid (>=99.9 % trace metals basis) and trisodium citrate dihydrate (>=99.0 %) were ordered from Sigma Aldrich. Ethylenediaminetetraacetic acid tetrasodium salt hydrate (EDTA) and citric acid monohydrate (>=99.5 %) was from Merck. a-Methoxypoly(ethylene glycol)-o-(11-mercaptoundecanoate) (PEGMUA) was synthesized as described previously. 30 Ultrapure water (18.2 M*cm, Millipore) was used for all procedures.

Syntheses of AuNPs. INVERSE METHOD.

23 97.1 mg (0.33 mmol) trisodium citrate dihydrate (SC) were dissolved in 150 ml water (c = 2.2 mM) and the solution was heated to reflux in a 250 ml three-necked flask equipped with a Dimrothcondenser. The boiling time before precursor-addition was [?]t = 15 min unless noted otherwise. 1 ml precursor solution (HAuCl4*3H2O in water, c = 25 mM) was then quickly injected under