- Nano Express
- Open Access
Synthesis of Gold Nanoparticles with Buffer-Dependent Variations of Size and Morphology in Biological Buffers
- Syed Rahin Ahmed†1,
- Sangjin Oh†2,
- Rina Baba3,
- Hongjian Zhou4,
- Sungu Hwang5,
- Jaebeom Lee2Email author and
- Enoch Y. Park1, 6Email author
© Ahmed et al. 2016
- Received: 30 December 2015
- Accepted: 28 January 2016
- Published: 4 February 2016
The demand for biologically compatible and stable noble metal nanoparticles (NPs) has increased in recent years due to their inert nature and unique optical properties. In this article, we present 11 different synthetic methods for obtaining gold nanoparticles (Au NPs) through the use of common biological buffers. The results demonstrate that the sizes, shapes, and monodispersity of the NPs could be varied depending on the type of buffer used, as these buffers acted as both a reducing agent and a stabilizer in each synthesis. Theoretical simulations and electrochemical experiments were performed to understand the buffer-dependent variations of size and morphology exhibited by these Au NPs, which revealed that surface interactions and the electrostatic energy on the (111) surface of Au were the determining factors. The long-term stability of the synthesized NPs in buffer solution was also investigated. Most NPs synthesized using buffers showed a uniquely wide range of pH stability and excellent cell viability without the need for further modifications.
- Gold nanoparticles
- Good’s buffer
- Synthesis route
- MD simulation
- Cell viability
Gold nanoparticles (Au NPs) are one of the most intensely studied materials in the field of nanotechnology. Recent research interest in Au NPs has stemmed from their peculiar localized surface plasmon resonance (SPR) behavior as well as their size- and shape-dependent physicochemical characteristics, properties that are not observed in bulk Au [1–3]. The surface chemistry of Au NPs permits binding with thiols and amines [4, 5], allowing for easy tagging of the NPs with various proteins and biomolecules. These properties have led to important biomedical applications including selective targeting [6–8], cellular imaging [9, 10], and biosensing [11–14]. Although Au NPs are commonly believed to be chemically inert, recent evidence reveals their high catalytic activity that emerges at significantly reduced particle sizes [15–17], and more importantly, with deliberately tailored shapes introducing high-index facets on the NP surfaces. Au NPs are required in many commercial and industrial applications, with novel emerging applications dramatically increasing global demand for Au NPs. These uses include biomolecule- and/or biopolymer-conjugated Au NPs acting as bio-markers and bio-delivery vehicles in medicine and pharmaceuticals, as anti-aging components in cosmetic products [18–20], for permanent coloration of valuable wool or cotton textiles , as novel coatings and paints created from polymer/gold nanocomposites , as non-volatile memory devices and metal printing inks in electronics [23, 24], and as substrates in surface-enhanced Raman scattering studies [25, 26]. Au NPs do not affect the functional activity of biomolecules after binding, which in turn can be used for the detection of specific target analytes . Because of all the above listed advantages, Au NPs are used in the development of lateral flow assays, a one-step in situ screening test for the analyte.
In particular, more attention must be given to completely biocompatible and agglomeration-free syntheses of Au NPs with diverse biomedical applications in order to meet the increasing demand. Current Au NP preparations are limited by phase transfer, as surface modification causes difficulty in transferring Au NPs to a designated solvent. Many applications of Au NPs require the particles’ introduction to biological buffer systems, and researchers have often failed to transfer Au NPs from their aqueous system to physiological media due to the NPs’ instability, seriously limiting their therapeutic and diagnostic applications [28, 29]. In this process, Au NPs typically lose their original plasmonic band, becoming dark brown precipitates. The ability to develop simple, reliable, and accessible methods to efficiently control and manipulate Au NP size and morphology in physiological media remains one of the most important endeavors in this field of research.
Organic biological buffers can advantageously replace mineral buffers in many biological research and analysis applications. Several biological buffers such as PIPES, MES, and TAPSO are useful for in vitro cell culturing, enzyme assays, protein crystallization (bicine), medicine (triethanolamine, TEA), and some electrophoretic applications (e.g., bis-tris propane, TAPS) (see the “Methods” section to find full names of buffers). Universally applicable buffers for biochemistry must be water-soluble and should not produce chelates or possess complex-forming tendencies with metal ions. However, no buffer is truly and completely inert in biological systems. For example, the Good’s buffers that contain piperazine rings (such as PIPES) can generate nitrogen-centered free radicals. Some buffers have also shown significant affinities with metal ions , resulting in the formation of metal complexes with appreciable association constants. While Habib et al.  reported the successful synthesis of Au NPs in MES and HEPES buffers, fine-tuning of the structures and sizes of the NPs remains an ongoing challenge, and the study of Au NP formation with different buffers is also necessary.
In this study, we have developed 11 synthetic methods to obtain Au NPs, 10 of which were developed using biological buffers. Here, all reagents used in these syntheses also acted as reducing and particle-stabilizing agents during synthesis. It is interesting to note that no additional chemicals were required for further stabilization of these NPs. Molecular dynamics (MD) simulations and electrochemical analysis were performed in order to better understand the surface stability of Au NPs co-functionalized with different reagents. We also characterized the resulting Au NPs and discussed their properties.
Synthesis of Au NPs Using Different Buffers
Each synthesis was performed in a 50-mL conical flask under ambient conditions. The flask was washed using King’s solution and a large volume of DI water prior to each synthesis. The concentration of Au, reaction temperature, and reaction time were varied depending on the reducing ability of the buffer used in each process. In these experiments, NP syntheses using mixtures of two or three different buffers were not considered, as these may give more complicated results that could not be explained using current theoretical understanding and simulations, owing to the many electrolytes present in a solution of even a single buffer. In addition, there is a lack of biological or chemical research employing combined buffer solutions for practical reasons. The detail of the synthesis using different buffers is as follows.
Four milliliters of 100 mM aqueous PIPES buffer and 1 mL of 20 mM aqueous HAuCl4 were placed in 36 mL DI water and vigorously stirred at 25 °C. The solution turned deep pink within 1 min, indicating particle formation, where the buffer serves as both reducing and capping agent without heating or cooling.
Four milliliters of 100 mM aqueous MES buffer and 1 mL of 20 mM aqueous HAuCl4 were placed in 36 mL DI water under vigorous stirring at 100 °C for 10 min. The deep pink solution was then cooled while stirring to obtain Au NPs.
One milliliter of 20 mM aqueous HAuCl4 and 4 mL of 100 mM aqueous PIPES-SS buffer were placed in 36 mL DI water under vigorous stirring at 150 °C for 10 min. The solution turned light orange within 5 min, indicating particle formation.
One milliliter of 20 mM aqueous HAuCl4 was mixed with 36 mL DI water and boiled at 100 °C for 5 min. Then, 4 mL of 0.1 M aqueous TAPSO buffer was added, and the particles formed within 30 s.
One milliliter of 20 mM aqueous HAuCl4 was mixed with 36 mL DI water and boiled at 100 °C for 5 min. Then, 4 mL of 0.5 M aqueous TAPS buffer was added; the reaction was allowed to continue for 1 h.
One milliliter of 20 mM aqueous HAuCl4 was mixed with 36 mL DI water and boiled at 100 °C for 5 min. Then, 4 mL of 0.05 M aqueous TES buffer was added, and the reaction continued for 10 min.
One milliliter of 20 mM aqueous HAuCl4 and 4 mL of 100 mM aqueous TEA buffer were placed in 36 mL DI water under vigorous stirring at 25 °C. The solution turned deep blue within 30 s, indicating particle formation.
One milliliter of 20 mM aqueous HAuCl4 was mixed with 36 mL DI water and boiled at 100 °C for 5 min. Then, 4 mL of 0.05 M aqueous bicine buffer was added, and the reaction was allowed to continue for 1 min.
One milliliter of 20 mM aqueous HAuCl4 and 4 mL of 100 mM aqueous bis-tris methane buffer were placed in 36 mL DI water under vigorous stirring at 25 °C. The solution turned a deep pink color within 30 s, indicating particle formation.
One milliliter of 20 mM aqueous HAuCl4 and 4 mL of 100 mM and aqueous bis-tris propane buffer were placed in 36 mL DI water under vigorous stirring at 100 °C for 5 min. The solution turned light pink within 5 min, indicating particle formation.
One milliliter of 20 mM aqueous HAuCl4 was placed in 36 mL DI water under vigorous stirring at 150 °C for 20 min. Then, 4 mL of 100 mM aqueous PSTT was added with continued stirring. The solution turned light pink within 5 min, indicating particle formation.
pH Stability, Spectroscopic, and Microscopic Analysis of Synthesized NPs
After synthesizing NPs using the above methods, aggregation tests were performed across a wide range of pH values. Aqueous Au NP solutions at different pH values were prepared by mixing adequate amounts of NH4OH in demineralized distilled water to form basic solutions (pH 8–9), whereas acidic media (pH 2–6) was prepared with HCl in water. The pH stability for each solution was monitored using a digital pH meter (Model HM-25R, DKK-TOA, Tokyo, Japan). Ultraviolet-visible absorption spectra were also collected (Infinite M 200, Tecan, Männedorf, Switzerland). Transmission electron microscopy (TEM) images were obtained at an acceleration voltage of 120 kV (TEM-1400, JEOL, Tokyo, Japan). Chemical reactions and surface functional groups were monitored using FT-IR spectroscopy (FT-IR 6300, JASCO Corp., Tokyo, Japan).
Electrochemical Analysis of NP Synthesis
Electrochemical analyses were performed using cyclic voltammetry (IVIUM soft, Ivium Technology, Eindhoven, Netherlands) to monitor the reduction ability of the buffers. An aqueous solution of 0.1 M buffer was used for each measurement with 0.2 M sodium sulfate as a supporting electrolyte. The working electrode was a glassy carbon (GC) electrode, which was polished with 0.3 μm alumina powder on soft lapping pads and deionized water to achieve a mirror-like surface. An Ag/AgCl electrode (3.0 M NaCl) was used as the reference electrode, and a platinum wire was used as the counter electrode. All the electrodes were purchased from ALS Co., Ltd., Tokyo, Japan. The cell volume was 20 mL, and the scan rate was 0.1 V · s−1. Cyclic voltammograms were recorded in the potential range from −0.1 to 1.5 V. All measurements were performed at room temperature (the range of 20 to 25 °C) and ambient pressure.
Computational Simulation of Buffer-Induced Synthesis of Au NPs
The interactions of buffers and their mixtures with the Au NPs were determined using computational simulations. The dimensions of the Au surface used for Au/buffer molecule interface simulations were 8.65 × 8.65 × 9.42 Å for Au (111). First, buffer models were built with the amorphous cell module in the material studio. A packing model featuring a density of 1.0 g cm−3 and containing one buffer molecule was constructed by the amorphous cell module. A 5000-step energy minimization was performed at the initial stage to eliminate undesirable contacts (e.g., overlapping or close contact). Molecular dynamic calculations were performed using three models in order to obtain a stable configuration, and the calculations were conducted for 100 ps at 298 K under the canonical ensemble (NVT) where the amount of substance is defined as N, volume as V, and temperature as T. These models were based on the experimental conditions for the reduction of Au NPs by the different buffer solutions.
Cytotoxicity evaluation of the Au NPs was performed using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, as described by Mossman . Approximately 2 × 103 HEK 294T cells in their exponential growth phase were seeded in a flat-bottomed 96-well polystyrene-coated plate and incubated for 48 h at 37 °C in a 5 % CO2 incubator. After incubation, the medium was discarded and 100 μL fresh medium was added per well to the cells, after thorough washing with sterile phosphate-buffered saline (PBS). Next, 10 μL of Au NPs from each sample was added to the plate, in which the NP amount was selected from the previous optimization experiments for biocompatibility . After 24 h of further incubation, 10 μL of MTT reagent was added to each well, and a final incubation period of 4 h progressed. The media were then discarded from the wells, and 200 μL dimethyl sulfoxide was added to solubilize the formazan crystals that had formed in the interim. Readings were recorded using a Bio-Rad enzyme-linked immunosorbent assay reader at 490 nm, with subtraction for plate absorbance at 530 nm.
Redox potentials of buffers in a 0.2 M sodium sulfate electrolyte solution
Reduction peak (V)
Oxidation peak (V)
UV-Visible Absorption Spectra of the Synthesized Au NPs
Electron Microscopy Analysis of NP Morphology
In our buffer-based synthesis systems, these buffers displayed significant affinity to a number of metal ions, resulting in the formation of metal complexes. However, these buffer complexes are not stable and are readily oxidized by Au(III). This oxidation is accompanied by the simultaneous reduction of Au(III) to Au(II)/Au(I), and finally to Au(0), which results in the formation of Au NPs. NP formation can be completed within a few seconds to a few hours, depending on the buffer chemical, and the formed particles are stable for periods ranging from a few days to several months. BTM, containing five hydroxyl groups, was found to induce the spontaneous, instant formation of Au NPs at room temperature. The second fastest Au NP formations occurred when using the TEA and PIPES buffers. The three hydroxyl groups in TEA and the –SO3H groups and piperazine rings in PIPES and PIPES-SS were able to reduce Au(III) to Au(0) within a few minutes at room temperature. Note that this short reaction time may induce less size and shape homogeneity in the formed NPs, as it provides insufficient time to allow for Ostwald ripening. The other buffers contain either electron-donating groups or fewer electron-withdrawing groups, which result in slower oxidation reactions; these require heating at 100 °C to accelerate the formation of Au NPs. Unreacted buffer salts may introduce additional confusion to the research in this field. Additional file 1: Figure S4a depicts unreacted buffer salts with cubic structures. After washing and re-dispersing in water, these salts form urchin-like structures (Additional file 1: Figure S4b), which ultimately break into many small parts (Additional file 1: Figure S4e).
Zeta Potential and pH Stability of Au NPs
Binding Interaction of Reducing Agents and Au Surface by MD Simulation
Interaction energies between Au NPs and surfactants (units: kcal · mol−1)
where E Au is the energy of the Au surface, E reagent is the energy of the reagent molecules, and E total is the energy of the Au surface with the organism. Note that high interaction energy indicates high adhesive strength between the reagent and the Au surface and a more stable model in general. The interaction energies between Au and the reagents were calculated using the COMPASS force field as shown in Table 2. The simulation results produced negative values for the interaction energies of all systems, clearly indicating that the reagents were easily combined with the surface of the Au crystal. Moreover, among all models, the interaction energy of the PIPES-Au model was the highest, indicating that the strongest interaction existed between PIPES and the Au (111) surface. The results of these MD simulations provide strong evidence to help explain the relationship between the reduction of Au NPs by different reagents and the stability of these formed NPs. In particular, both theoretical and experimental observations agreed on the long-term stability of Au NPs formed using PIPES buffer.
Biocompatibility of Au NPs
The present work investigated one-step syntheses of Au NPs using common biological buffers. The synthesized Au NPs were characterized using TEM, dynamic light scattering, UV-vis spectroscopy, and electrochemical experiments. Theoretical investigations were also performed using molecular dynamic simulations in order to understand the interaction between the reagents and the Au surface. The results revealed that Au NPs synthesized using the PIPES buffer have the highest zeta potential and highest interaction energy, producing the most stable NPs of the group. An MTT assay was performed to check cytotoxicity of Au NPs on HEK 293 cells, which indicated lack of any noticeable toxicity of NPs. Such Au NPs can provide new opportunities for safe and convenient applications in molecular imaging, drug delivery, therapy, and biosensors.
This work was supported in part by the Promotion of Nanobio-Technology Research to support Aging and Welfare Society funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the International Research & Development Program of the National Research Foundation of Korea (NRF-2014K2A2A4001081) and the Korean Government (MSIP) (No.2015R1A5A7036513).
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