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One pot, rapid and efficient synthesis of water dispersible gold nanoparticles using alphaamino acids
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 435608 (http://iopscience.iop.org/0957-4484/25/43/435608) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 128.59.222.12 This content was downloaded on 03/03/2015 at 09:23
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 25 (2014) 435608 (7pp)
doi:10.1088/0957-4484/25/43/435608
One pot, rapid and efficient synthesis of water dispersible gold nanoparticles using alpha-amino acids Nishima Wangoo1, Sarabjit Kaur2, Manish Bajaj2, D V S Jain2 and Rohit K Sharma2 1
Centre for Nanoscience & Nanotechnology (U.I.E.A.S.T.), Panjab University, Sector-14, Chandigarh-160014, India 2 Department of Chemistry & Centre for Advanced Studies in Chemistry, Panjab University, Sector-14, Chandigarh-160014, India E-mail: [email protected] and [email protected] Received 18 June 2014, revised 4 September 2014 Accepted for publication 9 September 2014 Published 10 October 2014 Abstract
A detailed study on the synthesis of spherical and monodispersed gold nanoparticles (AuNPs) using all of the 20 naturally occurring α-amino acids has been reported. The synthesized nanoparticles have been further characterized using various techniques such as absorbance spectroscopy, transmission electron microscopy, dynamic light scattering and nuclear magnetic resonance. Size control of the nanoparticles has been achieved by varying the ratio of the gold ion to the amino acid. These monodispersed water soluble AuNPs synthesized using non-toxic, naturally occurring α-amino acids as reducing and capping/stabilizing agents serve as a remarkable example of green chemistry. S Online supplementary data available from stacks.iop.org/NANO/25/435608/mmedia Keywords: gold nanoparticles, amino acids, reducing agents (Some figures may appear in colour only in the online journal) 1. Introduction
surfaces can be functionalized also makes these nanoparticles attractive for various applications. As far as the synthesis of the AuNPs is concerned, a number of procedures are available using both polar and nonpolar solvents, thereby displaying different characteristics of the final product. By far, the citrate reduction method by Turkevich [9] in mid 20th century is the most popular and standard protocol for the synthesis of monodispersed aqueous AuNPs. The method reported by Burst and Schiffrin [10] describing the synthesis of small particles based on fast chemical reduction of gold ions at an oil–water interface followed by adsorption of thiolated molecules, also proved to be a landmark in the chemistry of the synthesis of AuNPs. A variety of reducing agents (e.g. sodium borohydride, hydrazine etc) and capping agents (e.g. surfactants, small molecules, polymers etc) can be used for the synthesis and surface modification of the AuNPs. A large number of
The synthesis of nanomaterials of different kinds and morphologies is an area of extensive research due to its potential applications in numerous fields ranging from optoelectronics [1] to biosensors [2], catalysis [3], cancer therapy [4], bioengineering [5] and cosmetics [6, 7]. In particular, noble metals have generated significant interest due to their unique optical and electronic properties. These properties depend on their size and shape, and particularly in case of biological applications [8], on the surface functionalities. Among these, metallic nanoparticles in general, and gold nanoparticles (AuNPs) in particular, have been gaining a lot of attention in various biomedical applications. In addition to this, AuNPs have good biocompatibility as well as noncytotoxicity which have made them leading candidates in diagnosis and therapeutics. Further, the ease with which their 0957-4484/14/435608+07$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Nanotechnology 25 (2014) 435608
N Wangoo et al
different chemical moieties which can act both as reducing and capping agents simultaneously have also been developed. Most of these methods are extremely expensive and also involve the use of toxic, hazardous chemicals, which may pose potential environmental and biological risks. Since noble metal nanoparticles are widely applied to areas of human contact, there is a growing need to develop environmentally friendly processes for nanoparticle synthesis that do not use toxic chemicals. Consequently, the potential area of interest for various biological applications are functionalized nanoparticles synthesized by green methods so that the by-product of the system remains non-toxic [11]. For this purpose, a variety of compounds having amine functionality have been explored which includes simple primary amines [12], aromatic amines [13], amino acids and multifunctional amines [14]. L-Lysine has been employed for successful synthesis of water dispersible AuNPs [15]. Recently, glutamic acid and histidine have also been reported to employed as stabilizer in the synthesis of anisotropic structures, that are, nanochains or nanowires using sodium borohydride as reductant in the same step [16]. Isoleucine, serine, ornithine and histidine have been used in the assembly formation of AuNPs leading to nanometric compounds with anticancer potential [17]. Aspartic acid has been used to provide a very stable colloidal gold solution with high monodispersity [18] and has also been studied to control the morphology of nanostructures formed under conditions of different pH and evaporation rates [19]. Tryptophan has been reported as a spontaneous reductant as well as capping agent to give water dispersible AuNPs and it was observed that tryptophan capped and tryptophan reduced AuNPs were different in terms of functionality bound to nanoparticle surface [20]. Reports are also available in the literature which employ tryptophan as the reductant with additional stabilizing agents [21]. An investigation into the particle sizes and morphologies of AuNPs produced using the amino acids such as L-tyrosine, glycyl-L-tyrosine and L-arginine under alkaline conditions has been recently carried out [22]. The thiol containing amino acids like cysteine has also been studied for capping AuNPs which employs the use of strong Au–S interactions [23]. In a recent report, the synthesis of L-glutamic acid and L-arginine capped AuNPs at different pH have also been demonstrated [24]. The authors observed that the interactions between Larginine, L-glutamic acid and AuNPs were of electrostatic and covalent/coordinate types, respectively at physiological pH using agarose gel electrophoresis technique. Similarly, the use of amino acids such as glutamic acid, phenylalanine and tryptophan as reductant as well as capping agent has also been reported [25, 26]. The same authors also explored the binding interactions between AuNPs and amino acids through quantum calculations confirming strong interactions between amino acids and nanoparticle surface. AuNPs have also been synthesized using tyrosine based oligopeptides [27]. Typtophan-reduced as well as tryptophan-capped AuNPs were synthesized and used for studying the interaction of tryptophan with nanoparticle surface [21, 28]. Moreover, synthesis of phenylalanine capped as well as reduced AuNPs [29] along with glutamic acid reduced monodispersed AuNPs [26] have
Scheme 1. Synthesis of gold nanoparticles using amino acids as reducing agents.
been reported. There are some reports in the literature where protein molecules have been used in metal nanoparticles synthesis [30]. Similarly, peptides which are the basic constituents of the proteins have also been used as a reducing and/or capping agent [31]. Although there are various reports available in literature mentioning the use of amino acids in gold nanostructure synthesis or stabilization, to the best of our knowledge, there is no complete and detailed study available highlighting the reducing, stabilizing and size control capabilities of all twenty natural amino acids. Keeping this in mind, we have carried out a systematic study on the synthesis of AuNPs using amino acids along with a thorough physicochemical investigation (scheme 1).
2. Experimental details 2.1. Materials and methods
Tetrachloroauric acid (HAuCl4), glutamic acid, phenylalanine, tryptophan, asparagine monohydrate, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, tyrosine, aspartic acid, glutamine, histidine, lysine and arginine were obtained from Sigma, India and used as received. All the glassware as well as magnetic beads used in the experiment were cleaned using aqua regia. 2.2. Synthesis of amino acid-reduced AuNPs
For the synthesis of the AuNPs, 10 mL aqueous solution of 0.01% (w/v) tetrachloroauric acid was brought to boil under constant stirring and subsequently, 10 mL of an aqueous solution of the amino acid of varying molarities was added to it under rapid stirring. Depending upon the type of amino 2
Nanotechnology 25 (2014) 435608
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Surprisingly, cysteine led to the formation of black precipitates despite having a nucleophilic thiol group on its side chain. This may be due to the complexation of cysteine initially to the chloroaurate salt via sulphur atom. Aliphatic amino acids such as glycine and alanine led to very low nanoparticle yield as observed through absorption studies. The hydrophobic amino acids like valine and leucine provided high yields of AuNPs as evident from the intense red colour and relatively small size as observed from dynamic light scattering (DLS) studies. On the other hand, isoleucine demonstrated faint red colour depicting the formation of relatively fewer AuNPs. Methionine and proline, however, were incapable of reducing Au3+ to Au0. It was further observed that all the aromatic amino acids were able to reduce the gold salt at very low concentrations and tend to aggregate at higher concentrations. In case of tyrosine, relatively larger particle size was obtained. Similar results were obtained with tryptophan which demonstrated rapid color change from colourless to blue. Asparagine and glutamine, having the same amide group at the side chain, displayed contrasting results. Asparagine showed excellent results with the formation of stable and highly monodispersed red colored particles, whereas glutamine could not replicate the same results and failed to show good reducing properties. The amino acids with acidic side chains showed excellent reducing and capping abilities. Both aspartic acid and glutamic acid yielded particles with size range of 10–50 nm at all recorded concentrations, including concentration as low as 1 mM. In sharp contrast to acidic amino acids, histidine, lysine and arginine did not reduce the gold salt at all and therefore, failed to yield any nanoparticles. The above results are consistent with a strong interaction of the amino acid molecules with the AuNP surface.
Figure 1. Reduction capabilities of all of the 20 natural amino acids in the concentration range from 0.1 mM to 5.0 mM. (Red: monodispersed nanoparticles; blue: no reduction; green: incomplete reduction; yellow: aggregated product).
acid, the color of the resulting solution either changed to pink/ red/purple or it did not change at all. The resulting nanoparticles were then washed with deionized water by repeated centrifugation at 6000 rpm for 30 min at room temperature and analyzed using various techniques. 2.3. Characterization of AuNPs
The UV–visible absorbance spectra of the nanoparticles were recorded using a JascoV-530 UV–vis spectrophotometer. NMR spectra were measured using Bruker AVANCE III400 MHz in either deuterated methanol or water. Transmission electron microscopy (TEM) studies were carried out using Hitachi (Model H-7500) transmission electron microscope operating in accelerating voltage of 120 kV. Samples for TEM studies were prepared by placing a drop of the nanoparticles solution on carbon-coated TEM grids and were allowed to dry for 5 min at room temperature before analysis. Particle size distribution and zeta potential was analyzed using Malvern Zetasizer.
3.2. Absorption studies
AuNPs were further analyzed for their plasmon resonance peaks by UV–vis analysis in the range of 400–800 nm. In all the recorded spectra, a strong absorption in the range between 519 nm and 530 nm was observed which corresponds to excitation of metal surface plasmons (figure 2). This resonance confirmed the synthesis of the monodispersed nanoparticles. It was further observed that the increase in the concentration of the amino acid led to a blue shift in the absorption maximum indicating a corresponding decrease in the size (figure 1). It was also observed that very low concentrations of the amino acids (⩽0.1 mM) led to aggregation of the nanoparticles as evident from the colour and the broad absorption peak. This phenomenon of change in colour from red to purple or blue due to aggregation has been very well studied and also used for the various sensing methods employing AuNPs [2].
3. Results and discussion 3.1. Reduction studies
The primary focus of our experimental investigations is on understanding the ability of amino acids in the synthesis of monodispersed AuNPs. The ability of all the amino acids to reduce the chloroaurate solution to form AuNPs was tested under the same conditions (figure 1). Amino acids possessing nucleophilic side chains such as serine and threonine demonstrated good reducing abilities.
3.3. TEM studies
The shape and size of the nanoparticles was analysed using TEM. The diameter of the NPs ranged from 10 nm to 25 nm depending upon the type and concentration of the amino acid 3
Nanotechnology 25 (2014) 435608
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Figure 2. UV–visible absorbance spectra of amino acid capped AuNPs.
led to the synthesis of relatively larger particles. Zeta potential measurements were also carried out for the samples and it was observed that the particles have a net negative charge on their surface (see table S1 in electronic supplementary information). The sufficiently high magnitude of the zeta potential suggests that the nanoparticles obtained using this methodology are fairly stable in nature.
used (figure 3). In general, the AuNPs obtained were fairly uniform in size. Also, it is important to mention that we obtained only spherical particles as evident from the TEM pictures (figure 3). 3.4. DLS and zeta potential studies
The effect of the variation in the size of the nanoparticles by changing the concentration of the amino acids was also analyzed by carrying out DLS studies (figure 4). It was observed that similar to the conventional citrate based synthesis of AuNPs, amino acids were also able to reduce the chloroaurate solution in a concentration dependent manner with higher concentrations leading to smaller particles and vice versa. This also is in agreement with the absorption spectra where shift in the surface plasmon resonance peak was also observed. It is quite evident from the size distribution data (figure 4) that the amino acids—asparagine, aspartic acid and glutamic acid produced smaller size of the AuNPs whereas amino acids such as tyrosine, serine and threonine
3.5. Proton NMR studies
In order to understand the effect of binding of the amino acids to the nanoparticles, proton NMR (1H NMR) studies were carried out. The comparison of chemical shift values (in ppm) of α and β protons of pure amino acids and amino acid capped AuNPs as obtained from 1H NMR spectra show a distinct splitting and shifting (table 1). The NMR signals of the amino acid capped AuNPs indicated an overall downfield shift in α as well as β protons. This may be attributed to the shifting of electron density from the amino acid to nanoparticles leading to de-shielding of α and β protons of the amino acid. In other 4
Nanotechnology 25 (2014) 435608
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Figure 3. TEM images of AuNPs reduced by L-Glutamic acid (a), L-Phenylalanine (b), L-Aspartic acid (c), L-Asparagine (d), L-Serine (e), L-Leucine (f), L-Valine (g), L-Threonine (h) and L-Tyrosine (i).
4. Conclusions
words, the nanoparticles attract electron density from the either the amino group and/or the carboxyl group of the amino acid leading to downfield shift. Apart from the apparent shifting of NMR signals, crucial evidence confirming the proximity of the metal centre to the concerned amino acid is the consistent metal induced peak broadening observed in the spectra of all the amino acid reduced AuNPs which is clearly visible in the spectra of all the samples. Considering the downfield shift and unsymmetrical pattern observed in the 1H NMR peaks, it can be safely interpreted that the amino acids interact strongly with the nanoparticle surface.
In conclusion, size tunable synthesis of mono-dispersed water soluble AuNPs by using non-toxic, naturally occurring amino acids both as reducing and capping/stabilising agents has been described. The size control could be easily achieved by varying the ratio of the gold ion to the amino acid. The synthesized AuNPs were analyzed by UV–vis spectroscopy, DLS, TEM and NMR Out of the 20 amino acids, glutamic acid, aspartic acid, asparagine, serine, threonine, isoleucine, leucine, tyrosine, valine showed good nanoparticle yield. Moreover, threonine and tyrosine provided good size 5
Nanotechnology 25 (2014) 435608
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Table 1. Comparison of chemical shift values (in ppm) of α and β protons of pure amino acids and amino acid capped AuNPs as obtained
from 1H NMR spectra (see figure S1–S8 in electronic supplementary information for detailed 1H NMR spectra).
1. 2. 3. 4. 5. 6. 7. 8.
Glutamic acid Aspartic acid Phenylalanine Leucine Valine Asparagine Serine Threonine
α-proton (pure amino acids)
α-proton (amino acid capped AuNPs)
β-proton (pure amino acids)
β-proton (amino acid capped AuNPs)
3.6259–3.6576 3.8707–3.8999 3.8470–3.8799 3.5927–3.6265 3.4695–3.4803 3.8707–3.9007 3.7923–3.8812 4.1049–4.1666
3.6599–3.6913 3.9447–3.9731 3.7971–3.9022 3.6293–3.6634 3.5337–3.5445 3.8686–3.9149 3.8217–3.8708 4.1237–4.1854
2.3335–2.4633 2.7236–2.8729 2.9666–3.1871 1.5361–1.6404 2.1046–2.1857 2.7001–2.8609 3.7035–3.7271 3.4597–3.4721
2.3437–2.4670 2.8250–2.9428 2.9754–3.1956 1.5714–1.6779 2.1595–2.2407 2.7098–2.8689 3.7676–3.7790 3.4786–3.4909
*R represents the side chain functionality of the respective amino acid.
References [1] Shipway A N, Katz E and Willner I 2000 Chemphyschem 1 18–52 [2] Saha K, Agasti S S, Kim C, Li X and Rotello V M 2012 Chem. Rev. 112 2739–79 [3] Mitsudome T and Kaneda K 2013 Green Chem. 15 2636–54 [4] Jain S, Hirst D G and O’ Sullivan J M 2012 Br. J. Radiol. 85 101–13 [5] Polito L, Monti D, Caneva E, Delnevo E, Russo G and Prosperi D 2008 Chem. Commun. 621–3 [6] Wiechers J W and Musee N 2010 J. Biomed. Nanotechnology 6 408–31 [7] Popov A P, Zvyagin A V, Lademann J, Roberts M S, Sanchez W, Priezzhev A V and Myllylä R 2010 J. Biomed. Nanotechnology 6 432–51 [8] Salata O V 2004 J. Nanobiotechnology 2 1–6 [9] Turkevich J, Stevenson P C and Hillier J 1951 Discuss. Faraday Soc. 11 55–75 [10] Brust M, Walker M, Bethell D, Schiffrin D J and Whyman R 1994 J. Chem. Soc. Chem. Commun. 801–2 [11] Giljohann D A, Seferos D S, Daniel W L, Massich M D, Patel P C and Mirkin C A 2010 Angew. Chem. Int. Ed. 49 3280–94 [12] Newman J D S and Blanchard G J 2006 Langmuir 22 5882–7 [13] Subramaniam C, Tom R T and Pradeep T 2005 J. Nanopart. Res. 7 209–17 [14] Kang E T, Ting Y P and Neoh K G 1993 Polymer 34 4994–6 [15] Selvakannan P R, Mandal S, Phadtare S, Pasricha R and Sastry M 2003 Langmuir 19 3545–9 [16] Polavarapu L and Xu Q 2008 Nanotechnology 19 1–6 [17] Atta A H, El-Shenawya A I, Refatd M S and Elsabawy K M 2013 J. Mol. Struct. 1039 51 [18] Mandal S, Selvakannan P R, Phadtare S, Pasricha R and Sastry M Proc. Indian Acad. Sci. Chem. Sci. 114 513 [19] Shao Y, Jin Y and Dong S 2004 Chem. Commun. 1104–5 [20] Iosin M, Baldeck P and Astilean S 2010 J. Nanopart. Res. 12 2843–9 [21] Akbarzadeh A, Zare D, Farhangi A, Mehrabi M R and Norouzian D 2009 Am. J. Appl. Sci. 6 691–5 [22] Bhargava S K, Booth J M, Agrawal S, Coloe P and Kar G 2005 Langmuir 21 5949–56 [23] Aryal S, Remant B K C, Dharmaraj N, Bhattarai N, Kim C H and Kim H Y 2006 Spectrochim. Acta A 63 160–3
Figure 4. Variation in size of AuNPs with change in concentration of amino acids as observed from dynamic light scattering.
tunability as can be seen from the absorption spectra and DLS data where increase in the concentration lead to the smaller size of particles. NMR studies confirmed the layering of amino acid on to the nanoparticle surface which can be further used for binding with peptides, proteins and other functionalities in order to be applicable for biological and medical applications.
Acknowledgments This study was supported by the Department of Science & Technology (DST) INSPIRE, India [IFA12-CH-52] and Science Education & Research Board (SERB) of India grant (F. No. SB/SO/BB/0040/2013). Sarabjit Kaur and Manish Bajaj thank Council of Scientific & Industrial Research (CSIR), India and University Grants Commission (UGC), India, respectively for research fellowships.
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[28] Selvakannan P R, Mandal S, Phadtare S, Gole A, Pasricha R, Adyanthaya S D and Sastry M 2004 J. Colloid Interface Sci. 269 97–108 [29] Nayak N C and Shin K 2006 J. Nanosci. Nanotechnology 6 3512–6 [30] Das S K, Dickinson C, Lafir F, Brougham D F and Marsili E 2012 Green Chem. 14 1322–34 [31] Tan Y N, Lee J Y and Wang D I C 2010 J. Am. Chem. Soc. 132 5677–86
[24] Zare D, Akbarzadeh A, Barkhi M, Khoshnevisan K, Bararpour N, Noruzi M and Tabatabaei M 2012 Synth. React. Inorg. Met. Org. Chem. 42 266–72 [25] Zare D, Khoshnevisan K, Barkhi M and Tahami H V 2014 J. Exp. Nanosci. 9 1–9 [26] Wangoo N, BhasinK K, Mehta S K and Suri C R 2008 J. Colloid Interface Sci. 323 247–54 [27] Si S, Bhattacharjee R, Banerjee R A and Mandal T K 2006 Chem. Eur. J. 12 1256–65
7
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One pot, rapid and efficient synthesis of water dispersible gold nanoparticles using alphaamino acids
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 435608 (http://iopscience.iop.org/0957-4484/25/43/435608) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 128.59.222.12 This content was downloaded on 03/03/2015 at 09:23
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 25 (2014) 435608 (7pp)
doi:10.1088/0957-4484/25/43/435608
One pot, rapid and efficient synthesis of water dispersible gold nanoparticles using alpha-amino acids Nishima Wangoo1, Sarabjit Kaur2, Manish Bajaj2, D V S Jain2 and Rohit K Sharma2 1
Centre for Nanoscience & Nanotechnology (U.I.E.A.S.T.), Panjab University, Sector-14, Chandigarh-160014, India 2 Department of Chemistry & Centre for Advanced Studies in Chemistry, Panjab University, Sector-14, Chandigarh-160014, India E-mail: [email protected] and [email protected] Received 18 June 2014, revised 4 September 2014 Accepted for publication 9 September 2014 Published 10 October 2014 Abstract
A detailed study on the synthesis of spherical and monodispersed gold nanoparticles (AuNPs) using all of the 20 naturally occurring α-amino acids has been reported. The synthesized nanoparticles have been further characterized using various techniques such as absorbance spectroscopy, transmission electron microscopy, dynamic light scattering and nuclear magnetic resonance. Size control of the nanoparticles has been achieved by varying the ratio of the gold ion to the amino acid. These monodispersed water soluble AuNPs synthesized using non-toxic, naturally occurring α-amino acids as reducing and capping/stabilizing agents serve as a remarkable example of green chemistry. S Online supplementary data available from stacks.iop.org/NANO/25/435608/mmedia Keywords: gold nanoparticles, amino acids, reducing agents (Some figures may appear in colour only in the online journal) 1. Introduction
surfaces can be functionalized also makes these nanoparticles attractive for various applications. As far as the synthesis of the AuNPs is concerned, a number of procedures are available using both polar and nonpolar solvents, thereby displaying different characteristics of the final product. By far, the citrate reduction method by Turkevich [9] in mid 20th century is the most popular and standard protocol for the synthesis of monodispersed aqueous AuNPs. The method reported by Burst and Schiffrin [10] describing the synthesis of small particles based on fast chemical reduction of gold ions at an oil–water interface followed by adsorption of thiolated molecules, also proved to be a landmark in the chemistry of the synthesis of AuNPs. A variety of reducing agents (e.g. sodium borohydride, hydrazine etc) and capping agents (e.g. surfactants, small molecules, polymers etc) can be used for the synthesis and surface modification of the AuNPs. A large number of
The synthesis of nanomaterials of different kinds and morphologies is an area of extensive research due to its potential applications in numerous fields ranging from optoelectronics [1] to biosensors [2], catalysis [3], cancer therapy [4], bioengineering [5] and cosmetics [6, 7]. In particular, noble metals have generated significant interest due to their unique optical and electronic properties. These properties depend on their size and shape, and particularly in case of biological applications [8], on the surface functionalities. Among these, metallic nanoparticles in general, and gold nanoparticles (AuNPs) in particular, have been gaining a lot of attention in various biomedical applications. In addition to this, AuNPs have good biocompatibility as well as noncytotoxicity which have made them leading candidates in diagnosis and therapeutics. Further, the ease with which their 0957-4484/14/435608+07$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Nanotechnology 25 (2014) 435608
N Wangoo et al
different chemical moieties which can act both as reducing and capping agents simultaneously have also been developed. Most of these methods are extremely expensive and also involve the use of toxic, hazardous chemicals, which may pose potential environmental and biological risks. Since noble metal nanoparticles are widely applied to areas of human contact, there is a growing need to develop environmentally friendly processes for nanoparticle synthesis that do not use toxic chemicals. Consequently, the potential area of interest for various biological applications are functionalized nanoparticles synthesized by green methods so that the by-product of the system remains non-toxic [11]. For this purpose, a variety of compounds having amine functionality have been explored which includes simple primary amines [12], aromatic amines [13], amino acids and multifunctional amines [14]. L-Lysine has been employed for successful synthesis of water dispersible AuNPs [15]. Recently, glutamic acid and histidine have also been reported to employed as stabilizer in the synthesis of anisotropic structures, that are, nanochains or nanowires using sodium borohydride as reductant in the same step [16]. Isoleucine, serine, ornithine and histidine have been used in the assembly formation of AuNPs leading to nanometric compounds with anticancer potential [17]. Aspartic acid has been used to provide a very stable colloidal gold solution with high monodispersity [18] and has also been studied to control the morphology of nanostructures formed under conditions of different pH and evaporation rates [19]. Tryptophan has been reported as a spontaneous reductant as well as capping agent to give water dispersible AuNPs and it was observed that tryptophan capped and tryptophan reduced AuNPs were different in terms of functionality bound to nanoparticle surface [20]. Reports are also available in the literature which employ tryptophan as the reductant with additional stabilizing agents [21]. An investigation into the particle sizes and morphologies of AuNPs produced using the amino acids such as L-tyrosine, glycyl-L-tyrosine and L-arginine under alkaline conditions has been recently carried out [22]. The thiol containing amino acids like cysteine has also been studied for capping AuNPs which employs the use of strong Au–S interactions [23]. In a recent report, the synthesis of L-glutamic acid and L-arginine capped AuNPs at different pH have also been demonstrated [24]. The authors observed that the interactions between Larginine, L-glutamic acid and AuNPs were of electrostatic and covalent/coordinate types, respectively at physiological pH using agarose gel electrophoresis technique. Similarly, the use of amino acids such as glutamic acid, phenylalanine and tryptophan as reductant as well as capping agent has also been reported [25, 26]. The same authors also explored the binding interactions between AuNPs and amino acids through quantum calculations confirming strong interactions between amino acids and nanoparticle surface. AuNPs have also been synthesized using tyrosine based oligopeptides [27]. Typtophan-reduced as well as tryptophan-capped AuNPs were synthesized and used for studying the interaction of tryptophan with nanoparticle surface [21, 28]. Moreover, synthesis of phenylalanine capped as well as reduced AuNPs [29] along with glutamic acid reduced monodispersed AuNPs [26] have
Scheme 1. Synthesis of gold nanoparticles using amino acids as reducing agents.
been reported. There are some reports in the literature where protein molecules have been used in metal nanoparticles synthesis [30]. Similarly, peptides which are the basic constituents of the proteins have also been used as a reducing and/or capping agent [31]. Although there are various reports available in literature mentioning the use of amino acids in gold nanostructure synthesis or stabilization, to the best of our knowledge, there is no complete and detailed study available highlighting the reducing, stabilizing and size control capabilities of all twenty natural amino acids. Keeping this in mind, we have carried out a systematic study on the synthesis of AuNPs using amino acids along with a thorough physicochemical investigation (scheme 1).
2. Experimental details 2.1. Materials and methods
Tetrachloroauric acid (HAuCl4), glutamic acid, phenylalanine, tryptophan, asparagine monohydrate, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, tyrosine, aspartic acid, glutamine, histidine, lysine and arginine were obtained from Sigma, India and used as received. All the glassware as well as magnetic beads used in the experiment were cleaned using aqua regia. 2.2. Synthesis of amino acid-reduced AuNPs
For the synthesis of the AuNPs, 10 mL aqueous solution of 0.01% (w/v) tetrachloroauric acid was brought to boil under constant stirring and subsequently, 10 mL of an aqueous solution of the amino acid of varying molarities was added to it under rapid stirring. Depending upon the type of amino 2
Nanotechnology 25 (2014) 435608
N Wangoo et al
Surprisingly, cysteine led to the formation of black precipitates despite having a nucleophilic thiol group on its side chain. This may be due to the complexation of cysteine initially to the chloroaurate salt via sulphur atom. Aliphatic amino acids such as glycine and alanine led to very low nanoparticle yield as observed through absorption studies. The hydrophobic amino acids like valine and leucine provided high yields of AuNPs as evident from the intense red colour and relatively small size as observed from dynamic light scattering (DLS) studies. On the other hand, isoleucine demonstrated faint red colour depicting the formation of relatively fewer AuNPs. Methionine and proline, however, were incapable of reducing Au3+ to Au0. It was further observed that all the aromatic amino acids were able to reduce the gold salt at very low concentrations and tend to aggregate at higher concentrations. In case of tyrosine, relatively larger particle size was obtained. Similar results were obtained with tryptophan which demonstrated rapid color change from colourless to blue. Asparagine and glutamine, having the same amide group at the side chain, displayed contrasting results. Asparagine showed excellent results with the formation of stable and highly monodispersed red colored particles, whereas glutamine could not replicate the same results and failed to show good reducing properties. The amino acids with acidic side chains showed excellent reducing and capping abilities. Both aspartic acid and glutamic acid yielded particles with size range of 10–50 nm at all recorded concentrations, including concentration as low as 1 mM. In sharp contrast to acidic amino acids, histidine, lysine and arginine did not reduce the gold salt at all and therefore, failed to yield any nanoparticles. The above results are consistent with a strong interaction of the amino acid molecules with the AuNP surface.
Figure 1. Reduction capabilities of all of the 20 natural amino acids in the concentration range from 0.1 mM to 5.0 mM. (Red: monodispersed nanoparticles; blue: no reduction; green: incomplete reduction; yellow: aggregated product).
acid, the color of the resulting solution either changed to pink/ red/purple or it did not change at all. The resulting nanoparticles were then washed with deionized water by repeated centrifugation at 6000 rpm for 30 min at room temperature and analyzed using various techniques. 2.3. Characterization of AuNPs
The UV–visible absorbance spectra of the nanoparticles were recorded using a JascoV-530 UV–vis spectrophotometer. NMR spectra were measured using Bruker AVANCE III400 MHz in either deuterated methanol or water. Transmission electron microscopy (TEM) studies were carried out using Hitachi (Model H-7500) transmission electron microscope operating in accelerating voltage of 120 kV. Samples for TEM studies were prepared by placing a drop of the nanoparticles solution on carbon-coated TEM grids and were allowed to dry for 5 min at room temperature before analysis. Particle size distribution and zeta potential was analyzed using Malvern Zetasizer.
3.2. Absorption studies
AuNPs were further analyzed for their plasmon resonance peaks by UV–vis analysis in the range of 400–800 nm. In all the recorded spectra, a strong absorption in the range between 519 nm and 530 nm was observed which corresponds to excitation of metal surface plasmons (figure 2). This resonance confirmed the synthesis of the monodispersed nanoparticles. It was further observed that the increase in the concentration of the amino acid led to a blue shift in the absorption maximum indicating a corresponding decrease in the size (figure 1). It was also observed that very low concentrations of the amino acids (⩽0.1 mM) led to aggregation of the nanoparticles as evident from the colour and the broad absorption peak. This phenomenon of change in colour from red to purple or blue due to aggregation has been very well studied and also used for the various sensing methods employing AuNPs [2].
3. Results and discussion 3.1. Reduction studies
The primary focus of our experimental investigations is on understanding the ability of amino acids in the synthesis of monodispersed AuNPs. The ability of all the amino acids to reduce the chloroaurate solution to form AuNPs was tested under the same conditions (figure 1). Amino acids possessing nucleophilic side chains such as serine and threonine demonstrated good reducing abilities.
3.3. TEM studies
The shape and size of the nanoparticles was analysed using TEM. The diameter of the NPs ranged from 10 nm to 25 nm depending upon the type and concentration of the amino acid 3
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Figure 2. UV–visible absorbance spectra of amino acid capped AuNPs.
led to the synthesis of relatively larger particles. Zeta potential measurements were also carried out for the samples and it was observed that the particles have a net negative charge on their surface (see table S1 in electronic supplementary information). The sufficiently high magnitude of the zeta potential suggests that the nanoparticles obtained using this methodology are fairly stable in nature.
used (figure 3). In general, the AuNPs obtained were fairly uniform in size. Also, it is important to mention that we obtained only spherical particles as evident from the TEM pictures (figure 3). 3.4. DLS and zeta potential studies
The effect of the variation in the size of the nanoparticles by changing the concentration of the amino acids was also analyzed by carrying out DLS studies (figure 4). It was observed that similar to the conventional citrate based synthesis of AuNPs, amino acids were also able to reduce the chloroaurate solution in a concentration dependent manner with higher concentrations leading to smaller particles and vice versa. This also is in agreement with the absorption spectra where shift in the surface plasmon resonance peak was also observed. It is quite evident from the size distribution data (figure 4) that the amino acids—asparagine, aspartic acid and glutamic acid produced smaller size of the AuNPs whereas amino acids such as tyrosine, serine and threonine
3.5. Proton NMR studies
In order to understand the effect of binding of the amino acids to the nanoparticles, proton NMR (1H NMR) studies were carried out. The comparison of chemical shift values (in ppm) of α and β protons of pure amino acids and amino acid capped AuNPs as obtained from 1H NMR spectra show a distinct splitting and shifting (table 1). The NMR signals of the amino acid capped AuNPs indicated an overall downfield shift in α as well as β protons. This may be attributed to the shifting of electron density from the amino acid to nanoparticles leading to de-shielding of α and β protons of the amino acid. In other 4
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Figure 3. TEM images of AuNPs reduced by L-Glutamic acid (a), L-Phenylalanine (b), L-Aspartic acid (c), L-Asparagine (d), L-Serine (e), L-Leucine (f), L-Valine (g), L-Threonine (h) and L-Tyrosine (i).
4. Conclusions
words, the nanoparticles attract electron density from the either the amino group and/or the carboxyl group of the amino acid leading to downfield shift. Apart from the apparent shifting of NMR signals, crucial evidence confirming the proximity of the metal centre to the concerned amino acid is the consistent metal induced peak broadening observed in the spectra of all the amino acid reduced AuNPs which is clearly visible in the spectra of all the samples. Considering the downfield shift and unsymmetrical pattern observed in the 1H NMR peaks, it can be safely interpreted that the amino acids interact strongly with the nanoparticle surface.
In conclusion, size tunable synthesis of mono-dispersed water soluble AuNPs by using non-toxic, naturally occurring amino acids both as reducing and capping/stabilising agents has been described. The size control could be easily achieved by varying the ratio of the gold ion to the amino acid. The synthesized AuNPs were analyzed by UV–vis spectroscopy, DLS, TEM and NMR Out of the 20 amino acids, glutamic acid, aspartic acid, asparagine, serine, threonine, isoleucine, leucine, tyrosine, valine showed good nanoparticle yield. Moreover, threonine and tyrosine provided good size 5
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Table 1. Comparison of chemical shift values (in ppm) of α and β protons of pure amino acids and amino acid capped AuNPs as obtained
from 1H NMR spectra (see figure S1–S8 in electronic supplementary information for detailed 1H NMR spectra).
1. 2. 3. 4. 5. 6. 7. 8.
Glutamic acid Aspartic acid Phenylalanine Leucine Valine Asparagine Serine Threonine
α-proton (pure amino acids)
α-proton (amino acid capped AuNPs)
β-proton (pure amino acids)
β-proton (amino acid capped AuNPs)
3.6259–3.6576 3.8707–3.8999 3.8470–3.8799 3.5927–3.6265 3.4695–3.4803 3.8707–3.9007 3.7923–3.8812 4.1049–4.1666
3.6599–3.6913 3.9447–3.9731 3.7971–3.9022 3.6293–3.6634 3.5337–3.5445 3.8686–3.9149 3.8217–3.8708 4.1237–4.1854
2.3335–2.4633 2.7236–2.8729 2.9666–3.1871 1.5361–1.6404 2.1046–2.1857 2.7001–2.8609 3.7035–3.7271 3.4597–3.4721
2.3437–2.4670 2.8250–2.9428 2.9754–3.1956 1.5714–1.6779 2.1595–2.2407 2.7098–2.8689 3.7676–3.7790 3.4786–3.4909
*R represents the side chain functionality of the respective amino acid.
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Figure 4. Variation in size of AuNPs with change in concentration of amino acids as observed from dynamic light scattering.
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Acknowledgments This study was supported by the Department of Science & Technology (DST) INSPIRE, India [IFA12-CH-52] and Science Education & Research Board (SERB) of India grant (F. No. SB/SO/BB/0040/2013). Sarabjit Kaur and Manish Bajaj thank Council of Scientific & Industrial Research (CSIR), India and University Grants Commission (UGC), India, respectively for research fellowships.
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