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Synthesis of fluorescent metal nanoparticles in aqueous solution by photochemical reduction
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 045601 (http://iopscience.iop.org/0957-4484/25/4/045601) View the table of contents for this issue, or go to the journal homepage for more
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Nanotechnology Nanotechnology 25 (2014) 045601 (8pp)
doi:10.1088/0957-4484/25/4/045601
Synthesis of fluorescent metal nanoparticles in aqueous solution by photochemical reduction Prakash Kshirsagar1 , Shiv Shankar Sangaru2 , Virgilio Brunetti1 , Maria Ada Malvindi1 and Pier Paolo Pompa1 1
Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies@Unile, Via Barsanti, I-73010 Arnesano, LE, Italy 2 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia E-mail: [email protected] and [email protected] Received 12 June 2013, revised 22 September 2013 Accepted for publication 5 November 2013 Published 6 January 2014 Abstract
A facile green chemistry approach for the synthesis of sub-5 nm silver and gold nanoparticles is reported. The synthesis was achieved by a photochemical method using tyrosine as the photoreducing agent. The size of the gold and silver nanoparticles was about 3 and 4 nm, respectively. The nanoparticles were characterized using x-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy and photoluminescence spectroscopy. Both silver and gold nanoparticles synthesized by this method exhibited fluorescence properties and their use for cell imaging applications has been demonstrated. Keywords: metal nanoparticles, fluorescence, photoreduction, bio-imaging S Online supplementary data available from stacks.iop.org/Nano/25/045601/mmedia (Some figures may appear in colour only in the online journal)
1. Introduction
very effective antimicrobial agents [19]. Furthermore, in the quantum confinement regime, these metal NPs can also exhibit fluorescence properties [20]. Keeping in mind that currently it is highly desirable to develop green chemical methods for materials synthesis [21, 22], in this report we demonstrate such an efficient procedure to synthesize sub-5 nm monodispersed AuNPs and AgNPs in an aqueous medium. The synthesis is carried out photochemically using an amino acid, tyrosine, as the photoreducing agent. Synthesis of metal nanoparticles with amino acids or peptides including tyrosine has already been demonstrated but did not yield sub-5 nm NPs or NPs with a narrow size distribution as achieved with the current method [22, 23]. Moreover, we show preliminary data to demonstrate that these NPs are suitable for bio-imaging applications. Photoreduction has been widely used as a route for the synthesis of metal NPs either in solution or in polymeric films [24]. While sub-5 nm AuNPs and AgNPs, owing to their fluorescence properties [25], can
Silver (AgNPs) and gold nanoparticles (AuNPs) have been widely studied and applied in various fields of science and technology [1–5]. Their size dependent properties have also been well analyzed [6–9]. In particular, remarkable changes in the physical and chemical properties of metal NPs in the sub-5 nm size range have been observed [6, 10–12]. In the last two decades, many synthesis methods have been developed to control the size of NPs from a few nanometers to the submicron range in aqueous, organic and other media [13–15]. However, in the case of sub-5 nm NPs, synthesis protocols are still typically carried out in an organic or biphasic system. Even when carried out in an aqueous medium, the methods invariably use thiols, phosphines or some polymeric capping agents to achieve this size range [16–18]. Sub-5 nm metal particles have high catalytic activity [6] and, in the case of AgNPs, they are also known to have better ion release kinetics, making them 0957-4484/14/045601+08$33.00
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c 2014 IOP Publishing Ltd Printed in the UK
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of Ag2 SO4 , tyrosine and KOH was kept at 1:2.5:10. The irradiation time was 30 min. Synthesis of AuNPs was carried out similarly with a reaction solution consisting of HAuCl4 , tyrosine and KOH in the ratio 1:3:10 maintained at 15 ◦ C. The AgNPs and AuNPs were separated from the unbound tyrosine photoreaction products by first concentrating the entire as-prepared NP solutions to around 1 ml by rotary evaporation and then passing them through a column packed with Sephadex G-75 using water as the elutant.
be used for bio-imaging [26] and detection [27], their synthesis by a photochemical route has been demonstrated only recently. However, these syntheses were done in organic solvent [25] making it less advantageous for bio-applications [28], as almost invariably an additional step of surface modification is necessary to render them water soluble and biocompatible [21]. The photochemical synthesis to obtain such NPs in an aqueous medium with biocompatible reducing agents, therefore, has special significance as the NPs can then be readily used in physiological conditions. Additionally, for biolabeling applications, the size of the fluorophore is crucial for an efficient bio-conjugation and targeted delivery [29]. In an efficient bio-conjugation, a small fluorophore size minimizes possible steric hindrance that can interfere with biomolecule functions or access to certain specific cellular targets [29–31]. For example, when targeting neuronal synapses or the nucleus, one of the necessary criteria is that the NP–biconjugate ensemble is sufficiently small to efficiently access these regions [32, 33]. Though several methods have demonstrated the synthesis of metal nanoclusters, the presence of bio- or polymeric macromolecules on the nanoclusters negates the size advantage. As a result, though the achieved metal core size is very small, the actual hydrodynamic radius including the stabilizing molecules is comparatively large [34].
The NP solutions were characterized with UV–vis spectroscopy on a Cary 300 UV–visible spectrophotometer at a resolution of 1 nm using a 5 mm path length quartz cuvette. The NPs were imaged using transmission electron microscopy (TEM). Samples were prepared by drop casting on a carbon-coated copper grid, and imaged on a JEOL JEM1011 microscope operating at an accelerating voltage of 100 kV. X-ray diffraction (XRD) measurements were carried out on a Panalytical ˚ beam X’Pert PRO diffractometer with a Cu Kα (1.54 A) line. The XRD samples were prepared by drop casting the aqueous AgNP solution on a silicon wafer and drying in air. Fourier transform infrared (FTIR) spectra of tyrosine, AgNPs and AuNPs were carried out with a Bruker Vertex-70 FTIR spectrometer. For the FTIR spectra, samples were prepared by making KBr pellets of the pure tyrosine and lyophilized AgNP and AuNP samples. The unbound tyrosine photoproducts and metal NPs were separated by centrifuging a concentrated sample of metal NPs in an Amicon centrifugal filter with 3 kDa cut off. The separated NPs and the filtered-off solution containing the unbound photoproducts were lyophilized to get enough dry sample that could be used for preparing a KBr pellet. An additional control sample for FTIR was prepared from the basic tyrosine solution after photoirradiation for 30 min in the absence of any metal ions. All fluorescence measurements were recorded in photon counting mode using a 450-W Xenon lamp as the source of excitation and double monochromators in both excitation and emission (4 nm bandwidths). The emitted light was observed at right angles to the excitation radiation. Prior to the fluorescence measurements, the as-prepared AgNP and AuNP solutions were purified with an Amicon Ultra centrifugal filters having 3 kDa cutoff at a speed of 4000 rpm for 20 min and then redispersed in deionized water. Concentrations of NPs were evaluated by ICP-AES with a Varian Vista AX spectrometer. The metal NP samples were digested in aqua regia prior to ICP-AES measurements. Zeta potential (ζ ) and dynamic light scattering (DLS) characterizations of AgNPs and AuNPs were performed using a Zetasizer Nano-ZS instrument (Malvern) equipped with a 4.0 mV He–Ne 633 nm laser at a measurement temperature of 25 ◦ C. The sample solution was passed through Puradisc polypropylene FP 30/0.2 µM membrane (GE Healthcare) before measurements, and five acquisitions were made to get a mean value for the measurement. 2.2.2. Characterization.
2. Experimental section 2.1. Chemical
Tyrosine, silver sulfate (Ag2 SO4 ), chloroauric acid (HAuCl4 ), potassium hydroxide (KOH), fetal bovine serum, phosphate buffer, saline buffer, penicillin 1% (v/v), streptomycin, L -glutamine, non-essential amino acids, paraformaldehyde and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich. Gold (1000 ppm, code E3AU4) and silver (1000 ppm, code E3AGA) element reference solutions for inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis were purchased from Romil Ltd. All chemicals were used without any purification. Ultrapure deionized water (Millipore-elix water purification system, 18.2 M cm) was used for the preparation of all stock solutions. Glassware and the magnetic stirring-bars were washed thoroughly with aqua regia (HCl and HNO3 in a 3:1 volumetric ratio). Entire stock solutions should be freshly prepared. 2.2. Procedure
The reactions were typically carried out in a laboratory reactor system II (UV Consulting Peschl) fitted with a UV lamp (150 W medium pressure Hg vapor lamp, Heraeus TQ 150) surrounded by quartz tubing for water cooling. The reaction vessel was filled with 600 ml of 10−3 M KOH aqueous solution pre-cooled to 4 ◦ C and irradiated with the UV lamp. After the UV lamp started to glow steadily at peak intensity, tyrosine and Ag2 SO4 were added to it under vigorous stirring. The ratio 2.2.1. Synthesis of metal nanoparticles.
Human cervical carcinoma cells, HeLa (ICLC number HTL950239), MCF7 human breast 2.2.3. Cell culture.
2
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adenocarcinoma cells (ICLC number HTL95021), SH SY5Y human neuroblastoma cells (ICLC number HTL95013) and A549 human lung carcinoma cells (ICLC number HTL03001) were purchased from Interlab Cell Line Collection, IST, Genova, Italy. HuH7 human hepatoma cells were purchased from the American Tissue Type Culture Collection (LGC Standards S.r.l., Milano, Italy). All adherent cell lines were cultured in 75 cm2 flasks (Sarstedt) in high glucose Dulbecco’s modified Eagles medium (DMEM), supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) 10 000 U ml−1 penicillin, 10 000 U ml−1 streptomycin, 2 mM L-glutamine and 1% non-essential amino acids. For all the experiments, cells were maintained under standard cell culture conditions (5% CO2 , 95% humidity and 37 ◦ C) and harvested every 3 days. The uptake of NPs into cells was tracked by confocal microscopy. All cell lines were seeded onto thin glass slide bottomed Petri dishes at a density of 105 cells ml−1 and after 24 h were incubated with both AgNPs and AuNPs at a final concentration of 5 nM for 24 h at 37 ◦ C in 5% CO2 . On the following days, cell culture medium was removed from the cells and replaced by standard complete medium. After 24 h of incubation, samples were washed with phosphate buffered saline (PBS) buffer pH 7.4, harvested and then fixed in buffered 3.7% paraformaldehyde for 20 min. After PBS washing, samples were imaged by confocal microscopy (Leica TCS SP5 AOBS) using a 405 pulsed diode laser (PicoQuant GmbH) and the following conditions: 40× oil immersion objective (HCX PL APO CS, numerical aperture 40×/1.25 oil), pixel size of approximately 400 nm, pixel dwell time 2.3 µs, photomultiplier tube (PMT) detector, with a and 451–638 nm detection spectral range for AgNPs and 485–601 nm for AuNPs. 2.2.4. Confocal microscopy.
Figure 1. (a) UV–vis spectra of AgNP and AuNP suspensions
synthesized by photochemical reduction with tyrosine. The inset is the enlarged UV–vis spectrum of AuNP solution shown for clarity. (b) XRD pattern of the above mentioned AgNP and AuNP samples deposited on Si substrate. The solid bars at the bottom are the standard 2θ values for bulk fcc Ag (JCPDS 04-0783) and the vertical dashed lines are the standard 2θ values for bulk fcc Au (JCPDS 04-0784).
OPTIMA control software and elaborated with MARS data analysis software (BMG Labtech). To express the cytotoxicity, the average absorbance of the wells containing cell culture medium without cells was subtracted from the average absorbance of the solvent control, 5% DMSO or both AgNP and AuNP treated cells. The percentage of viable cells was calculated as previously reported [35–37].
The metabolic activity of MCF7 cells was measured after 24, 48 and 96 h of exposure to 0.5, 1, 5 nM of both AgNPs and AuNPs, using colorimetric assays based on the detection of the highly water-soluble tetrazolium salt WST-8 (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) (Cell Counting Kit-8 Fluka) [35–37]; these assays were performed in 96-well plates (Sarstedt) for each time. The cells were seeded in microplates at a density of 10 000 cells per well and cultured for 24, 48 and 96 h. Different amounts of both AgNPs and AuNPs dispersed in stock cell culture medium solution were added into different wells to obtain final NP concentrations of 0.5, 1, 5 nM. A final concentration of 5% DMSO in medium was used as the positive control for both cell lines; eight replicates were performed for each investigated point including the controls and blanks (medium only). A 10 ml aliquot of WST-8 solution was added to each well. The 96-well microplates were incubated for 3 h in a humidified atmosphere of 5% CO2 at 37 ◦ C. Subsequently, the orange WST-8 formazan product was measured at a wavelength of 460 nm in a FluoStar Optima (BMG Labtech) microplate reader. Data were collected by 2.2.5. WST-8 cytotoxicity assay.
3. Result and discussion
The photoactive molecule tyrosine was tested for its ability to reduce metal ions, such as Ag (I) and Au (III), to their zero oxidation state and thereby form metal NPs. Tyrosine with an amine group also has good affinity to bind onto a metal surface and with two readily ionizable groups, namely carboxyl and phenoxyl groups. It can stabilize NPs very efficiently. Figure 1(a) shows the UV–vis spectra of AgNPs and AuNPs synthesized by photoirradiation of aqueous Ag+ or AuCl− 4 ion solutions, respectively, in presence of tyrosine. The absorbance peak for AgNPs is observed at 408 nm. The plasmonic resonance band for the AuNPs is observed as a small step around 500 nm (inset figure 1(a)). The absorbance of the AuNPs is very weak compared to the AgNPs, as the molar extinction coefficient of the plasmon resonance peak of AgNPs is much higher than that of AuNPs [38]. The absence of a distinct and sufficiently intense absorbance band with 3
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a peak around or above 520 nm indicates the presence of very small AuNPs, close to the size range where quantum confinement effects begin to play a noticeable role [39–41]. Imaging conditions were as in section 2.2.4. The nanoparticles were further characterized by XRD (figure 1(b)). The XRD curves for AgNPs show Bragg peaks at 38.23, 44.17, 64.55 and 77.51◦ corresponding to the (111), (200), (220) and (311) lattice planes of standard face centered cubic (fcc) silver (JCPDS 04-0783). Similarly, for AuNPs Bragg peaks at 38.54, 44.11, 64.95 and 77.9◦ , can be observed close to the values for bulk fcc gold (JCPDS 04-0784). This confirms the successful formation of crystalline metallic NPs. In particular, in the case of AuNPs, an increase of about 0.3–0.4◦ in the 2θ value for the Bragg peaks corresponding to (111), (220) and (311) lattices and a decrease of about 0.3◦ for the (200) lattice can be noticed. The increase in the 2θ value indicates a very minor contraction [42, 43] (∼1–2 pm), which is usually observed with NPs within the 1–4 nm size range. However, a decrease in the 2θ value for the (200) lattice diffraction peak indicates that, unlike the other lattice planes, a minor expansion occurs within these sets of lattice planes of the AuNPs. The broadening of the Bragg diffraction peaks in the XRD pattern is characteristic of very small NPs [44, 45]. Moreover; the Bragg peaks corresponding to the AuNPs are broader than those of the AgNPs, indicating their relatively smaller size. Using the Scherrer formula [45], the size of AgNPs and AuNPs were calculated by considering the FWHM of the (220) Bragg peak, as this peak was distinct without any overlap with adjacent Bragg peaks. The particles size was calculated to be around 4.2 and 2.5 nm for AgNPs and AuNPs, respectively. As XRD gives information about the bulk sample, it can be unequivocally claimed that the sample primarily consists of such very small NPs. To confirm the above observations, TEM imaging of both AgNPs and AuNPs was carried out as shown in figure 2. At first glance, it can be observed that both samples have narrow size distributions. Silver nanoparticles are mostly less than 5 nm in size as presented in figure 2(a). The size distribution histogram of AgNPs calculated from the TEM images is shown in figure 2(c). From the Gaussian fit of the histogram, the mean particle size and its standard deviation were calculated to be around 4.0 ± 1 nm, in close agreement with XRD measurements. On the other hand, AuNPs as shown in figure 2(b) are smaller than the AgNPs. The particle size distribution histogram indicates a mean size of 3.4 ± 0.8 nm (figure 2(d)). This value for AuNPs is slightly higher than that calculated from the XRD measurements. Such a discrepancy could be attributed to the fact that very small NPs, around 1 nm, are too close to the resolution limit to be well resolved by TEM. These nanoparticles were also characterized by dynamic light scattering, and the mean particle size was about ∼6.2 nm and ∼2.4 nm for AgNPs and AuNPs, respectively. Considering the small size and reasonably narrow size distribution of these nanoparticles, it is worth mentioning that achieving such a degree of size distribution in an aqueous solution is usually difficult, in spite of the number of reports on photoreduction methods [46–48]. Moreover, the lack of thiols, surfactants
Figure 2. Representative TEM images of (a) AgNPs and (b) AuNPs
synthesized by photochemical reduction with tyrosine. (c), (d) The particle size distributions, calculated from TEM images, of AgNPs and AuNPs, respectively.
or bulky polymeric capping molecules in this method can minimize the limitations faced in efficient ligand exchange necessary for the desired surface modification [49, 50]. The NPs were also analyzed by FTIR measurements. In figure 3, the FTIR spectra of tyrosine, AgNPs, AuNPs and of unbound tyrosine derivative molecules after the photoreduction process are shown. In the pure tyrosine spectrum (figure 3(e), gray curve), the characteristic peaks are observed for the carboxylate group at 1591/1608 cm−1 and at 1363/1377 cm−1 corresponding to the asymmetric and symmetric stretching frequencies [51], respectively, and for the benzene skeletal CC stretching and the phenolic CO stretching at 1514 and 1245 cm−1 , respectively. The FTIR spectrum of the tyrosine photoreduction product (figure 3(f), light gray curve) obtained in absence of Ag+ and AuCl− 4 ions is almost identical to that of tyrosine itself. Only some broadening of the peaks corresponding to the carboxylate stretching frequencies is observed, which could be a result 4
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Figure 3. FTIR spectra of (a) AgNPs and (b) AuNPs synthesized by
photoreduction with tyrosine. Unbound photoreaction products of tyrosine (c) from the AgNP solution and spectra (d) from the AuNP solution after repeated washing by centrifugation and redispersion in deionized water. Spectra from pure tyrosine (e) and from the control photoirradiated product of tyrosine (f).
of the formation of potassium salt in the presence of KOH. The lack of any significant changes suggests that there are no identifiable structural changes occurring in the tyrosine molecule after photoirradiation in the absence of Ag+ and AuCl− 4 ions. In the spectrum of unbound photoreaction products of tyrosine (figures 3(c) and (d)), peaks in the region corresponding to carboxylate group, benzene skeletal CC and the CO stretch frequencies could still be observed. There are, however, changes in the fingerprint region that are difficult to attribute individually, probably indicating a change in the structure of the molecules with their functional groups intact. Importantly, a distinct peak above 1670 cm−1 corresponding to the carbonyl group of a quinone like derivative reportedly observed during thermal reduction with tyrosine is not observed with photoreduction [23]. Even in the case of FTIR spectra of AuNPs and AgNPs (figures 3(a) and (b)) a peak is only observed at around 1630 cm−1 . Hence, the reduction of the silver ion does not appear to primarily proceed through the oxidation pathway, as observed in thermal reduction process [23]. As formation of dityrosine is highly probable under UV irradiation [52, 53], it can be expected that the same occurs in the present case. Under alkaline conditions and UV irradiation, tyrosyl radicals are formed due to the release of solvated electrons [53] from the phenoxide group of tyrosine. These solvated electrons have a high probability of reducing the silver ion [54] and the tyrosyl radicals undergo crosslinking to yield dityrosine [55] or even higher tyrosine adducts such as trityrosine, tetra-tyrosine and pulcherosine [56–58]. Therefore the reaction pathway is likely to be UV
Tyr− −−→ Tyr• + e− solv
(1)
+ 0 e− solv + Ag −−→ Ag
(2)
Tyr• + Tyr• −−→ Tyr − Tyr.
(3)
Figure 4. (a) Fluorescence emission (solid curves) and excitation
spectra of the AgNP solution. Curve-1 and the dotted curve are the emission and excitation spectra of the unbound photoproducts of AgNPs synthesis. Emission was acquired with λexc at 314 nm and the excitation was acquired by monitoring λem at 415 nm. Curve-2 and the dashed curve are the emission and excitation spectra of the purified AgNPs by passing through Sephadex G-75 column. In (b) the emission spectrum was acquired with λexc at 550 nm and the excitation spectra with λem at 480 nm. Fluorescence emission (solid curves) and excitation spectra of AuNPs solution. Curve-1, curve-2 and curve-3 represent emission spectra with λexc at 310, 410 and 470 nm, respectively. The dotted curve, dash–dot curve and dashed curves are the excitation spectra acquired with λem at 420, 480 and 580 nm, respectively.
In the case of AuNPs, the CC stretch and CO stretch peaks in the FTIR spectra (figure 3(b)) are comparatively weak. This indicates that the photoproduct of AuNP synthesis also includes some oxidation products other than di-, trimeric, etc forms of tyrosine. Though it is difficult to precisely identify all the reaction products using FTIR, the formation of dityrosine or other tyrosine-adduct species can be collectively confirmed from their fluorescence properties. Figures 4(a) and (b) show the excitation and emission spectra of the purified AgNP and AuNP solutions and of their unbound tyrosine photoreaction products. Curve-1 of both figures 4(a) and (b) represents the emission spectra of the unbound 5
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Table 1. Details of zeta potential of AgNPs and AuNPs in both water and DMEM.
Nanoparticles AgNPs AuNPs
Zeta potential in water (mV) −28 ± 10 −25 ± 10
Zeta potential in DMEM (mV) −22 ± 18 −19 ± 08
photoreaction product of tyrosine molecules. These unbound molecules of AgNPs (figure 4(a)) have a strong emission at 415 nm (gray color; solid curve-1) with excitation maxima at 315 nm (gray color; dotted curve). Similarly, for unbound molecules of AuNPs (figure 4(b)) the corresponding emission and excitation peaks are observed at 418 nm (gray color; solid curve-1) and 311 nm (gray color; dotted curve), respectively. These fluorescence characteristics match very well those of dityrosine molecules [52, 59] whose emission characteristics have been demonstrated by Harms et al [60]. The observed similar emission feature therefore confirms the formation of such dimeric form of tyrosine in our photoreduction method. The emission band at around 410 nm is also observed from the purified AgNP and AuNP solutions, though with much reduced intensity, suggesting that they are also acting as stabilizing molecules. Curve-2 of figure 4(a), obtained from the AgNP fraction shows an emission peak at 550 nm (black color, solid curve) with an excitation peak at 480 nm (black color; dash curve), which possibly originates from the NPs themselves [11, 25, 61]. Similarly, curve-3 in figure 4(b) with an emission peak at 535 nm (black color, solid curve) possibly corresponds to AuNPs [10, 62, 63]. Additionally, as a common feature, the longer wavelength emission spectra for both AgNPs and AuNPs showed a gradual red shift with increasing λexc , and it was also accompanied by a decrease in intensity of the emission band. This behavior has been observed by several other research groups and could possibly be attributed to the size distribution of the NPs in the sample [64] (supporting figure S4 and S5 available at stacks.iop.org/Nano/ 25/045601/mmedia). However, contribution from tyrosine photoproducts also cannot be excluded from these emission characteristics. The excitation spectra for the AuNP solutions (figure 4(b)) interestingly show two overlapping bands at 412 and 471 nm. Excitation at these two wavelengths yields two main emission bands. While the emission band at 535 nm (curve-3, figure 4(b)) corresponds to excitation at 470 nm (black color, dash curve), the emission band at 480 nm (light gray, solid curve-2) corresponds to excitation at 410 nm (light gray, dash–dot line). We believe that the emission at 480 nm (curve-2, figure 4(b)) is a result of some higher oligomeric photoproducts other than simple di- or trimeric, etc forms of tyrosine [65]. Further, as this emission at 480 nm has strong overlap with the excitation band at 471 nm (black color, dash curve) of AuNPs, there is a high probability for fluorescence resonance energy transfer (FRET) between the two fluorophores leading to the appearance of a double band like structure in the excitation spectra of AuNPs (figure 4(b)). It may be recalled that the presence of photoproducts other than di-, trimeric, etc forms of tyrosine was also evident from the FTIR spectra. The differences in the oxidation products
of tyrosine in the two syntheses may be attributed to the fact that, unlike in the case of Ag+ ion reduction, which is a one-electron transfer process, AuCl− 4 is a three-electron process. Irrespective of the variation, however, the ability of these photoproducts to stabilize the metal nanoparticles is remarkable. In fact, the as-prepared solution of metal NPs could be readily concentrated from about 1 l of solution to about 1 ml (up to NP concentrations of about ∼1.5–2 mM) or even lyophilized to a powder and still be redispersed [66]. From zeta potential (ζ ) measurement of both AgNPs and AuNPs in water and DMEM, the surface charge (zeta) of both particles were observed as given in table 1 (figure S3 available at stacks.iop.org/Nano/25/045601/mmedia). 3.1. Cell imaging
While a number of methods for fluorescent metal NPs have been reported, not all could be successfully applied for bioimaging. For instance, DNA mediated synthesis yields very stable fluorescent metal NPs; however, their internalization into cells for bio-imaging is difficult and could be facilitated only in the presence of some other carrier vehicles [67, 68]. To test the bio-imaging applicability of tyrosine photoreduced metal NPs, five different cell lines were selected, namely four cancer cell lines (A549, lung adenocarcinoma; HeLa, cervical carcinoma; HuH7, hepatocarcinoma; MCF7, breast cancers) and one a neuroblastoma cell line (SY5Y). All the cell lines were incubated with metal NPs at a final concentration of 5 nM for 24 h. Figure 5 shows the confocal images of the cells incubated with both AgNPs (figures 5(a)–(e), i.e. left panel) and AuNPs (figures 5(f)–(j), i.e. right panel), respectively. As can be noticed, a good fluorescence signal was obtained from all the cell lines. The observed fluorescence was localized in the cytoplasmic region around the nucleus, suggesting effective internalization of both kinds of NPs. Similar confocal imaging with a NP concentration of 0.5 nM incubated with A549, HeLa, HuH7 and SY5Y cell lines is shown in supporting information S6 (available at stacks.iop.org/Nano/25/045601/mmedia). Evidently, the tyrosine derivative molecules, apart from providing sufficient charge density for stabilization of the NPs in water (figure S3 available at stacks.iop.org/Nano/25/045601/mmedia), give them favorable surface characteristics, preventing significant aggregation of NPs in the cell culture medium (the effective hydrodynamic diameter of AuNPs and Ag NPs, is about ∼18 nm and ca. ∼20 nm, respectively, after 24 h of incubation, supporting figure S2 available at stacks.iop. org/Nano/25/045601/mmedia) due to protein corona effects, which in turn promote efficient cellular internalization. The mechanism of internalization is apparently very similar to that observed for citrate capped AuNPs [69] that have similar 6
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Figure 6. Toxicity assessments by WST-8 assay on MCF7 cells
incubated with increasing concentrations (0.5, 1 and 5 nM respectively) of (a) AgNPs and (b) AuNPs at different times (24, 48 and 96 h). Ctrl identifies the negative controls in cells grown in the absence of NPs; ctrl + is the positive control (5% DMSO).
AuNPs in DMEM. The non-specific binding of the serum protein from the culture medium could thus facilitate the internalization of the AgNPs and Au NPs without any aggregation in the present case. The low hydrodynamic radius of the as-synthesized NPs and the presence of carboxylic groups in tyrosine derivative capping could prove to be a convenient aspect for bio-conjugation and subsequent biolabeling applications. Toxicity assessment was carried out in MCF7 cells (figure 6). Experimental data indicate that, in the case of AuNPs, the induced toxicity was quite low, even at high concentrations and long incubation times. As expected, for AgNPs we observed a more pronounced toxicity, although their use up to 1 nM seems to not compromise the cells viability significantly. Overall, these data recommend the exploitation of the NPs in the short term (24/48 h) and with low doses in biological applications.
Figure 5. Representative confocal microscopy images (cells were
imaged in bright field) showing bioaccumulation of AgNPs (left panel) in (a) A549, (b) HeLa, (c) HuH7, (d) MCF7 and (e) SH SY5Y cells and AuNPs (right panel) in (f) A549, (g) HeLa, (h) HuH7, (i) MCF7 and (j) SH SY5Y cells. All cells were exposed to 5 nM of AgNPs and AuNPs, respectively, for 24 h at 37 ◦ C in 5% CO2 . A good fluorescence signal was obtained from the cytoplasmic regions around the nucleus, suggesting effective internalization of both kinds of NP.
4. Conclusion
In conclusion, we have shown that sub-5 nm fluorescent metallic NPs can be efficiently synthesized by a photoreduction method. The presented method is quite general and works efficiently for both AgNPs and AuNPs to yield good quality NPs. Additionally; it is a green chemistry approach that utilizes water as the solvent and tyrosine as an environmentally benign photoreducing agent. The synthesized NPs are stabilized by photo-oxidation products of tyrosine such as dityrosine, trityrosine, etc and show multiple
zeta potential values. However, unlike citrate molecules the stabilizing molecules in the tyrosine mediated synthesis are not so weakly bound to the surface to cause aggregation like that observed by Chitrani et al [69] for citrate capped 7
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fluorescence emission characteristics. Due to the simplicity of the process, large-scale synthesis of fluorescent NPs can also be conveniently achieved by this method.
[36] Brunetti V, Chimbli H, Fiammengo R, Galeone A, Malvindi M A, Vecchio G, Cingolani R, Nadaeu J and Pompa P P 2013 Nanoscale 5 307 [37] Malvindi M A, Brunetti V, Vecchio G, Galeone A, Cingolani R and Pompa P P 2012 Nanoscale 4 486 [38] Link S, Wang Z L and El-Sayed M A 1999 J. Phys. Chem. B 103 3529 [39] Alvarez M M, Khoury J T, Schaaff T G, Shafigullin M N, Vezmar I and Whetten R L 1997 J. Phys. Chem. B 101 3706 [40] Hodak J H, Henglein A and Hartland G V 2000 J. Phys. Chem. B 104 9954 [41] Quin H, Zhu M, Wu Z and Jin R 2012 Acc. Chem. Res. 9 1470 [42] Szczerba W, Riesemeier H and Thunemann A 2010 Anal. Bioanal. Chem. 398 1967 [43] Zanchet D, Tolentino H, Martins Alves M C, Alves O L and Ugarte D I 2000 Chem. Phys. Lett. 323 167 [44] Mari A, Imperatori P, Marchegiani G, Pilloni L, Mezzi A, Kaciulis S, Cannas C, Meneghini C, Mobilio S and Suber L 2010 Langmuir 26 15561 [45] Klug H P and Alexander L E 1974 X-Ray Diffraction Procedures: For Polycrystalline and Amorphous Materials 2nd edn (New York: Wiley) p 692 [46] Itakura T, Torigoe K and Esumi K 1995 Langmuir 11 4129 [47] Fong Y Y, Gascooke J R, Visser B R, Metha G F and Buntine M A 2010 J. Phys. Chem. C 114 15931 [48] Huang H H, Ni X P, Loy G L, Chew C H, Tan K L, Loh F C, Deng J F and Xu G Q 1996 Langmuir 12 909 [49] Schulz-Dobrick M, Vijaya Sarathy K and Jansen M 2005 J. Am. Chem. Soc. 127 12816 [50] Liu X, Li C, Xu J, Lv J, Zhu M, Guo Y, Cui S, Liu H and Li Y 2008 J. Phys. Chem. C 112 10778 [51] Hadzija O and Spoljar B 1995 Fresen. J. Anal. Chem. 351 692 [52] Lehrer S S and Fasman G D 1967 Biochemistry 6 757 [53] Bent D V and Hayon E 1975 J. Am. Chem. Soc. 97 2599 [54] Soroushian B, Lampre I, Belloni J and Mostafavi M 2005 Radiat. Phys. Chem. 72 111 [55] Heinecke J W, Li W, Francis G A and Goldstein J A 1993 J. Clin. Invest. 91 2866 [56] Skaff O, Jolliffe K A and Hutton C A 2005 J. Org. Chem. 70 7353 [57] Rothschild K J, Roepe P, Ahl P L, Earnest T N, Bogomolni R A, Das Gupta S K, Mulliken C M and Herzfeld J 1986 Proc. Natl Acad. Sci. 83 347 [58] Brady J D, Sadler I H and Fry S C 1998 Phytochemistry 47 349 [59] Jacob J S, Cistola D P, Hsu F F, Muzaffar S, Mueller D M, Hazen S L and Heinecke J W 1996 J. Biol. Chem. 271 19950 [60] Harms G S, Pauls S W, Hedstrom J F and Johnson C K 1997 J. Fluoresc. 7 283 [61] Yu X and Wang Q 2011 Microchim. Acta 173 293 [62] Huang C C, Yang Z, Lee K H and Chang H T 2007 Angew. Chem. Int. Edn 46 6824 [63] Zheng J, Nicovich P R and Dickson R M 2007 Annu. Rev. Phys. Chem. 58 409 [64] Diez I, Ras R, Kanyuk M and Demchenko A 2013 Phys. Chem. Chem. Phys. 15 979 [65] Sionkowska A and Kaminska A 1999 J. Photochem. Photobiol. A 120 207 [66] Kshirsagar P, Sangaru S S, Malvindi M A, Martiradonna L, Cingolani R and Pompa P P 2011 Colloids Surf. A 392 264 [67] Lattore A and Somoza A 2012 Chem.Bio.Chem. 13 951 [68] Choi S, Dickson R and Yu J 2012 Chem. Soc. Rev. 41 1867 [69] Chitrani B D, Ghazani A A and Chan W C W 2006 Nano Lett. 6 662
References [1] Wang G, Wang Y, Chen L and Choo J 2010 Biosens. Bioelectron. 25 1859 [2] Pradeep T and Anshup 2009 Thin Solid Films 517 6441 [3] Sepulveda B, Angelome P C, Lechuga L M and Liz-Marzan L M 2009 Nano Today 4 244 [4] Cuenya B R 2010 Thin Solid Films 518 3127 [5] Shankar S S and Deka S 2011 Sci. Adv. Mater. 3 169 [6] Hutchings G J 2008 Dalton Trans. 41 5523 [7] Jain P K, Huang X, El-Sayed I H and El-Sayed M A 2008 Acc. Chem. Res. 41 1578 [8] Warrier P and Teja A 2011 Nanoscale Res. Lett. 6 247 [9] Dienerowitz M, Mazilu M and Dholakia K 2008 J. Nanophoton. 2 021875 [10] Wilcoxon J P, Martin J E, Parsapour F, Wiedenman B and Kelley D F 1998 J. Chem. Phys. 108 9137 [11] Diez I and Ras R H A 2011 Nanoscale 3 1963 [12] Yu L and Andriola A 2010 Talanta 82 869 [13] Zheng N, Fan J and Stucky G D 2006 J. Am. Chem. Soc. 128 6550 [14] Kwon K, Lee K Y, Kim M, Lee Y W, Heo J, Ahn S J and Han S W 2006 Chem. Phys. Lett. 432 209 [15] Zhang C, Zhang J, Han B, Zhao Y and Li W 2008 Green Chem. 10 1094 [16] Wilcoxon J P and Abrams B L 2006 Chem. Soc. Rev. 35 1162 [17] Stoeva S, Klabunde K J, Sorensen C M and Dragieva I J 2002 J. Am. Chem. Soc. 124 2305 [18] Wu Z, Chen J and Jin R 2011 Adv. Funct. Mater. 21 177 [19] Liu J, Sonshine D A, Shervani S and Hurt R A 2010 ACS Nano 4 6903 [20] Zheng J, Nicovich P and Dickson R 2007 Annu. Rev. Phys. Chem. 58 409 [21] Dahl J A, Maddux B L S and Hutchison J E 2007 Chem. Rev. 107 2228 [22] Tan Y N, Lee J Y and Wang D I C 2010 J. Am. Chem. Soc. 132 5677 [23] Selvakannan P R, Swami A, Srisathiyanarayanan D, Shirude P S, Pasricha R, Mandale A B and Sastry M 2004 Langmuir 20 7825 [24] Sakamoto M, Fujistuka M and Majima T 2009 J. Photochem. Photobiol. C 10 33 [25] Maretti L, Billone P S, Liu Y and Scaiano J C 2009 J. Am. Chem. Soc. 131 13972 [26] He H, Xie C and Ren J 2008 Anal. Chem. 80 5951 [27] Sharma J, Yeh H C, Yoo H, Werner J H and Martinez J S 2011 Chem. Commun. 47 229 [28] Huang S, Bai M and Wang L 2013 Sci. Rep. 3 2023 [29] Lovric J, Bazzi H, Cuie Y, Fortin G, Winnik F and Maysinger D 2005 J. Mol. Med. 83 377 [30] Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R and Nann T 2008 Nature Methods 5 763 [31] Goldman E R, Clapp A R, Anderson G P, Uyeda H T, Mauro J M, Medintz I G and Mattoussi H 2004 Anal. Chem. 76 684 [32] Chen F and Gerion D 2004 Nano Lett. 4 1827 [33] Howarth M, Liu W, Puthenveetil S, Zheng Y, Marshall L F, Schmidt M M, Dane Wittrup K, Bawendi M G and Ting A Y 2008 Nature Methods 5 397 [34] Bronstein L and Shifrina Z 2011 Chem. Rev. 111 5301 [35] Sabella S, Brunetti V, Vecchio G, Galeone A, Maiorano G, Cingolani R and Pompa P P 2011 J. Nanopart. Res. 13 6821
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Synthesis of fluorescent metal nanoparticles in aqueous solution by photochemical reduction
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 045601 (http://iopscience.iop.org/0957-4484/25/4/045601) View the table of contents for this issue, or go to the journal homepage for more
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Nanotechnology Nanotechnology 25 (2014) 045601 (8pp)
doi:10.1088/0957-4484/25/4/045601
Synthesis of fluorescent metal nanoparticles in aqueous solution by photochemical reduction Prakash Kshirsagar1 , Shiv Shankar Sangaru2 , Virgilio Brunetti1 , Maria Ada Malvindi1 and Pier Paolo Pompa1 1
Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies@Unile, Via Barsanti, I-73010 Arnesano, LE, Italy 2 King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia E-mail: [email protected] and [email protected] Received 12 June 2013, revised 22 September 2013 Accepted for publication 5 November 2013 Published 6 January 2014 Abstract
A facile green chemistry approach for the synthesis of sub-5 nm silver and gold nanoparticles is reported. The synthesis was achieved by a photochemical method using tyrosine as the photoreducing agent. The size of the gold and silver nanoparticles was about 3 and 4 nm, respectively. The nanoparticles were characterized using x-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy and photoluminescence spectroscopy. Both silver and gold nanoparticles synthesized by this method exhibited fluorescence properties and their use for cell imaging applications has been demonstrated. Keywords: metal nanoparticles, fluorescence, photoreduction, bio-imaging S Online supplementary data available from stacks.iop.org/Nano/25/045601/mmedia (Some figures may appear in colour only in the online journal)
1. Introduction
very effective antimicrobial agents [19]. Furthermore, in the quantum confinement regime, these metal NPs can also exhibit fluorescence properties [20]. Keeping in mind that currently it is highly desirable to develop green chemical methods for materials synthesis [21, 22], in this report we demonstrate such an efficient procedure to synthesize sub-5 nm monodispersed AuNPs and AgNPs in an aqueous medium. The synthesis is carried out photochemically using an amino acid, tyrosine, as the photoreducing agent. Synthesis of metal nanoparticles with amino acids or peptides including tyrosine has already been demonstrated but did not yield sub-5 nm NPs or NPs with a narrow size distribution as achieved with the current method [22, 23]. Moreover, we show preliminary data to demonstrate that these NPs are suitable for bio-imaging applications. Photoreduction has been widely used as a route for the synthesis of metal NPs either in solution or in polymeric films [24]. While sub-5 nm AuNPs and AgNPs, owing to their fluorescence properties [25], can
Silver (AgNPs) and gold nanoparticles (AuNPs) have been widely studied and applied in various fields of science and technology [1–5]. Their size dependent properties have also been well analyzed [6–9]. In particular, remarkable changes in the physical and chemical properties of metal NPs in the sub-5 nm size range have been observed [6, 10–12]. In the last two decades, many synthesis methods have been developed to control the size of NPs from a few nanometers to the submicron range in aqueous, organic and other media [13–15]. However, in the case of sub-5 nm NPs, synthesis protocols are still typically carried out in an organic or biphasic system. Even when carried out in an aqueous medium, the methods invariably use thiols, phosphines or some polymeric capping agents to achieve this size range [16–18]. Sub-5 nm metal particles have high catalytic activity [6] and, in the case of AgNPs, they are also known to have better ion release kinetics, making them 0957-4484/14/045601+08$33.00
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c 2014 IOP Publishing Ltd Printed in the UK
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of Ag2 SO4 , tyrosine and KOH was kept at 1:2.5:10. The irradiation time was 30 min. Synthesis of AuNPs was carried out similarly with a reaction solution consisting of HAuCl4 , tyrosine and KOH in the ratio 1:3:10 maintained at 15 ◦ C. The AgNPs and AuNPs were separated from the unbound tyrosine photoreaction products by first concentrating the entire as-prepared NP solutions to around 1 ml by rotary evaporation and then passing them through a column packed with Sephadex G-75 using water as the elutant.
be used for bio-imaging [26] and detection [27], their synthesis by a photochemical route has been demonstrated only recently. However, these syntheses were done in organic solvent [25] making it less advantageous for bio-applications [28], as almost invariably an additional step of surface modification is necessary to render them water soluble and biocompatible [21]. The photochemical synthesis to obtain such NPs in an aqueous medium with biocompatible reducing agents, therefore, has special significance as the NPs can then be readily used in physiological conditions. Additionally, for biolabeling applications, the size of the fluorophore is crucial for an efficient bio-conjugation and targeted delivery [29]. In an efficient bio-conjugation, a small fluorophore size minimizes possible steric hindrance that can interfere with biomolecule functions or access to certain specific cellular targets [29–31]. For example, when targeting neuronal synapses or the nucleus, one of the necessary criteria is that the NP–biconjugate ensemble is sufficiently small to efficiently access these regions [32, 33]. Though several methods have demonstrated the synthesis of metal nanoclusters, the presence of bio- or polymeric macromolecules on the nanoclusters negates the size advantage. As a result, though the achieved metal core size is very small, the actual hydrodynamic radius including the stabilizing molecules is comparatively large [34].
The NP solutions were characterized with UV–vis spectroscopy on a Cary 300 UV–visible spectrophotometer at a resolution of 1 nm using a 5 mm path length quartz cuvette. The NPs were imaged using transmission electron microscopy (TEM). Samples were prepared by drop casting on a carbon-coated copper grid, and imaged on a JEOL JEM1011 microscope operating at an accelerating voltage of 100 kV. X-ray diffraction (XRD) measurements were carried out on a Panalytical ˚ beam X’Pert PRO diffractometer with a Cu Kα (1.54 A) line. The XRD samples were prepared by drop casting the aqueous AgNP solution on a silicon wafer and drying in air. Fourier transform infrared (FTIR) spectra of tyrosine, AgNPs and AuNPs were carried out with a Bruker Vertex-70 FTIR spectrometer. For the FTIR spectra, samples were prepared by making KBr pellets of the pure tyrosine and lyophilized AgNP and AuNP samples. The unbound tyrosine photoproducts and metal NPs were separated by centrifuging a concentrated sample of metal NPs in an Amicon centrifugal filter with 3 kDa cut off. The separated NPs and the filtered-off solution containing the unbound photoproducts were lyophilized to get enough dry sample that could be used for preparing a KBr pellet. An additional control sample for FTIR was prepared from the basic tyrosine solution after photoirradiation for 30 min in the absence of any metal ions. All fluorescence measurements were recorded in photon counting mode using a 450-W Xenon lamp as the source of excitation and double monochromators in both excitation and emission (4 nm bandwidths). The emitted light was observed at right angles to the excitation radiation. Prior to the fluorescence measurements, the as-prepared AgNP and AuNP solutions were purified with an Amicon Ultra centrifugal filters having 3 kDa cutoff at a speed of 4000 rpm for 20 min and then redispersed in deionized water. Concentrations of NPs were evaluated by ICP-AES with a Varian Vista AX spectrometer. The metal NP samples were digested in aqua regia prior to ICP-AES measurements. Zeta potential (ζ ) and dynamic light scattering (DLS) characterizations of AgNPs and AuNPs were performed using a Zetasizer Nano-ZS instrument (Malvern) equipped with a 4.0 mV He–Ne 633 nm laser at a measurement temperature of 25 ◦ C. The sample solution was passed through Puradisc polypropylene FP 30/0.2 µM membrane (GE Healthcare) before measurements, and five acquisitions were made to get a mean value for the measurement. 2.2.2. Characterization.
2. Experimental section 2.1. Chemical
Tyrosine, silver sulfate (Ag2 SO4 ), chloroauric acid (HAuCl4 ), potassium hydroxide (KOH), fetal bovine serum, phosphate buffer, saline buffer, penicillin 1% (v/v), streptomycin, L -glutamine, non-essential amino acids, paraformaldehyde and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich. Gold (1000 ppm, code E3AU4) and silver (1000 ppm, code E3AGA) element reference solutions for inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis were purchased from Romil Ltd. All chemicals were used without any purification. Ultrapure deionized water (Millipore-elix water purification system, 18.2 M cm) was used for the preparation of all stock solutions. Glassware and the magnetic stirring-bars were washed thoroughly with aqua regia (HCl and HNO3 in a 3:1 volumetric ratio). Entire stock solutions should be freshly prepared. 2.2. Procedure
The reactions were typically carried out in a laboratory reactor system II (UV Consulting Peschl) fitted with a UV lamp (150 W medium pressure Hg vapor lamp, Heraeus TQ 150) surrounded by quartz tubing for water cooling. The reaction vessel was filled with 600 ml of 10−3 M KOH aqueous solution pre-cooled to 4 ◦ C and irradiated with the UV lamp. After the UV lamp started to glow steadily at peak intensity, tyrosine and Ag2 SO4 were added to it under vigorous stirring. The ratio 2.2.1. Synthesis of metal nanoparticles.
Human cervical carcinoma cells, HeLa (ICLC number HTL950239), MCF7 human breast 2.2.3. Cell culture.
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adenocarcinoma cells (ICLC number HTL95021), SH SY5Y human neuroblastoma cells (ICLC number HTL95013) and A549 human lung carcinoma cells (ICLC number HTL03001) were purchased from Interlab Cell Line Collection, IST, Genova, Italy. HuH7 human hepatoma cells were purchased from the American Tissue Type Culture Collection (LGC Standards S.r.l., Milano, Italy). All adherent cell lines were cultured in 75 cm2 flasks (Sarstedt) in high glucose Dulbecco’s modified Eagles medium (DMEM), supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) 10 000 U ml−1 penicillin, 10 000 U ml−1 streptomycin, 2 mM L-glutamine and 1% non-essential amino acids. For all the experiments, cells were maintained under standard cell culture conditions (5% CO2 , 95% humidity and 37 ◦ C) and harvested every 3 days. The uptake of NPs into cells was tracked by confocal microscopy. All cell lines were seeded onto thin glass slide bottomed Petri dishes at a density of 105 cells ml−1 and after 24 h were incubated with both AgNPs and AuNPs at a final concentration of 5 nM for 24 h at 37 ◦ C in 5% CO2 . On the following days, cell culture medium was removed from the cells and replaced by standard complete medium. After 24 h of incubation, samples were washed with phosphate buffered saline (PBS) buffer pH 7.4, harvested and then fixed in buffered 3.7% paraformaldehyde for 20 min. After PBS washing, samples were imaged by confocal microscopy (Leica TCS SP5 AOBS) using a 405 pulsed diode laser (PicoQuant GmbH) and the following conditions: 40× oil immersion objective (HCX PL APO CS, numerical aperture 40×/1.25 oil), pixel size of approximately 400 nm, pixel dwell time 2.3 µs, photomultiplier tube (PMT) detector, with a and 451–638 nm detection spectral range for AgNPs and 485–601 nm for AuNPs. 2.2.4. Confocal microscopy.
Figure 1. (a) UV–vis spectra of AgNP and AuNP suspensions
synthesized by photochemical reduction with tyrosine. The inset is the enlarged UV–vis spectrum of AuNP solution shown for clarity. (b) XRD pattern of the above mentioned AgNP and AuNP samples deposited on Si substrate. The solid bars at the bottom are the standard 2θ values for bulk fcc Ag (JCPDS 04-0783) and the vertical dashed lines are the standard 2θ values for bulk fcc Au (JCPDS 04-0784).
OPTIMA control software and elaborated with MARS data analysis software (BMG Labtech). To express the cytotoxicity, the average absorbance of the wells containing cell culture medium without cells was subtracted from the average absorbance of the solvent control, 5% DMSO or both AgNP and AuNP treated cells. The percentage of viable cells was calculated as previously reported [35–37].
The metabolic activity of MCF7 cells was measured after 24, 48 and 96 h of exposure to 0.5, 1, 5 nM of both AgNPs and AuNPs, using colorimetric assays based on the detection of the highly water-soluble tetrazolium salt WST-8 (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) (Cell Counting Kit-8 Fluka) [35–37]; these assays were performed in 96-well plates (Sarstedt) for each time. The cells were seeded in microplates at a density of 10 000 cells per well and cultured for 24, 48 and 96 h. Different amounts of both AgNPs and AuNPs dispersed in stock cell culture medium solution were added into different wells to obtain final NP concentrations of 0.5, 1, 5 nM. A final concentration of 5% DMSO in medium was used as the positive control for both cell lines; eight replicates were performed for each investigated point including the controls and blanks (medium only). A 10 ml aliquot of WST-8 solution was added to each well. The 96-well microplates were incubated for 3 h in a humidified atmosphere of 5% CO2 at 37 ◦ C. Subsequently, the orange WST-8 formazan product was measured at a wavelength of 460 nm in a FluoStar Optima (BMG Labtech) microplate reader. Data were collected by 2.2.5. WST-8 cytotoxicity assay.
3. Result and discussion
The photoactive molecule tyrosine was tested for its ability to reduce metal ions, such as Ag (I) and Au (III), to their zero oxidation state and thereby form metal NPs. Tyrosine with an amine group also has good affinity to bind onto a metal surface and with two readily ionizable groups, namely carboxyl and phenoxyl groups. It can stabilize NPs very efficiently. Figure 1(a) shows the UV–vis spectra of AgNPs and AuNPs synthesized by photoirradiation of aqueous Ag+ or AuCl− 4 ion solutions, respectively, in presence of tyrosine. The absorbance peak for AgNPs is observed at 408 nm. The plasmonic resonance band for the AuNPs is observed as a small step around 500 nm (inset figure 1(a)). The absorbance of the AuNPs is very weak compared to the AgNPs, as the molar extinction coefficient of the plasmon resonance peak of AgNPs is much higher than that of AuNPs [38]. The absence of a distinct and sufficiently intense absorbance band with 3
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a peak around or above 520 nm indicates the presence of very small AuNPs, close to the size range where quantum confinement effects begin to play a noticeable role [39–41]. Imaging conditions were as in section 2.2.4. The nanoparticles were further characterized by XRD (figure 1(b)). The XRD curves for AgNPs show Bragg peaks at 38.23, 44.17, 64.55 and 77.51◦ corresponding to the (111), (200), (220) and (311) lattice planes of standard face centered cubic (fcc) silver (JCPDS 04-0783). Similarly, for AuNPs Bragg peaks at 38.54, 44.11, 64.95 and 77.9◦ , can be observed close to the values for bulk fcc gold (JCPDS 04-0784). This confirms the successful formation of crystalline metallic NPs. In particular, in the case of AuNPs, an increase of about 0.3–0.4◦ in the 2θ value for the Bragg peaks corresponding to (111), (220) and (311) lattices and a decrease of about 0.3◦ for the (200) lattice can be noticed. The increase in the 2θ value indicates a very minor contraction [42, 43] (∼1–2 pm), which is usually observed with NPs within the 1–4 nm size range. However, a decrease in the 2θ value for the (200) lattice diffraction peak indicates that, unlike the other lattice planes, a minor expansion occurs within these sets of lattice planes of the AuNPs. The broadening of the Bragg diffraction peaks in the XRD pattern is characteristic of very small NPs [44, 45]. Moreover; the Bragg peaks corresponding to the AuNPs are broader than those of the AgNPs, indicating their relatively smaller size. Using the Scherrer formula [45], the size of AgNPs and AuNPs were calculated by considering the FWHM of the (220) Bragg peak, as this peak was distinct without any overlap with adjacent Bragg peaks. The particles size was calculated to be around 4.2 and 2.5 nm for AgNPs and AuNPs, respectively. As XRD gives information about the bulk sample, it can be unequivocally claimed that the sample primarily consists of such very small NPs. To confirm the above observations, TEM imaging of both AgNPs and AuNPs was carried out as shown in figure 2. At first glance, it can be observed that both samples have narrow size distributions. Silver nanoparticles are mostly less than 5 nm in size as presented in figure 2(a). The size distribution histogram of AgNPs calculated from the TEM images is shown in figure 2(c). From the Gaussian fit of the histogram, the mean particle size and its standard deviation were calculated to be around 4.0 ± 1 nm, in close agreement with XRD measurements. On the other hand, AuNPs as shown in figure 2(b) are smaller than the AgNPs. The particle size distribution histogram indicates a mean size of 3.4 ± 0.8 nm (figure 2(d)). This value for AuNPs is slightly higher than that calculated from the XRD measurements. Such a discrepancy could be attributed to the fact that very small NPs, around 1 nm, are too close to the resolution limit to be well resolved by TEM. These nanoparticles were also characterized by dynamic light scattering, and the mean particle size was about ∼6.2 nm and ∼2.4 nm for AgNPs and AuNPs, respectively. Considering the small size and reasonably narrow size distribution of these nanoparticles, it is worth mentioning that achieving such a degree of size distribution in an aqueous solution is usually difficult, in spite of the number of reports on photoreduction methods [46–48]. Moreover, the lack of thiols, surfactants
Figure 2. Representative TEM images of (a) AgNPs and (b) AuNPs
synthesized by photochemical reduction with tyrosine. (c), (d) The particle size distributions, calculated from TEM images, of AgNPs and AuNPs, respectively.
or bulky polymeric capping molecules in this method can minimize the limitations faced in efficient ligand exchange necessary for the desired surface modification [49, 50]. The NPs were also analyzed by FTIR measurements. In figure 3, the FTIR spectra of tyrosine, AgNPs, AuNPs and of unbound tyrosine derivative molecules after the photoreduction process are shown. In the pure tyrosine spectrum (figure 3(e), gray curve), the characteristic peaks are observed for the carboxylate group at 1591/1608 cm−1 and at 1363/1377 cm−1 corresponding to the asymmetric and symmetric stretching frequencies [51], respectively, and for the benzene skeletal CC stretching and the phenolic CO stretching at 1514 and 1245 cm−1 , respectively. The FTIR spectrum of the tyrosine photoreduction product (figure 3(f), light gray curve) obtained in absence of Ag+ and AuCl− 4 ions is almost identical to that of tyrosine itself. Only some broadening of the peaks corresponding to the carboxylate stretching frequencies is observed, which could be a result 4
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Figure 3. FTIR spectra of (a) AgNPs and (b) AuNPs synthesized by
photoreduction with tyrosine. Unbound photoreaction products of tyrosine (c) from the AgNP solution and spectra (d) from the AuNP solution after repeated washing by centrifugation and redispersion in deionized water. Spectra from pure tyrosine (e) and from the control photoirradiated product of tyrosine (f).
of the formation of potassium salt in the presence of KOH. The lack of any significant changes suggests that there are no identifiable structural changes occurring in the tyrosine molecule after photoirradiation in the absence of Ag+ and AuCl− 4 ions. In the spectrum of unbound photoreaction products of tyrosine (figures 3(c) and (d)), peaks in the region corresponding to carboxylate group, benzene skeletal CC and the CO stretch frequencies could still be observed. There are, however, changes in the fingerprint region that are difficult to attribute individually, probably indicating a change in the structure of the molecules with their functional groups intact. Importantly, a distinct peak above 1670 cm−1 corresponding to the carbonyl group of a quinone like derivative reportedly observed during thermal reduction with tyrosine is not observed with photoreduction [23]. Even in the case of FTIR spectra of AuNPs and AgNPs (figures 3(a) and (b)) a peak is only observed at around 1630 cm−1 . Hence, the reduction of the silver ion does not appear to primarily proceed through the oxidation pathway, as observed in thermal reduction process [23]. As formation of dityrosine is highly probable under UV irradiation [52, 53], it can be expected that the same occurs in the present case. Under alkaline conditions and UV irradiation, tyrosyl radicals are formed due to the release of solvated electrons [53] from the phenoxide group of tyrosine. These solvated electrons have a high probability of reducing the silver ion [54] and the tyrosyl radicals undergo crosslinking to yield dityrosine [55] or even higher tyrosine adducts such as trityrosine, tetra-tyrosine and pulcherosine [56–58]. Therefore the reaction pathway is likely to be UV
Tyr− −−→ Tyr• + e− solv
(1)
+ 0 e− solv + Ag −−→ Ag
(2)
Tyr• + Tyr• −−→ Tyr − Tyr.
(3)
Figure 4. (a) Fluorescence emission (solid curves) and excitation
spectra of the AgNP solution. Curve-1 and the dotted curve are the emission and excitation spectra of the unbound photoproducts of AgNPs synthesis. Emission was acquired with λexc at 314 nm and the excitation was acquired by monitoring λem at 415 nm. Curve-2 and the dashed curve are the emission and excitation spectra of the purified AgNPs by passing through Sephadex G-75 column. In (b) the emission spectrum was acquired with λexc at 550 nm and the excitation spectra with λem at 480 nm. Fluorescence emission (solid curves) and excitation spectra of AuNPs solution. Curve-1, curve-2 and curve-3 represent emission spectra with λexc at 310, 410 and 470 nm, respectively. The dotted curve, dash–dot curve and dashed curves are the excitation spectra acquired with λem at 420, 480 and 580 nm, respectively.
In the case of AuNPs, the CC stretch and CO stretch peaks in the FTIR spectra (figure 3(b)) are comparatively weak. This indicates that the photoproduct of AuNP synthesis also includes some oxidation products other than di-, trimeric, etc forms of tyrosine. Though it is difficult to precisely identify all the reaction products using FTIR, the formation of dityrosine or other tyrosine-adduct species can be collectively confirmed from their fluorescence properties. Figures 4(a) and (b) show the excitation and emission spectra of the purified AgNP and AuNP solutions and of their unbound tyrosine photoreaction products. Curve-1 of both figures 4(a) and (b) represents the emission spectra of the unbound 5
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Table 1. Details of zeta potential of AgNPs and AuNPs in both water and DMEM.
Nanoparticles AgNPs AuNPs
Zeta potential in water (mV) −28 ± 10 −25 ± 10
Zeta potential in DMEM (mV) −22 ± 18 −19 ± 08
photoreaction product of tyrosine molecules. These unbound molecules of AgNPs (figure 4(a)) have a strong emission at 415 nm (gray color; solid curve-1) with excitation maxima at 315 nm (gray color; dotted curve). Similarly, for unbound molecules of AuNPs (figure 4(b)) the corresponding emission and excitation peaks are observed at 418 nm (gray color; solid curve-1) and 311 nm (gray color; dotted curve), respectively. These fluorescence characteristics match very well those of dityrosine molecules [52, 59] whose emission characteristics have been demonstrated by Harms et al [60]. The observed similar emission feature therefore confirms the formation of such dimeric form of tyrosine in our photoreduction method. The emission band at around 410 nm is also observed from the purified AgNP and AuNP solutions, though with much reduced intensity, suggesting that they are also acting as stabilizing molecules. Curve-2 of figure 4(a), obtained from the AgNP fraction shows an emission peak at 550 nm (black color, solid curve) with an excitation peak at 480 nm (black color; dash curve), which possibly originates from the NPs themselves [11, 25, 61]. Similarly, curve-3 in figure 4(b) with an emission peak at 535 nm (black color, solid curve) possibly corresponds to AuNPs [10, 62, 63]. Additionally, as a common feature, the longer wavelength emission spectra for both AgNPs and AuNPs showed a gradual red shift with increasing λexc , and it was also accompanied by a decrease in intensity of the emission band. This behavior has been observed by several other research groups and could possibly be attributed to the size distribution of the NPs in the sample [64] (supporting figure S4 and S5 available at stacks.iop.org/Nano/ 25/045601/mmedia). However, contribution from tyrosine photoproducts also cannot be excluded from these emission characteristics. The excitation spectra for the AuNP solutions (figure 4(b)) interestingly show two overlapping bands at 412 and 471 nm. Excitation at these two wavelengths yields two main emission bands. While the emission band at 535 nm (curve-3, figure 4(b)) corresponds to excitation at 470 nm (black color, dash curve), the emission band at 480 nm (light gray, solid curve-2) corresponds to excitation at 410 nm (light gray, dash–dot line). We believe that the emission at 480 nm (curve-2, figure 4(b)) is a result of some higher oligomeric photoproducts other than simple di- or trimeric, etc forms of tyrosine [65]. Further, as this emission at 480 nm has strong overlap with the excitation band at 471 nm (black color, dash curve) of AuNPs, there is a high probability for fluorescence resonance energy transfer (FRET) between the two fluorophores leading to the appearance of a double band like structure in the excitation spectra of AuNPs (figure 4(b)). It may be recalled that the presence of photoproducts other than di-, trimeric, etc forms of tyrosine was also evident from the FTIR spectra. The differences in the oxidation products
of tyrosine in the two syntheses may be attributed to the fact that, unlike in the case of Ag+ ion reduction, which is a one-electron transfer process, AuCl− 4 is a three-electron process. Irrespective of the variation, however, the ability of these photoproducts to stabilize the metal nanoparticles is remarkable. In fact, the as-prepared solution of metal NPs could be readily concentrated from about 1 l of solution to about 1 ml (up to NP concentrations of about ∼1.5–2 mM) or even lyophilized to a powder and still be redispersed [66]. From zeta potential (ζ ) measurement of both AgNPs and AuNPs in water and DMEM, the surface charge (zeta) of both particles were observed as given in table 1 (figure S3 available at stacks.iop.org/Nano/25/045601/mmedia). 3.1. Cell imaging
While a number of methods for fluorescent metal NPs have been reported, not all could be successfully applied for bioimaging. For instance, DNA mediated synthesis yields very stable fluorescent metal NPs; however, their internalization into cells for bio-imaging is difficult and could be facilitated only in the presence of some other carrier vehicles [67, 68]. To test the bio-imaging applicability of tyrosine photoreduced metal NPs, five different cell lines were selected, namely four cancer cell lines (A549, lung adenocarcinoma; HeLa, cervical carcinoma; HuH7, hepatocarcinoma; MCF7, breast cancers) and one a neuroblastoma cell line (SY5Y). All the cell lines were incubated with metal NPs at a final concentration of 5 nM for 24 h. Figure 5 shows the confocal images of the cells incubated with both AgNPs (figures 5(a)–(e), i.e. left panel) and AuNPs (figures 5(f)–(j), i.e. right panel), respectively. As can be noticed, a good fluorescence signal was obtained from all the cell lines. The observed fluorescence was localized in the cytoplasmic region around the nucleus, suggesting effective internalization of both kinds of NPs. Similar confocal imaging with a NP concentration of 0.5 nM incubated with A549, HeLa, HuH7 and SY5Y cell lines is shown in supporting information S6 (available at stacks.iop.org/Nano/25/045601/mmedia). Evidently, the tyrosine derivative molecules, apart from providing sufficient charge density for stabilization of the NPs in water (figure S3 available at stacks.iop.org/Nano/25/045601/mmedia), give them favorable surface characteristics, preventing significant aggregation of NPs in the cell culture medium (the effective hydrodynamic diameter of AuNPs and Ag NPs, is about ∼18 nm and ca. ∼20 nm, respectively, after 24 h of incubation, supporting figure S2 available at stacks.iop. org/Nano/25/045601/mmedia) due to protein corona effects, which in turn promote efficient cellular internalization. The mechanism of internalization is apparently very similar to that observed for citrate capped AuNPs [69] that have similar 6
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Figure 6. Toxicity assessments by WST-8 assay on MCF7 cells
incubated with increasing concentrations (0.5, 1 and 5 nM respectively) of (a) AgNPs and (b) AuNPs at different times (24, 48 and 96 h). Ctrl identifies the negative controls in cells grown in the absence of NPs; ctrl + is the positive control (5% DMSO).
AuNPs in DMEM. The non-specific binding of the serum protein from the culture medium could thus facilitate the internalization of the AgNPs and Au NPs without any aggregation in the present case. The low hydrodynamic radius of the as-synthesized NPs and the presence of carboxylic groups in tyrosine derivative capping could prove to be a convenient aspect for bio-conjugation and subsequent biolabeling applications. Toxicity assessment was carried out in MCF7 cells (figure 6). Experimental data indicate that, in the case of AuNPs, the induced toxicity was quite low, even at high concentrations and long incubation times. As expected, for AgNPs we observed a more pronounced toxicity, although their use up to 1 nM seems to not compromise the cells viability significantly. Overall, these data recommend the exploitation of the NPs in the short term (24/48 h) and with low doses in biological applications.
Figure 5. Representative confocal microscopy images (cells were
imaged in bright field) showing bioaccumulation of AgNPs (left panel) in (a) A549, (b) HeLa, (c) HuH7, (d) MCF7 and (e) SH SY5Y cells and AuNPs (right panel) in (f) A549, (g) HeLa, (h) HuH7, (i) MCF7 and (j) SH SY5Y cells. All cells were exposed to 5 nM of AgNPs and AuNPs, respectively, for 24 h at 37 ◦ C in 5% CO2 . A good fluorescence signal was obtained from the cytoplasmic regions around the nucleus, suggesting effective internalization of both kinds of NP.
4. Conclusion
In conclusion, we have shown that sub-5 nm fluorescent metallic NPs can be efficiently synthesized by a photoreduction method. The presented method is quite general and works efficiently for both AgNPs and AuNPs to yield good quality NPs. Additionally; it is a green chemistry approach that utilizes water as the solvent and tyrosine as an environmentally benign photoreducing agent. The synthesized NPs are stabilized by photo-oxidation products of tyrosine such as dityrosine, trityrosine, etc and show multiple
zeta potential values. However, unlike citrate molecules the stabilizing molecules in the tyrosine mediated synthesis are not so weakly bound to the surface to cause aggregation like that observed by Chitrani et al [69] for citrate capped 7
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fluorescence emission characteristics. Due to the simplicity of the process, large-scale synthesis of fluorescent NPs can also be conveniently achieved by this method.
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