Size-Controlled Green Synthesis of Highly Stable and Uniform Small

Apr 18, 2017 - Department of Chemistry and Physics, Florida Gulf Coast University, Fort Myers, Florida 33965, United States. § Department of Mechanic...
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Size-Controlled Green Synthesis of Highly Stable and Uniform Small to Ultrasmall Gold Nanoparticles by Controlling Reaction Steps and pH Bo Yang,†,§ Ju Chou,*,‡ Xiaoqing Dong,§ Chengtun Qu,† Qingsong Yu,§ Kerry J. Lee,∥ and Natalie Harvey‡ †

College of Chemistry and Chemical Engineering, Xi’an Shiyou University, Xi’an, Shaanxi 710065, China Department of Chemistry and Physics, Florida Gulf Coast University, Fort Myers, Florida 33965, United States § Department of Mechanical and Aerospace Engineering, University of MissouriColumbia, Columbia, Missouri 65211, United States ∥ Department of Biology, Florida Gulf Coast University, Fort Myers, Florida 33965, United States ‡

ABSTRACT: Synthesis of gold nanoparticles (AuNPs) with controllable particle size and stable dispersion through green chemistry without using toxic regents is crucial for biomedical applications. In this study, spherical AuNPs with controllable particle size in the range of 8 to 18 nm were synthesized by the reduction of HAuCl4 using only fruit juices/extract without adding any other chemicals. By controlling the chemical reaction steps and adjusting the pH of the solution at a later stage of the reaction, the sizes of the spherical AuNPs were fine tuned to 4.5 ± 2.0 nm, 5.9 ± 2.5 nm, and 6.0 ± 1.5 nm with fruit juices/extract of A. deliciosa, P. persica, and M. domestica, respectively. For the first time, spherical ultrasmall AuNPs of 2.6 ± 1.1 nm with uniform distribution were successfully achieved using M. Acuminate extract at pH 10 and 11. The ultrasmall and small AuNPs were imaged again by transmission electron microscopy (TEM) after 4 months stored at the room temperature and 72 h incubation in 1 mM NaCl, which is typically found in biological media. No aggregation was observed in the above AuNP solutions after four months and incubation for 72 h in NaCl. These results indicate that highly stable AuNPs synthesized through green chemistry can be used in cells or embryos and hold great promise in biological applications.

1. INTRODUCTION Due to their extremely small size, large surface to volume ratio, attractive electronic/optical/thermal properties, and catalytic properties, nanoparticles are of great interest in the fields of physics, chemistry, biology, medicine, material science, and also many other interdisciplinary fields.1,2 Many of their important applications include catalysis,3 biological labeling,4 drug and gene delivery,5 tissue engineering, and optoelectronics.6,7 To date, various metal nanoparticles have been fabricated with controllable particle shape (spherical, hexagonal, triangular, and rod like) and size (1.0−150 nm) for many novel applications.8 Gold nanoparticles (AuNPs), also known as colloidal gold, are the most widely used noble metal nanoparticles in biological systems due to their stability, less toxicity, and biocompatibility.9 The properties and applications of AuNPs strongly depend on their sizes, shapes, and morphology. Therefore, many research works have been reported with the aim of synthesizing AuNPs into desired shape and size in a controllable manner.10−13 In general, the synthesis of AuNPs is based on a redox reaction between Au3+ and a reducing agent and then followed by protection of the freshly formed nanoparticles with a © XXXX American Chemical Society

protecting or stabilizing agent to prevent their aggregation. Most of these techniques often involve the use of reducing chemicals such as sodium borohydride,14 hydrazine,15 and triethylamine16 as well as a protecting agent, which are often toxic. When the chemically synthesized AuNPs were used in biological tests such as toxicity in various types of cells, the results often varied.17−22 Some studies show a toxic effect, and other studies report no significant cytotoxicity. One important factor that leads to inconclusive results is that the chemicals present in AuNP solution were not removed before these tests.9 Additionally, AuNPs could undergo size and shape changes in biological media if they are not stable. A nontoxic route to make size-/shape-controllable AuNPs without using toxic chemicals and being stable in biological media is preferable for biological applications. One of the most promising methods is through green chemistry using natural products such as natural plants (leaves, roots, rinds, and fruits) that contain various natural antioxidants with reducing power Received: January 14, 2017 Revised: March 15, 2017

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The Journal of Physical Chemistry C to be able to reduce Au3+ to AuNPs.23−28 The green synthesis of AuNPs by using fruit juice/extract as both reducing and stabilizing agents has received significant attention because they are nontoxic, easily available, and renewable. AuNPs formed using such a method seem to be stable, and the rate of synthesis is very fast. As a nontoxic method, synthesis using plant extracts has thus opened a new era in fast production of AuNPs. Despite the advantages of using fruit extracts as both reducing and stabilizing agents, the green synthesis of AuNPs has not been widely applied yet due to the difficulty of controlling the particle sizes and shapes. To date, AuNPs with controllable size, shape, and high stability in biological media have not been achieved through the green chemistry route. It is reported that the sizes of AuNPs can be affected by solution pH.29,30 In this study, the uniform AuNPs were produced by controlling both the reaction step and the pH in order to better control their morphology, shape, and size. As a result, stable ultrasmall to small AuNPs with controllable particle size and shape were successfully obtained with various freshly prepared fruit juices/extract.

solution was adjusted from 3 (control group) to 6, 8, 10, and 11 by adding different volumes of 0.2 M NaOH (0, 20, 30, 80, and 100 μL, respectively) to the reaction mixture. Then 0.20 mL of preheated fresh fruit juice or extract was added into the solution, and the mixture was heated in a boiled water bath for 5 min after the reaction started. Again, the synthesis of AuNPs in different pH conditions was repeated three times by each juice or extract for reproducibility. 2.5. Incubation of AuNPs in 1 mM NaCl. The AuNPs synthesized in this study were stored at room temperature for four months and were incubated in 1 mM NaCl for 72 h. After storing for 4 months and the incubation, the AuNPs were characterized again for their stability by TEM. 2.6. Analysis and Characterization of AuNPs by UV− vis, EDS, TEM, and HRTEM. UV−visible (UV−vis) absorption spectra of the AuNP solutions were recorded on a double beam Shimadzu UV−vis spectrophotometer in the wavelength range of 400−750 nm. A 1.0 cm polystyrene cuvette was used for all UV−vis measurements. A scanning electron microscope (SEM) (JEOL JSM-6360A) equipped with energy-dispersive spectrometer (EDS) was used for the elemental analysis of Au nanoparticles. TEM images of the AuNPs were obtained using a JEOL JEM-1400 120 kV TEM (JEOL USA) to examine the morphology of the AuNPs in terms of size and shape. The HRTEM images were taken on an FEI Tecnai G2 F30 TEM (FEI USA) under 300 keV to image the atomic structure of the AuNPs. The TEM and HRTEM samples were prepared by depositing 5.0 μL of AuNP onto carbon-coated copper grids by wiping away the excess solution with a filter paper 30 min later and were dried in air for another 10 min before measurements. Ten TEM images with the same magnification were taken per sample batch to ensure that there are enough images to represent a significant statistical amount of the tested AuNPs. 2.7. Image Processing. The image-analysis tool ImageJ 1.50i was used for average nanoparticle size and their size distribution analysis based on TEM images.27 The sizes of AuNPs were calculated based on ten TEM images containing at least 100 NPs to obtain statistically significant mean and standard deviation values.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Materials. Hydrogen tetrachloraurate(III) trihydrate (HAuCl4, 30 wt %) was purchased from SigmaAldrich. The working solution of 0.75 mM HAuCl4 was freshly prepared by diluting 2 mM HAuCl4 stock solution with deionized water. Sodium hydroxide (NaOH) was purchased from Fisher Scientific, and 0.2 M NaOH solution was prepared and used to adjust the pH of reaction solutions. Fresh fruit juices/extract were freshly prepared from Actini diadeliciosa (A. deliciosa), Malus domestica (M. domestica), Prunus persica (P. persica), and Musa acuminate (M. acuminate), and all fresh fruits were purchased from a local grocery market. 2.2. Fruit Juice/Extract. The fruits were squeezed to obtain juices, which were filtered using Whatman filter paper (No.5) to remove larger pulp. Each filtered juice sample was centrifuged at 12 000 rpm for 5 min. The clear filtrates of the fruit juice were then collected in an 8 mL disposable vial for further testing. For M. acuminate, because of its low water content, a fruit extract was obtained by the following procedure. An amount of 200 g of M. acuminate was first mixed with 100 mL of deionized water, and then the mixture was boiled for 5 min to extract the antioxidant from the fruit. After it was cooled, the solution was filtered using Whatman filter paper (No.5) and centrifuged as described above. The clear filtrate of M. acuminate extract was collected in an 8 mL disposable vial for further testing. All the fruit juices/extract were freshly prepared and used for the synthesis of AuNPs in the same day. 2.3. Synthesis of AuNPs Using Various Fresh Fruit Juices/Extract. An amount of 0.80 mL of deionized water was added into 2.00 mL of 0.75 mM gold hydrochloride solution in a small vial. The Au3+ solution was heated in a boiling water bath for 2 min, and then 0.20 mL of a preheated (in boiling water bath) fresh fruit juice or extract was added into the above Au3+ solution. The reaction mixture was heated for 5 min in the boiling water bath after the reaction started (the color of Au3+ solution changed). Three trials of synthesis of AuNPs were performed for each fruit juice or extract for reproducibility. 2.4. Effect of pH on AuNP Size Distribution and Morphology. In order to keep the same total volume of the reaction mixtures as 3.0 mL as mentioned in Section 2.3, 0.80, 0.78, 0.77, 0.72, and 0.70 mL of deionized water was added into 2.00 mL of 0.75 mM Au3+ solution. The pH of the Au3+

3. RESULTS AND DISCUSSION 3.1. Formation of AuNPs Using Fresh Fruit Juices/ Extract at Different pHs. A dilute gold ion (Au3+) solution is almost colorless. When a fresh fruit extract was added to a hot Au3+ solution at 100 °C, however, the color of the reaction mixture started to change from colorless to pink, or purple, or red, depending on the fruit juice or extract used. Four fruit juices/extract including A. deliciosa (kiwi), M. domestica (apple), P. persica (peach), and M. acuminate (banana) were tested in the same way in this study. After 5 min reaction, the UV−vis absorption spectra of the reaction mixtures of Au3+ with the four fruit juices/extract were recorded. The reaction mixture of Au3+ with the four fruit juices/extract all had similar UV−vis spectra and all gave an absorption peak with the maximum wavelength (λmax) ranging from 535 to 549 nm. An example of the UV−visible spectra of AuNPs in the A. deliciosa juice is shown in Figure 1a (control). It is well-known that colloidal AuNPs have surface plasmon absorption in the range of 520− 550 nm depending on their particle sizes.23,27,30 Thus, the absorption peak observed in the four fruit juices/extract indicates the formation of AuNPs with different particle sizes. Among the four fruit juices/extracts tested in this study, AuNPs B

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Figure 1. UV−vis absorption spectra of AuNPs formed in A. deliciosa at different pHs.

formed from M. acuminate extract show the lowest λmax at 535 nm, while the AuNPs from the M. domestica juice have the highest λmax at 549 nm. The experimental results indicate that the four fruit juices/extracts used were all able to serve as a reducing agent to reduce Au3+ to AuNPs without adding any other stabilizing/capping agent. In order to investigate the pH effect on the formation of AuNPs in terms of the shape, the size, and particle distribution, the four fruit juices/extract were used to synthesize AuNPs at different pHs of 6, 8, 10, and 11 by adding different amounts of OH− before the reaction started. The AuNP solutions formed in A. deliciosa at different pHs were analyzed using a UV−vis spectrophotometer, and the absorption spectra of the AuNPs obtained at different pHs were shown in Figure 1b−e. The maximum absorption wavelength λmax of the AuNPs shifted slightly to a shorter wavelength (also called blue shift) with increasing solution pH. The λmax was shifted from 538 to 533 nm when the pH was increased from 3 (control) to 11. It was also observed that the absorbance intensity of AuNPs changed with the increase of the solution pH. As the pH increased, the absorbance intensity of the AuNP solution decreased. Compared to the absorbance intensity of 1.2 at pH = 3, only half peak absorbance intensity (∼0.6) was observed at pH 11. The blue shift of the λmax and the decrease in the absorbance intensity were also observed on the AuNPs formed from the other three fruit juices/extract with increasing solution pHs as listed in Table 1. 3.2. TEM Characterization and EDS Analysis of AuNPs. To investigate the size and morphology of AuNPs, the same AuNP colloidal solutions formed in the four fruits juices/extract at different pHs were characterized by TEM. Figure 2 shows the TEM images of AuNPs formed in A. deliciosa at pH of 3 (control), 6, 8, 10, and 11, respectively. Most of the AuNPs prepared in these solutions were spherical as shown in Figure 2,

Figure 2. TEM images of AuNPs formed by A. deliciosa fruit juices in different pHs. (a) Control, pH = 3; (b) pH = 6; (c) pH = 8; (d) pH = 10; (e) pH = 11.

while other shapes such as triangle were occasionally observed mainly from the control sample (without adding any OH−). The sizes of the AuNPs also varied with pH and decreased with increasing solution pH. The particle sizes in different pHs were measured by ImageJ software, and they decreased from 18.2 ± 10.4 nm (control group) to 9.5 ± 1.6 nm (at pH = 11). Not only the size of nanoparticles decreased with increasing pH, but also the size distribution of AuNPs was improved significantly. When the pH was increased to ≥8, all the AuNPs fabricated became spherical, and none of the large particles (diameter >20 nm) were observed at pH ≥ 10. As discussed above, at higher pH, the size of AuNPs decreased. Thus, the blue shift of the λmax observed previously

Table 1. Summary of the Peak Wavelength (λmax), Intensity, and Sizes of the AuNPs Obtained from All Four Fruit Juices at Different Experimental Conditions adjust pH before reactiona

control group fruit source A. deliciosa M. domestica P. persica M. acuminate a

size (nm)

λmax (nm)

abs.

± ± ± ±

538 549 539 535

1.186 1.410 1.397 1.000

18.2 15.7 15.0 8.0

10.4 6.0 3.0 2.7

adjust pH after reactiona

size (nm)

λmax (nm)

Abs.

size (nm)

λmax (nm)

abs.

± ± ± ±

533 532 535 529

0.661 1.155 0.540 0.543

4.5 6.0 5.9 2.6

± ± ± ±

528 531 525 521

0.970 1.268 1.071 0.881

9.5 12.2 9.3 5.3

1.6 3.8 2.6 2.1

2.0 1.5 2.0 1.1

All values on pH changes were taken at pH 11 for both before and after the reaction. C

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be due to the small quantity of AuNPs produced since the absorbance is directly proportional to the concentration of AuNPs. 3.3. Tuning AuNP Size and Size Distribution. The morphology and sizes of AuNPs were affected by the solution pH as discussed. High pH could benefit the decrease of the particle sizes and form only spherical particles with narrow size distribution. However, a negative impact to the quantity of Au nanoparticles was also observed by using all four fruit juices/ extract. At high pH by adding hydroxyl (OH−) ions into the reaction mixtures, the Au3+ ions could react with OH− ions to form gold hydroxide [Au(OH)3] which is highly insoluble (Ksp = 1.0 × 10−53). Under such an experimental condition, very few free Au3+ ions are available to react with the reducing agent in fruit juices/extract and therefore would decrease the formation of AuNPs and thus decrease the absorbance as observed in the UV−vis spectra. To tune the particle size of AuNPs and also to increase the quantity of AuNPs, more free Au3+ ions are required to react with the reducing agent in fruit juices/extract. To prevent Au3+ ions from forming gold hydroxide, the sequence of adding OH− was further adjusted in this study. First, the fruit juice/extract was added into Au3+ solution in order to make sure that the reaction mixture contained more free Au3+ ions to participate in the redox reaction. Then, the OH− ions were added into the reaction mixture to adjust the solution pH. With such new sequence, more free Au3+ ions are available to react with the reducing agent to form AuNPs in the early stage of the reaction, and the reaction mixture is then adjusted to a higher pH to control the growth of particle sizes. To compare with the previous experiments (adding OH− first), although the same amount of OH− ions was added into each mixture of Au3+ ions with the fruit juices/extract, the effect of the OH− ions on the nanoparticle formation/growth stages is different with this new sequence. Figure 4 shows the TEM images of AuNPs fabricated by using the four fruit juices/extract by adding OH− ions at a later reaction stage. As shown in Figure 4, only spherical nanoparticles with uniform distribution were observed on all four fruit juices/extract. There were no large particles observed on the four TEM images as shown in Figure 4a−d, indicating that no aggregation of AuNPs occurred at such an experimental condition. For A. deliciosa, with the new sequence (adjusting the pH after the reaction started), all spherical nanoparticles were well dispersed in the solution at pH = 11. Compared to the TEM image (Figure 2) obtained by adjusting pH before the reaction, the new TEM image (Figure 4a) shows an even smaller nanoparticle. The sizes of AuNPs were changed from the previous 9.5 ± 1.6 to 4.5 ± 2.0 nm with the new OH− adding sequence. TEM results suggested that once the OH− was added into Au3+ solution only small nanoparticles formed in the early reaction stage, and they stopped to grow into large particles at high pHs. Simultaneously the size distribution of AuNPs is narrow. The UV−vis absorption spectra of the above AuNP solution made from A. deliciosa juice at different pHs were shown in Figure 5A. The absorption spectra of AuNPs formed at pH = 10 and 11 show a shaper absorption peak than that in Figure 1d and 1e, indicating that the size distribution of AuNPs was narrowed. The slight blue shift on the λmax was also observed when the solution pH increased, which again is consistent with TEM results. In addition, the absorbance intensity of AuNPs at pH = 10 increased from previous 0.5 (Figure 1d) to 0.9 (Figure

from the UV−visible spectra was associated with the size decrease of AuNPs. Sizes or the diameters of the AuNPs varied with the solution pH. As pH increased, the size of AuNPs decreased. However, the absorbance intensity of the AuNP solution decreased. The decrease in the absorbance intensity of AuNPs indicates that the increase of pH might have a negative effect on the particle quantity, which was also observed in other research groups.31 An energy-dispersive spectroscopy (EDS) analysis was performed on Au nanoparticles fabricated from A. deliciosa at pH = 11. As shown in Figure 3, a strong signal and a weak

Figure 3. EDS spectra of the fabricated AuNPs obtained from A. deliciosa at pH = 11.

signal for the element gold were observed around 2.2 and 9.7 keV, respectively. The EDS analysis confirmed the presence of gold in the Au nanoparticles produced from the fruit. The same AuNP colloidal solutions formed in the other three fruit juices/extract at different pH were also characterized by TEM. The same phenomenon was also observed in these three fruit juices/extract. For the control group, most of the AuNPs observed are spherical, and their mean sizes and standard deviation were obtained from their TEM images and were listed in Table 1. The sizes of AuNPs formed from the four fruit juices/extract ranged from 8.0 ± 2.7 to 18.2 ± 10.4 nm, and their sizes varied and depended on the fruits used. At high pH (10 or 11), TEM examination shows that only spherical nanoparticles were formed, and no other shapes of nanoparticles were produced. No larger AuNPs (above 20 nm) formed in these AuNP solutions. Similar to A. deliciosa, the size of the AuNPs formed in the other three fruit juices/extract varied with pH, and they all decreased when increasing the pH as listed in Table 1. In addition, slightly narrower size distribution was also observed in the three fruits tested. For example, the sizes of AuNPs produced from the M. acuminate extract decreased from 8.0 ± 2.7 nm at pH = 3 (control group) to 5.3 ± 2.1 nm at pH = 10. Thus, adjusting pH could control the growth of AuNPs and prevent the aggregation of AuNPs. Additionally, the blue shift of λmax is consistent with TEM results observed. Among the four fruit juices/extract tested in the pH study, the smallest nanoparticles of 5.3 ± 2.1 nm were produced from the M. acuminate extract at pH = 11. Based on all UV−vis spectra of AuNPs from the four fruit juices/extract, again the blue shift of λmax was due to the formation of smaller particles of AuNPs confirmed from their TEM images. The decrease of the absorbance intensity could D

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Figure 5. (A) UV−vis spectra of AuNP solutions obtained from A. deliciosa at different pHs adjusted after the reaction: (a) control, pH = 3; (b) pH = 6; (c) pH = 8; (d) pH = 10; (e) pH = 11. (B) UV−vis spectra of AuNP solutions obtained from M. acuminate at different conditions: (a) control; (b) pH 10 adjusted before the reaction; (c) pH 10 adjusted after the reaction. Figure 4. TEM (a−d) and HRTEM (e) images of AuNPs formed in different fruit juices/extract at pH 11. (a) A. deliciosa; (b) M. domestica; (c) P. persica. Scale bar for a, b, c: 20 nm. (d) M. acuminate, scale bar 10 nm; (e) M. acuminate, scale bar 5 nm.

The formation of the ultrasmall AuNPs produced from M. acuminate extract was further supported by the blue shift of λmax in the UV−vis absorption spectra shown in Figure 5B. The wavelength of the λmax was shifted from 535 (curve a, control) to 521 nm (curve c) when the solution pH was changed from 3 to 10. Again the absorbance intensity was significantly increased from 0.5 (curve b, adjusting the pH before the reaction) to 0.9 (curve c, adjusting the pH after the reaction). These results indicate that the size of nanoparticles decreased and the quantities of AuNPs increased when adjusting the pH at a later state of the chemical reaction. In this study, different fruit juices/extract were used to synthesize different sizes of AuNPs by two different synthesis routes. As shown in Table 1, various sizes of AuNPs were obtained from 18.2 ± 10.4 nm (A. deliciosa), 15.7 ± 6.0 nm (M. domestica), 15.0 ± 3.0 nm (P. persica), and 8.0 ± 2.7 nm (M. acuminate) by only using each fruit juice/extract itself. No capping or stabilizing agent was added in the synthesis, but a large particle size distribution of AuNPs was observed. However, when the solution pH was adjusted to a basic solution by adding OH− ions at a later reaction stage, narrower size distribution of 4.5 ± 2.0 nm, 6.0 ± 1.5 nm, and 5.9 ± 2.0 nm of AuNPs was successfully synthesized from the A. deliciosa, M. domestica, and P. persica, respectively, as listed in Table 1. More importantly, as shown in Figure 4d, e, ultrasmall AuNPs (2.6 ± 1.1 nm) with uniform size distribution were obtained from M. acuminate extract by increasing pH after the reaction started.

5A, curve d) as expected since more free Au3+ ions were available to participate in the redox reaction to form AuNPs before OH− ions were added. The same trend with pH change was also observed with the AuNPs prepared using the other three fruit juices/extract when adjusting the pH at a later reaction stage. TEM images of AuNPs produced by M. domestica, P. persica, and M. acuminate juices/extracts were shown in Figure 4b, c, and d, respectively. Again only spherical AuNPs were produced, and uniform particle size distribution was obtained with all three fruit juices/ extract. The sizes of Au particles were tuned to 6.0 ± 1.5 nm for M. domestica and 5.9 ± 2.0 nm for P. persica, respectively, as listed in Table 1. For the first time, ultrasmall gold nanoparticles of 2.6 ± 1.1 nm were successfully obtained by M. acuminate extract as shown in Figure 4d. The ultrasmall AuNPs were further examined by a highresolution TEM, and a HRTEM image is shown in Figure 4e. The HRTEM image of the nanoparticles clearly shows the lattice fringes, indicating its high crystallinity. The HRTEM was used to obtain the lattice constant for the fabricated Au nanoparticles from green synthesis. The interplanar distance of planes measured from the HRTEM was 4.100 Å, which is almost identical to the pure gold lattice constant (4.080 Å). For the first time, ultrasmall gold nanoparticles were produced from the fruit extract without adding any other chemicals. E

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Since Au nanoparticles were synthesized by fruit juices/ extract, no additional reducing and stabilizing agents were added. There are no extra chemicals present in AuNP solutions, and they can be used without further wash or purification for biological applications; thus AuNPs produced in this study would be ideal for biological tests. We further characterized AuNPs of the above four AuNPs in 1 mM NaCl solution which is a typical concentration used in most biological medias.33,34 After 72 h incubation in 1 mM NaCl at room temperature, AuNPs were imaged by TEM again, and their size distributions were obtained and listed in Table 2. The TEM images of AuNPs showed all spherical shapes in all four fruit juices/ extract, and no shape and significant size changes were observed. The results indicate that no aggregation was observed after 72 h incubation in the biological media, thus these small and ultrasmall AuNPs remained the same size and shape in the typical biological media and are able to be used in biological tests. The synthesis methods reported here could also open up wonderful new opportunities for green synthesis for other types of metal nanoparticles for biological and medical purposes.

Several important aspects should be pointed out for the synthesis methods used to make AuNPs in this study. First, fruit juices/extracts are from natural plants, which have no toxic effect and will be ideal for biological and medical applications. In addition, the antioxidants in the fruit juices/extracts also act as a capping or stabilizing agent.27,30 Thus, no additional capping agent or other chemical is needed. Second, the size and morphology of AuNPs are dependent on the fruit juice/extract used. The size, shape, and size distribution of AuNPs can be improved by adjusting the solution pH. AuNPs with only spherical shape and uniform size distribution can be been obtained when the solution pH was adjusted to pH = 10 or 11 after the formation process of AuNPs started. Third, as discussed above, based on the thermodynamic and kinetic properties of Au3+ and OH−, the sequence of adding OH− is very important in the synthesis of AuNPs. The OH−ions should be added to the reaction mixture after the reaction starts to make sure more Au3+ ions are available to form AuNPs. Finally, when adding OH− ions into the reaction mixture, the size of the AuNPs formed in the early reaction stage can be well controlled to avoid further growth, and thus the aggregation of AuNPs was prevented to obtain only small nanoparticles. El-Kassas reported that the hydroxyl functional group of polyphenols from plant extracts might have stabilized gold nanoparticles formed.32 It is possible that OH− addition might promote the hydroxyl functional group of polyphenols from fruit juices/extract to prevent the AuNPs from forming large nanoparticles. At the basic solution, Au(OH)3 could also form and could contribute to preventing the aggregation of AuNPs. Since the hydroxyl functional group and/or Au(OH)3 formed prevents AuNPs from aggregation and stabilizes the small AuNPs, they might serve as a stabilizing agent to promote the fabrication of only small, spherical Au nanoparticles. As aforementioned, the chemical and physical properties of AuNPs and their applications all heavily depend on their sizes, shapes, morphology, and dispersion stability. The synthesis methods developed in this study have produced only spherical, uniform AuNPs with different sizes from ultrasmall (2.6 nm) to 10 nm. Therefore, the ecofriendly methods for the production of AuNPs have their advantages in achieving the goal with desired shapes and smaller nanoparticles, which are especially important for biological and medical applications. 3.4. Stability of AuNPs at Room Temperature and Their Characterization in Biological Media. The dispersion stability of the AuNPs synthesized in this study was tested, and their sizes and shape were examined by TEM again. After they were stored at room temperature for four months, the four sizes of AuNPs were obtained by TEM and summarized in Table 2. Among the four fruits tested, all AuNPs produced show constant size without any aggregation over a period of four months. No shape change was ever observed from their TEM images. These TEM results indicate that the AuNPs fabricated in this study are highly stable at room temperature.

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CONCLUSIONS Green synthesis method with fresh fruit juice/extract was used to make AuNPs in this study. Four fruit juices/extract tested were all able to serve as a reducing agent to reduce Au3+ to make AuNPs without adding any other stabilizing/capping agent. The morphology and particle size of AuNPs were found to be dependent on the type of fruit juice/extract used and can be fine-tuned by adjusting the solution pH at different reaction stages. High pH could benefit the decrease of the nanoparticle sizes and form only spherical nanoparticles with narrow size distribution. By adding the OH− ions at a later stage of the reaction, the aggregation of AuNPs was prevented to obtain highly stable small and ultrasmall gold nanoparticles with narrow distribution. Based on t test, there was no statistically significant difference in AuNP size before and after chronic storage at room temperature and no statistically significant difference before and after the incubation in the 1 mM NaCl solution, which is commonly used in biological medias The green synthesis developed for fabricating small and ultrasmall AuNPs not only provides a sound basis for their biological applications but also improves the scientific understanding for gold nanoparticle fabrication in nanoscale.

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Corresponding Author

*E-mail: [email protected]. ORCID

Ju Chou: 0000-0003-3770-8179 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the Key Laboratory Scientific Research Program of Shaanxi Provincial Education Department (No. 14JS085) and Small Grant for Preliminary Study of Florida Gulf Coast University (No. 16120).

Table 2. Mean Size and Standard Deviation of AuNPs after 4 Months and 72 h Incubation in 1 mM NaCl fruit source A. deliciosa M. domestica P. persica M. acuminate

after 4 months (nm) 4.0 5.9 5.8 2.6

± ± ± ±

1.1 1.1 1.5 0.6

after 72 h incubation (nm) 4.5 6.0 5.9 2.6

± ± ± ±

AUTHOR INFORMATION

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2.0 1.5 2.0 1.1

REFERENCES

(1) Islam, N. U.; Ahsan, F.; Khan, I.; Shah, M. R.; Khan, M. A. Green synthesis and biological activities of gold nanoparticles functionalized

F

DOI: 10.1021/acs.jpcc.7b00434 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C with citrusreticulata, citrus aurantium, citrus sinensis and citrusgrandis. J. Chem. Soc. Pak 2015, 37, 721−731. (2) Chow, E. (Editor). Gold Nanoparticles: Properties, Characterization and Fabrication; 2010. (3) Chou, J.; Zhang, S.; Sun, S.; McFarland, E. Benzene formation at 70 degrees C by coupling of propylene on supported Pd nanoclusters. Angew. Chem., Int. Ed. 2005, 44, 4735−4739. (4) Wang, F.; Tan, W. B.; Zhang, Y.; Fan, X.; Wang, M. Luminescent nanomaterials for biological labelling. Nanotechnology 2006, 17, R1− R13. (5) Huang, H.; Quan, Y.; Wang, X.; Chen, T. Gold Nanoparticles of diameter 13 nm induce apoptosis in rabbit articular chondrocytes. Nanoscale Res. Lett. 2016, 11, 249−258. (6) Elia, P.; Zach, R.; Hazan, S.; Kolusheva, S.; Porat, Z.; Zeiri, Y. Green synthesis of gold nanoparticles using plant extracts as reducing agents. Int. J. Nanomed. 2014, 9, 4007−4021. (7) Tanabe, K. Optical radiation efficiencies of metal nanoparticles for optoelectronic applications. Mater. Lett. 2007, 61, 4573−4575. (8) Leifert, A.; Pan-Bartnek, Y.; Simon, U.; Jahnen-Dechent, W. Molecularly stabilized ultrasmall gold nanoparticles: synthesis, characterization and bioactivity. Nanoscale 2013, 5, 6224−6242. (9) Browning, L. M.; Lee, K. J.; Huang, T.; Nallathamby, P. D.; Lowman, J. E.; Xu, X. N. Random walk of single gold nanoparticles in zebrafish embryos leading to stochastic toxic effects on embryonic developments. Nanoscale 2009, 1, 138−152. (10) Li, Q.; Lu, B.; Zhang, L.; Lu, C. Synthesis and stability evaluation of size-controlled gold nanoparticles via nonionic fluorosurfactant-assisted hydrogen peroxide reduction. J. Mater. Chem. 2012, 22, 13564−13570. (11) Han, S.; Lee, J.; Ahn, K. H.; Kim, Y. S.; et al. Size-dependent clearance of gold nanoparticles from lungs of Sprague−Dawley rats after short-term inhalation exposure. Arch. Toxicol. 2015, 89, 1083− 1094. (12) Balasubramanian, S. K.; Poh, K.; Ong, C.; Kreyling, W. G.; Ong, W. Y.; Yu, L. E. The effect of primary particle size on biodistribution of inhaled gold nano-agglomerates. Biomaterials 2013, 34, 5439−5452. (13) Villiers, C. L.; Freitas, H.; Couderc, R.; Villiers, M.; Marche, P. Analysis of the toxicity of gold nanoparticles on the immune system: effect on dendritic cell functions. J. Nanopart. Res. 2010, 12, 55−60. (14) Deraedt, C.; Salmon, L.; Gatard, S.; Ciganda, R.; Hernandez, R.; Ruiz, J.; Astruc, D. Sodium borohydride stabilizes very active gold nanoparticle catalysts. Chem. Commun. 2014, 50, 14194−14196. (15) Hussain, S. T.; Iqbal, M.; Mazhar, M. Size control synthesis of starch capped-gold nanoparticles. J. Nanopart. Res. 2009, 11, 1383− 1391. (16) Mandal, T. K.; Fleming, M. S.; Walt, D. R. Preparation of polymer coated gold nanoparticles by surface-confined living radical polymerization at ambient temperature. Nano Lett. 2002, 2, 3−7. (17) Chen, Y. H.; Tsai, C. Y.; Huang, P. Y.; Chang, M. Y.; Cheng, P. C.; Chou, C. H.; Chen, D. H.; Wang, C. R.; Shiau, A. L.; Wu, C. L. Methotrexate Conjugated to Gold Nanoparticles Inhibits Tumor Growth in a Syngeneic Lung Tumor Model. Mol. Pharmaceutics 2007, 4, 713−722. (18) Murphy, C. J.; Gole, A. M.; Stone, J. W.; Sisco, P. N.; Alkilany, Am M.; Goldsmith, E. C.; Baxter, S. C. Gold nanoparticles in biology: beyond toxicity to cellular imaging. Acc. Chem. Res. 2008, 41, 1721− 1730. (19) Perrault, S. D.; Walkey, C.; Jennings, T.; Fischer, H. C.; Chan, W. Mediating tumor targeting efficiency of nanoparticles through design. Nano Lett. 2009, 9, 1909−1915. (20) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity. Small 2005, 1, 325−327. (21) Pan, Y.; Neuss, S.; Leifert, A.; Fischler, M.; Wen, F.; Simon, U.; Schmid, G.; Brandau, W.; Jahnen-Dechent, W. Size-dependent cytotoxicity of gold nanoparticles. Small 2007, 3, 1941−1949. (22) Ray, P. C.; Yu, H.; Fu, P. P. Toxicity and environmental risks of nanomaterials: challenges and future needs. J. Environ. Sci. Health, Part C: Enivron. Carcinog. Ecotoxicol. Rev. 2009, 27, 1−35.

(23) Dauthal, P.; Mukhopadhyay, M. Phyto-synthesis and structural characterization of catalytically active gold nanoparticles biosynthesized using Delonixregia leaf extract. 3 Biotechnol. 2016, 6, 118. (24) Suman, T. Y.; Rajasree, S. R.; Ramkumar, R.; Rajthilak, C.; Perumal, P. The Green synthesis of gold nanoparticles using an aqueous root extract of Morindacitrifolia L. Spectrochim. Acta, Part A 2014, 118, 11−16. (25) Krishnaswamy, K.; Vali, H.; Orsat, V. Value-adding to grape waste: Green synthesis of gold nanoparticles. J. Food Eng. 2014, 142, 210−220. (26) Wadhwani, S. A.; Shedbalkar, U. U.; Singh, R.; Karve, M. S.; Chopade, B. A. Novel polyhedral gold nanoparticles: green synthesis, optimization and characterization by environmental isolate of Acinetobacter sp. SW30. World J. Microbiol. Biotechnol. 2014, 30, 2723−2731. (27) Chou, J.; Li, X.; Yin, Y.; Indrisek, N. Determination of antioxidant activities in fruit juices based on rapid colorimetric measurement and characterisation of gold nanoparticles. Int. J. Environ. Anal. Chem. 2015, 95, 531−541. (28) Iravani, S. Green synthesis of metal nanoparticles using plants. Green Chem. 2011, 13, 2638−2650. (29) Hieda, J.; Saito, N.; Takai, O. Size-regulated gold nanoparticles fabricated by a discharge in reverse micelle solutions. Surf. Coat. Technol. 2008, 202, 5343−5346. (30) Scampicchio, M.; Wang, J.; Blasco, A. J.; Arribas, A. S.; Mannino, S.; Escarpa, A. Nanoparticle-based assays of antioxidant activity. Anal. Chem. 2006, 78, 2060−2063. (31) Singh, P.; Kim, Y.; Zhang, D.; Yang, D. Biological synthesis of nanoparticles from plants and microorganisms. Trends Biotechnol. 2016, 3, 588−599. (32) El-Kassas, H. Y.; El-Sheekh, M. M. Cytotoxic activity of biosynthesized gold nanoparticles with an extract of the red seaweed Corallinaof f icinalis on the MCF-7 human breast cancer cell line. Asian Pac. J. Cancer Prev. 2014, 15, 4311−4317. (33) Lee, K.; Nallathamby, P.; Browning, L.; Osgood, C.; Xu, X. In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS Nano 2007, 1, 133−143. (34) Lee, K.; Browning, L.; Nallathamby, P.; Osgood, C.; Xu, X. Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish. Nanoscale 2013, 5 (23), 11625−11636.

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DOI: 10.1021/acs.jpcc.7b00434 J. Phys. Chem. C XXXX, XXX, XXX−XXX