Temperature Dependent Synthesis of Tryptophan-Functionalized Gold

Research Article pubs.acs.org/journal/ascecg

Temperature Dependent Synthesis of Tryptophan-Functionalized Gold Nanoparticles and Their Application in Imaging Human Neuronal Cells Dae-Young Kim,† Min Kim,‡ Surendra Shinde,† Rijuta Ganesh Saratale,§ Jung-Suk Sung,‡ and Gajanan Ghodake*,† †

Department of Biological and Environmental Science, College of Life Science and Biotechnology, Dongguk University-Seoul, 32, Dongguk-ro, Ilsandong-gu, Goyang-si, Gyonggido 10326, Republic of Korea ‡ Department of Life Science, College of Life Science and Biotechnology, Dongguk University-Seoul, 32, Dongguk-ro, Ilsandong-gu, Goyang-si, Gyonggido 10326, Republic of Korea § Research Institute of Biotechnology & Medical Converged Science, Dongguk University-Seoul, 32, Dongguk-ro, Ilsandong-gu, Goyang-si, Gyonggido 10326, Republic of Korea S Supporting Information *

ABSTRACT: The growth kinetics and temporal evolution of the UV−vis spectrum of gold nanoparticles (AuNPs) by following tryptophan reduction in different temperature conditions were studied systematically. The results revealed the productivity and overall reaction mechanism were mostly determined by the temperature, which was in turn affected by the concentration of tryptophan. Two considerably different reaction pathways were observed. The first pathway occurred at ambient temperature (35 °C) and consisted of three corresponding steps: nucleation, growth, and oriented attachment. The second pathway occurred above the ambient temperature (45, 65, and 95 °C) and was responsible for the well-known nucleation−growth route. The second pathway was used to develop a facile synthetic route for the preparation of functionalized AuNPs with the size about 20 nm. Consequently, the stability and functionalization of the AuNPs were demonstrated using dilution studies, zeta potential, and FTIR measurements. The imaging of human neuronal (SH-SY5Y) cells showed that the fluorescent signal from the tryptophanfunctionalized AuNPs was significantly brighter than that from autofluorescence of the cells. The strong signal, resistance to photobleaching, excellent stability, ease of synthesis, simplicity of functionalization, and biocompatibility make AuNPs an attractive option for imaging and biomedical applications. KEYWORDS: Biosynthesis, Gold nanoparticles, Tryptophan, Functionalization, Autofluorescence, Biocompatibility, Imaging

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INTRODUCTION Noble metal nanoparticles (NPs) have received much attention due to their unique properties arising from their size, shape, and assembly.1,2 Different synthesis routes such as green chemistry, template synthesis, microwave irradiation, organic solvents, and biological agents result in different features in the nanoproduct.3−5 However, the sustainability of the engineered nanoparticles is hindered by the energy-intensive processes, hazardous solvents, and toxic chemical wastes involved.6 Therefore, improving the quality of metal NPs in aqueous environments is still challenging with regard to the size, shape, and dispersion.7 Synthesis of AuNPs with small sizes and © XXXX American Chemical Society

excellent stability is essential for biomedical applications. In recent years, citrate (a weak base) has been studied extensively for use in controlling nucleation and growth based on the reactant concentration, reaction temperature, and solution pH.7,8 Thus, developing new methods for the synthesis of AuNPs is necessary to understand the nucleation process, growth, and functions of the nanoproduct. Thus, we speculated that the role of temperature in the growth of AuNPs could be useful for the Received: April 11, 2017 Revised: July 11, 2017 Published: August 15, 2017 A

DOI: 10.1021/acssuschemeng.7b01101 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Synthesis, Stabilization, and Functionalization of Gold Nanoparticles. Tryptophan mediated synthesis of AuNPs was carried out under different temperature conditions (35, 45, 65, and 95 °C) while varying the initial concentration of the precursor reactant tryptophan. The tryptophan concentration was varied from 0.0315 to 1.75 mM, whereas the concentrations of HAuCl4 (0.44 mM) and NaOH (2.5 mM) were kept fixed. The temporal evolution absorbance and UV−vis spectrum were recorded to elucidate the effect of temperature. The capability of tryptophan to reduce HAuCl4 chemically into AuNPs was assessed by tuning two different synthesis pathways. An improved understanding of the nanosynthesis process is necessary to develop methods that can maximize atom economy, similar to organic synthesis processes.23 Therefore, the maximum productivity of the dispersed AuNPs with the minimum amount of the tryptophan precursor was demonstrated successfully at both 65 and 95 °C. The stability of the AuNPs was tested using successive dilution with water and using zeta potential measurements. The overall methodology described herein for the preparation of the AuNPs meets all 12 principles of green chemistry, as no “man-made” reagents, other than the HAuCl4 and dilute NaOH, are used. The focus of this study is to highlight the need for a greener nanosynthesis process that accounts for the production of nanomaterials at optimum conditions and the need for an alternative to existing complicated and hazardous methods.24 Characterization of Gold Nanoparticles. The absorbance and UV−vis spectra of the AuNPs were obtained using the Optizen spectrophotometer-2120. UV−vis spectral data were used to determine the kinetics, productivity, and SPR bands of the respective samples prepared at 35, 45, 65, and 95 °C up to 72 h of incubation. Dynamic light scattering (DLS) measurements were used to identify the dependability and the size of AuNPs samples obtained at increasing concentrations of tryptophan using Brookhaven BI-9000. High resolution-transmission electron microscopy (HR-TEM) was used to examine the size, shape, dispersion, and self-assembly of the AuNPs prepared at 95 and 35 °C. HR-TEM samples were prepared using carbon-coated copper grids (300 mesh), and imaging was done using Tecnai G2. All sizing analysis was performed using licensed Adobe Photoshop. Zeta potential measurement was performed using Otsuka equipment for different AuNPs samples obtained using increasing concentrations of tryptophan at both 95 and 35 °C. AuNPs samples were used for DLS and zeta potential measurement after diluting 5 mL of the freshly prepared AuNPs to 10 mL of distilled water. The effect of dilution on the chemical and optical properties of the AuNPs solution was investigated. To confirm the stability of AuNPs under dilution, the plasmon resonance wavelength (λmax) and bandwidth (Δλ) were monitored after each addition of 0.25 mL of doubly ionized water to 1 mL of AuNPs solution. The fluorescence spectra were collected using spectrofluorometer (Jasco FP8200, Japan). The emission curves were recorded from 300 to 500 nm upon excitation at 310, 320, 330, 340, and 350 nm Cell Culture and Fluorescence Imaging. Human neuronal (SHSY5Y) cells were cultured in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin in an incubator at 37 °C in which at 5% CO2 level was kept constant. Typically, SH-SY5Y cells were seeded onto the coverslip in the 6 well cell culture (SPL, Korea) dish coated with poly L-lysine for 3 h, and then AuNPs were added into the medium grown for 17 h at 37 °C in an incubator under 5% CO2. AuNPs are allowed to uptake into cells via the endocytic pathway during cell differentiation and proliferation processes for 24 h at 100 ppm. The treated and nontreated SH-SY5Y cells on the coverslip were rinsed with PBS buffer (pH 7.4) and mounted with Dako fluorescence mounting medium (Dako, U.S.A.). The SH-SY5Y cells were incubated with tryptophan-capped AuNPs and imaged by using confocal microscopy excited with ultraviolet light in the range 330−350 and detected through a blue/cyan filter in the range 450−460. Cell imaging was accomplished by a Nikon confocal microscope at different pH conditions (4.0, 7.0, and 9.0). The nontreated SH-SY5Y cells were also observed to evaluate their autofluorescence. Fortunately, there was not serious interference from the autofluorescence from cells under the same imaging conditions. Image acquisition and processing were performed with the Nikon imaging software.

size controlled synthesis of AuNPs with excellent yield. Thus, the first part of this study was focused on the optical and structural properties of AuNPs in addition to achieving excellent stability from gold-tryptophan interactions. Fluorescence imaging is a powerful tool in biomedical applications and has been widely studied with a variety of fluorescent probes. Fluorescent probes presently in use are typically based on traditional organic dyes and on the sizedependent properties of quantum dots.9 Fluorescent probes must show a good response, biocompatibility, and high stability and sensitivity. Photostability in fluorescent dyes is desirable; however, increasing resistance to the photobleaching of the chromophores is quite challenging.10,11 Quantum dots are inherently toxic, and additional capping has been recommended to improve their biocompatibility.12 There have been reports on the functionalization of AuNPs with phosphors, dyes, and peptides to achieve emission in the visible range of the electromagnetic spectrum.13−15 Nevertheless, tryptophan, phenylalanine, and tyrosine are responsible for the characteristic optical properties of proteins.16,17 The fluorescence properties of the amino acid tryptophan originate from transitions between the indole group energy states.18 Tryptophan is an essential amino acid with the ability to absorb and emit electromagnetic radiation in the ultraviolet range.19 Joshi et al. proposed in their density functional theory that the carboxyl and indole functional groups of tryptophan are involved in the capping of tryptophan onto the AuNPs surface.20 The functionalization of metal nanoparticles with tryptophan enhances the lifetime of the excited electronic states.21 Moreover, tryptophan has few advantages such as solubility in water, biocompatibility, and high reducing/ stabilizing functionality.22 Thus, the fluorophore (tryptophan) is used in the green synthesis of AuNPs and in the development of fluorescent characteristics through surface chemistry. The ability of AuNPs capped by tryptophan molecules to enhance autofluorescence of the neuronal cells is one advantage of the present approach. A significant aim of this study was to elucidate the kinetics of the AuNPs synthesis in a quantitative manner to enable the understanding of the optical properties and atom economy of the synthesis process. The productivity of the AuNPs and temperature dependence of the surface plasmon resonance (SPR) bands were studied at 35, 45, 65, and 95 °C in aqueous solution. To identify the desired nanoproduct, the size, shape, and zetapotential profiles of the AuNPs at an increasing tryptophan concentration of the AuNPs were investigated. Synthesis and functionalization of AuNPs with a small size about 20 nm was achieved using tryptophan with excellent yield and stability at 95 °C. Capping of tryptophan on the AuNPs surface made it possible to obtain functionalized AuNPs that showed excitation at ∼330 nm and emission from 450 to 460 nm. This study clearly demonstrates the unique capabilities of tryptophan capped AuNPs under diverse conditions most applicable to the in vitro imaging. Thus, AuNPs entered in living human neuronal cells were observed during the cell differentiation and proliferation process.

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MATERIALS AND METHODS

Chemicals and Reagents. Chloroauric acid (HAuCl4·4H2O, 99.99%), tryptophan, and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich Chemicals, U.S.A. Tryptophan solution was freshly prepared prior to each experiment. All experiments were verified at least three times and were found to be highly reproducible. B


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Figure 1. UV−vis spectra of gold nanoparticles at four different temperature conditions indicated in the figure. (a) Effect of temperature conditions on the UV−vis spectrum of the gold nanoparticles with 0.125 mM tryptophan. (b) Effect of temperature conditions on the absorbance of the gold nanoparticles with 0.125 mM tryptophan. (c) Effect of temperature conditions on the UV−vis spectrum of the gold nanoparticles with 0.5 mM tryptophan. (d) Effect of temperature conditions on the absorbance of the gold nanoparticles with 0.5 mM tryptophan.

Figure 2. UV−vis spectra of gold nanoparticles at four different temperature conditions indicated in the figure. (a) Effect of temperature conditions on the UV−vis spectrum of the gold nanoparticles with 1.125 mM tryptophan. (b) Effect of temperature conditions on the absorbance of the gold nanoparticles with 1.125 mM tryptophan. (c) Effect of temperature conditions on the UV−vis spectrum of the gold nanoparticles with 1.75 mM tryptophan. (d) Effect of temperature conditions on the absorbance of the gold nanoparticles with 1.75 mM tryptophan.

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evolution patterns, and the size of the final gold nanoparticles (AuNPs). To explore this premise, further experiments were planned and executed in triplicate. Results from the experiments, described in the next two subsections, support the proposed

RESULTS AND DISCUSSION

Effect of Temperature on the Productivity of Gold Nanoparticles. The temperature of the reaction solution can play an essential role in the reaction rate, UV−vis spectral C


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Figure 3. (a) Performance of the gold nanoparticles was monitored at four different temperatures indicated in the figure while increasing tryptophan concentration. (b) The size of the gold nanoparticles was monitored by using DLS measurements for four different temperature conditions indicated in the figure while increasing tryptophan concentration.

hypothesis. Before discussing the results further, it should be pointed out that alkaline pH is suitable to initiate the classical nucleation and growth route. Reaction at acidic pH was too fast to be controlled and formed large clumps of AuNP aggregates within a fraction of a minute. Thus, it is inadvisible to perform the reaction in acidic conditions. On the other hand, the reaction slowed down at alkaline pH; however, the classical nucleation and growth route could be observed. Sodium citrate a weak base has been reported to increase the reactivity of gold complexes at increasing pH, thus increasing the reduction potential of reaction solutions.25 It was also reported that the reactivity of Ag complexes was improved significantly at alkaline pH conditions by adding NaOH with citrate reduction.26 The results are presented for the reactions adjusted to pH values above neutral with the addition of NaOH (2.5 mM). The reaction conditions were varied by temperature (35, 45, 65, and 95 °C), but the HAuCl4/NaOH ratio (0.44:2.5 mM) was kept constant. The influence of temperature on the UV−vis spectra of the AuNPs was studied with four different tryptophan concentrations (0.125, 0.5, 1.125, and 1.75 mM). The UV−vis spectral results reflect the effect of tryptophan and temperature conditions. Figure 1a shows the UV−vis spectral profile having single narrow SPR bands at a low tryptophan concentration (0.125 mM). The UV−vis absorbance in Figure 1b clearly shows the difference in reaction rates; at 35 and 45 °C it had lower productivity as compared to 65 and 95 °C. Figure 1c shows the UV−vis spectral profile at a tryptophan concentration of 0.5 mM with respect to the temperature conditions. The rate of nanoparticle synthesis was significantly improved at 45 °C as compared to the rate at 35 °C. Figure 1d clearly shows that the absorbance of the AuNPs at 65 and 95 °C was equal in terms of productivity. The UV−vis spectrum in Figure 2a clearly shows the differences in the intensity of the AuNPs obtained with a tryptophan concentration of 1.125 mM. The results indicate that the rate of AuNPs synthesis was almost similar at 45 and 65 °C. Figure 2b shows the productivity for the AuNPs was almost equal, and thus the reaction rate can be competitive at 45 and 65 °C. Figure 2c shows a linear UV−vis spectral profile for the tryptophan concentration of 1.75 mM. Finally, all of the tested concentrations of tryptophan exhibited temperature dependence. Figure 2d shows that the reaction rate increased linearly from 45 to 95 °C but did not improve at 35 °C for all of the tested concentrations of tryptophan. Thus, AuNPs synthesis with tryptophan can be controlled by changing the temperature. Similar spectral results, small size, and spherical morphology

have been reported for AuNPs at elevated temperatures using the citrate method.27 Implementing Green Chemistry Principles. Green chemistry aims to maximize material efficiency by optimizing the conversion of reactants into the desired products through the implementation of the atom economy principle (P2) and the real-time monitoring principle (P11). Excellent atom economy with the formation of dispersed AuNPs was observed at elevated temperatures of 65 and 95 °C (Figure 3a). As seen clearly in Figure 3a, it was essential to invest excessive atom economy in the form of the molecular atoms of tryptophan at 45 °C to generate AuNPs with maximum productivity. On the other hand, with the consumption of high atom economy, we confirmed the formation of well-defined clusters of AuNPs at 35 °C. These results clarify that the reaction rate tuned by temperature plays a vital role in defining the reaction pathway, productivity, and atom economy. Thus, the overall results presented in this report suggest that a high reaction rate is essential to produce dispersed and small AuNPs. The development of highly selective transformation pathways for dispersed and self-assembled AuNPs was achieved using the temperature approach. It is of prime importance to identify the reaction rate, maximize the yield, and minimize byproducts. Thus, data from this type of monitoring may be useful to optimize reaction conditions (e.g., temperature, time, and reactant concentration), particularly in future experiments. Furthermore, eco-friendly products that are less expensive and free of aggressive chemical reagents could be more desirable for the conversion of metallic salts into their corresponding NPs using a “bottom-up” approach.28 The concept of atom economy readily applies to all chemical synthesis reactions; however, there is a large scope to implement green chemistry principles in nanoparticle synthesis.29,30 The synthesis of nanomaterials that have minimal negative impacts on the environment is desired to reduce the effect of nanotechnology on environmental sustainability.24,31 Size and Shape of the Gold Nanoparticles. Finally, the size profile of the AuNPs was evaluated with DLS measurements and demonstrated good agreement with the spectral results. The effect of tryptophan concentration on the size of the AuNPs was examined from 0.031 to 1.75 mM. High-temperature conditions, both 65 and 95 °C, cannot support the dispersion of freshly formed AuNPs in a concentration of tryptophan lower than 0.125 mM, as can be seen in the DLS profile (Figure 3b). The tryptophan concentrations examined, from 0.125 to 1.75 mM, showed similar trends in DLS measurements. Thus, small and dispersed AuNPs could only be obtained at 65 and 95 °C and D


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Figure 4. Representative HR-TEM image of the dispersed gold nanoparticles obtained from 0.0315 to 1.75 mM tryptophan at 95 °C.

Figure 5. Representative TEM image of the self-assembled gold nanoparticles obtained from 0.0315 to 1.75 mM tryptophan at 35 °C.

aggregative growth and a model based on the initial phase of nucleation classical nucleation followed by isotropic growth and dispersion, Figure 4. HR-TEM imaging revealed that the formation of the AuNPs and their dispersion was dependent on the reactivity of gold ions, predominantly ordered by the concentration of tryptophan. However, the core size of the AuNPs after consuming all monomers remained constant as the concentration of tryptophan was increased (Figure 4). The aggregative growth has been observed, and it is supposed that the aggregate structure is a product of insufficient capping of the AuNPs in colloidal solution, particularly at elevated temperature (95 °C) and not arising from the drying process as reported previously.35 Similarly, there was an obvious decrease in the tendency to form aggregate and triplet-like morphology with the small sized AuNPs for reactions with higher tryptophan concentrations as noticed in size-distribution histograms (Figure S1). High tryptophan concentration lowers the reactivity of Au atoms as depositing on the AuNPs and thus suppress the

were found to be the most efficient temperature conditions. On the other hand, the DLS profile at 35 °C was significantly different from those at 45, 65, and 95 °C (Figure 3b). This result indicates that it is possible to control the self-assembling tendency of the AuNPs at ambient temperature. The control of size and morphology of functionalized AuNPs is significantly important for tuning the excitation of surface plasmons in surface enhanced fluorescence, surface-enhanced Raman scattering (SERS), and imaging.32−34 Size variation of the AuNPs with the tryptophan reduction method was observed at 95 °C as the initial concentration of tryptophan was modified. The HAuCl4/NaOH ratio was set at 0.44:2.5 mM for the results discussed here. The fixed auric acid and NaOH ratio reflects the effect of tryptophan concentration on the size, shape, and dispersion of the AuNPs. The effect of the tryptophan concentration ratio on the particle dispersion is noticeable from HR-TEM images of various samples presented in Figure 4. There are two possible growth mechanisms viz. E


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Figure 6. (a) Zeta potential measurements of the gold nanoparticles were monitored at 95 and 35 °C while increasing tryptophan concentration. (b) FTIR of the gold nanoparticles obtained at 95 and 35 °C. (c) UV−vis spectra observed in the ultraviolet region for gold nanoparticles obtained at 95 and 35 °C.

assembly of the AuNPs. HR-TEM images suggest that three overlying steps were involved in creating complex nanostructures: nucleation, kinetically favored deposition, and oriented attachment, as reported recently.38 The increase of the tryptophan concentration also supports a kinetically favored deposition of gold atoms and thus observed growth in the AuNP branches. Thus, it can be inferred that a kinetically favored growth route accessible at 35 °C is more facile for the anisotropic growth of the AuNPs.39 The dispersion and self-assembly of AuNPs identified in the HR-TEM results are in agreement with the DLS and UV−vis spectra, and similar results have been reported for protein functionalized AuNPs elsewhere.40 Temporal Evolution of the UV−vis Spectrum. The temporal evolution of the UV−vis absorption spectrum for the AuNPs was examined at 95 and 35 °C (Figure S3). For reactions at 95 °C, the rapid appearance of a significant absorbance in the wavelength range of 530 nm in the early reaction period (2 h) was consistent with considerable elongation, as shown in Figure S3a (see bottom rows). Predictably, the absorption spectrum for the AuNPs increased steadily, and a significant SPR peak appeared at 520−530 nm. The data produced herein can be used to determine the effect on both the size and concentration of AuNPs directly based on the progress in the UV−vis spectrum.41 The UV−vis absorption spectra for the reactions at low temperature (35 °C) are presented in all stages for the AuNPs in Figure S3b. A broad absorption peak at 600 nm was observed due to the formation of self-assembled AuNPs. This was mostly true in all repetitions, and the HR-TEM results were consistent with these observations. The AuNPs lost their dispersed nature, and the broadening of the SPR peak increased over time. The color of the solution underwent a gradual change from red to blue, and we observed a gradual increase in the intensity of the SPR band with time at 35 °C. After 48 h of incubation, the intensity of the observed SPR band did not decrease with further incubation,

aggregative growth. The proposed classical nucleation−growth route at 95 °C and the rate of the AuNPs dispersion increased as the concentration of tryptophan increased (Figure S1). The overall trend in the increasing of dispersion upon increasing the tryptophan concentration with the given precursor ratio indicates the potential application of the tryptophan in the preparation of dispersed AuNPs. Thus, tryptophan concentration is another parameter which significantly controls the average particle size and the dispersion of the AuNPs in a colloidal solution. This means that the dispersion rate actually increases as the tryptophan concentration increased, which is expected in the nucleation−growth route.36,37 The growth of AuNPs observed with increasing concentration of tryptophan, particularly at 95 °C, is consistent with the result of Peng et al., where high citrate concentration limits the growth of AuNPs.7 Figure 5 indicates the effect of tryptophan concentration on the properties of as-prepared AuNPs at 35 °C. Consistent with the UV−vis spectral results, only spherical AuNPs were achieved at low tryptophan concentration as observed in Figure 5. HRTEM images exhibited that the products from the addition of 0.031 to 0.250 mM tryptophan were spherical particles with the diameter about 20 nm. Thus, relatively small AuNPs with a narrow size distribution were observed in HR-TEM images for reactions with tryptophan concentrations ranging from 0.0315 to 0.250 mM (Figure S2). When the amount of tryptophan reached 0.5 mM, anisotropic AuNPs with more than three branches were observed. The size of AuNPs also increased to 60−80 nm with the addition of more tryptophan. Thus, the AuNPs prepared at 35 °C are also suitable to produce complex structures of the selfassembled AuNPs with a mean diameter of 80 ± 20 nm as presented in Figure S2. The obvious variation of AuNPs size and morphology by adjusting the tryptophan concentration was clearly possible via the supply of more Au(0) atoms in the growth system which was significant in producing urchin-like selfF


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form of tryptophan was suitable for stabilization of the AuNPs. The aromatic groups of tryptophan interacted with the surface of the AuNPs. It has been reported that the peak located at 280− 290 nm results from the excitation of π−π transitions in the aromatic (indole) group of tryptophan, which can also cause strong fluorescence and higher quantum yield.19,49 Both carboxyl and indole functional groups of tryptophan play a role in the surface chemistry of the particles.20,50,51 A facile route to prepare a DNA−gold complex has been reported involving the electrostatic self-assembly of AuNPs capped with tryptophan.52 Stability Studies. The optical properties of the AuNPs obtained at 95 °C were tested in a series of dilutions. The quality of UV−vis spectra was measured for every successive dilution by adding 0.25 mL of water to 1 mL of AuNPs to an optical density of approximately 1.5 and 0.9 at 95 and 35 °C, respectively. The bandwidth (Δλ) and wavelength (λmax) of SPR were closely monitored. The SPR band at Δλ and absorbance at λmax indicated that the dispersed AuNPs had excellent stability over a series of dilutions (Figure S4a). The absorption intensity was found to be linearly dependent on the concentration of AuNPs, in agreement with the Beer−Lambert law as shown in Figure S4b. Thus, it was vital to discover that the dilution effect does not alter the characteristic chemical and optical properties in the AuNP solutions. The decrease in the concentration of the AuNPs and absorption intensity at λmax was found to be linearly dependent; thus, both types of AuNPs followed the Beer−Lambert law as reported previously.53 The tryptophan-capped AuNPs were optically active and functional at diluted concentrations; thus, they have potential for imaging and biomedical applications.54−56 The rate of AuNPs aggregation and colloidal stability depends on the solution pH, ionic strength, and surface functionality.57 The stability of the AuNPs at different pH values and ionic strength was examined to determine their utility in biomedical applications. The in vitro stability of the AuNPs was assessed by monitoring their UV−vis spectra at different molar ratios of NaOH, HCl, and NaCl. The UV−vis spectral changes in AuNPs solution were investigated with increasing concentrations of diluted HCl, which caused the SPR band to shift to a longer wavelength (Figure S5a). The tryptophan-capped AuNPs were unstable at acidic conditions; thus, the size of the AuNP aggregates could be controlled through the interparticle interactions. The SPR band around 520 nm was affected by the aggregate size. The SPR band of AuNPs with smaller size about 20 nm had a narrow peak located at 530 nm; however, increasing the aggregate size may increase the intensity or cause a red-shift in the SPR wavelength (Figure S5a). A higher-order electron oscillation takes place in nanoparticles larger than 20 nm; thus, light absorption and scattering can be described by considering all oscillations.58 However, the SPR band remained intact in all formulations prepared with diluted NaOH, suggesting the AuNPs were stable in neutral and alkaline conditions (Figure S5b). These results indicated that AuNPs remains dispersed, thus demonstrating high in vitro stability with increasing ionic strength and at physiological pH values. It was observed that the AuNPs remained intact in ionic solutions, as tryptophan provides robust stabilization of the nanoparticle surfaces (Figure S5c). Biocompatibility, Fluorescent Properties, and Imaging Studies. In the past few decades, AuNPs have attracted much attention in biomedical and diagnostic fields due to their stability, biocompatibility, and characteristic optical properties.59,60 The cytotoxicity of AuNPs under in vitro conditions in SH-SY5Y cells was examined by monitoring the effect of AuNPs concentration

indicating the self-assembled AuNPs were extremely stable, Figure S3b. The evolution of metal nanoparticles is a central question in nanoscience research, since development of the SPR provides fundamental insights into revealing the origin of nanoparticles.42 These results were consistent with the properties of urchin-like AuNPs and anisotropic NPs prepared via kinetics-favored growth.43 Surface Chemistry of the Gold Nanoparticles. Zeta potential is an important parameter for measuring the stability, charge, and aggregation of NPs in the corresponding environment. The zeta potential of the AuNPs obtained with increasing tryptophan ratio with a fixed NaOH concentration (2.5 mM) was investigated systematically at 95 and 35 °C. As can be seen in Figure 6a, the negative charge on the AuNPs was decreased from −27 to −5 mV as the tryptophan concentration increased from 0.031 to 1.75 mM at 95 °C. There was a continual relationship between the reduction in negative charge and the increase in tryptophan concentration (Figure 6a); however, dispersed AuNPs were evidently produced. Thus, aggregation did not occur in the AuNPs solution due to enough charge and electrostatic repulsions. Based on our understanding, tryptophan is negatively charged in solution. The amount of capping of tryptophan on AuNPs determines its zeta potential. It is likely due to the amount of tryptophan capping per particle decreasing, thereby the zeta potential decreases. The increase in quantity of the AuNPs was observed for tryptophan at 1.38 and 1.75 mM (Figure 3a); however, the size of the AuNPs remains almost same. The mobility results suggest that the capping of tryptophan on AuNPs only occurs in the deprotonated state. Our studies show that tryptophan binds strongly to AuNPs; however, the carboxyl group in the tryptophan is required to be deprotonated for capping on AuNPs.44 Thus, amine groups form a covalent bond with the AuNPs, and the deprotonated state of carboxyl groups inhibit aggregation of NPs by coulomb repulsion mechanism.45 The use of tryptophan for surface functionalization of AuNPs and the resulting water-dispersible NPs have important implications in developing imaging applications and to form novel bioconjugates.46 In contrast, the negative charge on the AuNPs increased from −27 to −45 mV as the concentration of tryptophan increased from 0.031 to 1.75 mM, particularly for AuNPs produced at 35 °C. In a similar fashion, but in the opposite direction, a relationship between the negative charge on the self-assembled AuNPs and the increase of tryptophan concentration was observed (Figure 6a), either due to the slow kinetics or aggregate growth mechanism. Moreover, as-prepared urchin-like AuNPs were stable in the growth solution with their nanoscale building blocks, as the thermodynamic equilibrium of the AuNP structures was built up immediately after the nucleation and growth. Avoiding increased susceptibility of the particles to the local environment is challenging due to the large surface area and surface oxidation; thus, colloidal stability is very important.47 Nanoparticles were highly stable despite an average surface charge of −39 mV as shown by zeta potential measurements.48 The major transmittance peak at 1650 cm−1 signifies the presence of an amide I bond for both AuNPs obtained at 95 and 35 °C (Figure 6b). Healthy capping of the AuNPs was successfully established due to tryptophan ligands by anchoring the N−Au and O−Au via amide and carboxylic groups, respectively. From the FTIR spectra, the large peak at 3360 cm−1 is assigned to the stretching vibration of NH. An intense peak was observed for the as-prepared and centrifuged AuNPs in the UV region of 290−300 nm (Figure 6c). The deprotonated G


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biocompatibility and safety than the citric acid prepared AuNPs under both in vivo and in vitro conditions.63 Fluorescence spectra of tryptophan-functionalized AuNPs were recorded by measuring the emission intensity as a function of excitation wavelength. In an emission spectrum, a fixed wavelength is used to excite AgNPs solution, and the intensity of emitted radiation was monitored as a function of excitation wavelength (Figure S6). The excitation of the AuNPs solution at different wavelengths was established to select the larger excitation wavelength for a quantitative or qualitative analysis. Nevertheless, experimentally tryptophan functionalized AuNPs have strong enough emission to be detected by the confocal microscopy with blue/cyan filter of DAPI, even though emission intensity was decreased as increased excitation wavelength from 310 to 350 nm (Figure S6). When untreated human neuronal cells (SH-SY5Y) were first imaged with the blue/cyan filter set (DAPI), only a weak cytoplasmic autofluorescence was observed. AuNPs are efficient quenchers of the tryptophan fluorescence, and thus efficiency of quenching was used to determine the binding affinity and ease of functionalization with tryptophan residue.20 The fluorescence properties of proteins are typically dominated by tryptophan along with minor fluorescence from tyrosine and phenylalanine, essentially attributable to a tendency of excited-state of indole to donate electrons.64 Thus, we thought that the tryptophan capped AuNPs may be suitable to excite by ultraviolet light and emit a characteristic blue fluorescence, similar to most commonly used fluorescent DNA dye, known as DAPI. Previously, DAPI fluorescence was used as a tool for imaging cells and their appropriate areas; however, the

on cell proliferation. Untreated samples and cells treated with 20, 40, 60, 80, and 100 ppm of AuNPs for 24 h were subjected to the cell-viability determination (Figure 7). The AuNPs-treated SH-

Figure 7. Biocompatibility of the tryptophan capped AuNPs at increasing concentration 20, 40, 60, 80, and 100 ppm.

SY5Y cells showed more than 90% viability up to 80 ppm, indicating tryptophan capped AuNPs are biocompatible and safe. The results suggest that AuNPs do not show evidence of cytotoxicity up to 80 ppm. After treatment of the cells at 100 ppm, there is a marginal decrease in cell viability about 20% as can be seen in Figure 7. However, this slight decrease in the SHSY5Y cell number may be due to the stress placed on the cells. Higher concentrations of AuNPs induce cytotoxicity due to their negative surface charge and higher amount of uptake by cells.61,62 HEPES buffer prepared AuNPs reported excellent and better

Figure 8. (a) Autofluorescence image of SH-SY5Y cells. (b) Fluorescence image of SH-SY5Y cells treated with AuNPs at acidic conditions (pH 4.0). (c) Fluorescence image of SH-SY5Y cells treated with AuNPs at neutral condition (pH 7.0). (d) Fluorescence image of SH-SY5Y cells treated with AuNPs at alkaline condition (pH 9.0). H


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and have a few bright spots inside the cytoplasm, and it is easy to reveal that these spots were originated from the fluorescent AuNPs. Furthermore, the blue fluorescence emitting from the tryptophan conjugated AuNPs in SH-SY5Y cells can be easily visualized by the naked eyes. The minor cellular autofluorescence allows cell images to be collected with more clarity; thus, images can be shown with extremely improved contrast. These results indicate that the small AuNPs crosses the cytoplasmic membrane more easily at neutral and alkaline pH conditions, and thus a larger amount of AuNPs were accumulated in SH-SY5Y cells as revealed in Figure 8c,d. Furthermore, the AuNPs possessed excellent biocompatibility and were a good fluorescent probe for cell imaging at acidic, neutral, and alkaline pH conditions (Figure 8b−d). To the best of our knowledge, this is the first report on fluorescent imaging of SH-SY5Y cells using tryptophan capped AuNPs as a biocompatible fluorescent probe. The principle of this method is that the fluorescence emission of tryptophan can be detected in the visible range 450−460 nm upon excitation in the ultraviolet region (330 nm), which is appropriate to avoid spectral overlap of the SPR band of AuNPs located at 530 nm.68 Therefore, this simple, rapid, and sensitive imaging strategy may provide a new insight for probing their uptake, biocompatibility, and toxicity by a label-free fluorescence method.69−71 Most of the QDs having excellent photostability are appropriate for in vitro and in vivo imaging; however, they are composed of heavy metals that are toxic, making them unsuitable in developing imaging applications.72,73 Therefore, colloidal AuNPs can be an alternative in developing imaging applications due to ease of synthesis, colloidal stability, facile conjugation procedure, and excellent biocompatibility.3,74

photoconversion effect is a serious concern regarding hazards and false-positive results.65 Typical fluorescence images of the control and treated SHSY5Y cells are presented in Figure 8. As we can see in Figure 8a, the intensity of the fluorescence from the untreated cells was insignificant. In Figure 8a, autofluorescence images of SH-SY5Y adhering to the coverslips is presented. The fluorescent signals from the cells were attributed to the autofluorescence. Fortunately, such endogenous weak autofluorescence was not seriously confusing cell images obtained using tryptophan capped AuNPs as fluorescent probes. The distribution of the fluorescent probe within cells is of prime importance in a wide range of diagnostic and biochemical research.32 Thus, it is of great implication to further examine the fluorescent performances of AuNPs in imaging cells and explore their applications in chemical or biological fields. The following results confirm the unique utility of tryptophan capped AuNPs for simultaneous imaging, uptake, and toxicity study of nonradioactive metallic probes, such as gold, within the context of in vitro imaging. The SH-SY5Y cells were grown at a normal rate when incubated with AuNPs, and subsequently, particle uptake, accumulation, and toxicity were examined by a newly developed imaging technique. Tryptophan capped AuNPs are sensitive to acidic pH conditions; thus, fluorescence imaging of SH-SY5Y cells treated with AuNPs under different pH conditions was also performed. The fluorescence images of SH-SY5Y cells incubated with AuNPs at different pH conditions were acquired in the blue emission range (450−460 nm). A decrease in the fluorescence intensity of the aggregated AuNPs was observed at acidic pH 4.0 (Figure 8b). This effect was attributed to the synergistic effect of quenching the emission of the tryptophan and red-shift in the SPR band by the aggregated AuNP surfaces. From the image of SH-SY5Y cells (Figure 8b), we can see that the AuNP aggregates on the cell wall were partly accumulated inside the cytoplasm of the cells. In acidic conditions aggregation restricts the uptake of AuNPs; thus, imaging of the cell membrane was established. The AuNPs succeed to uptake inside the cells and get accumulated in the cell organelles and nucleus, indicating fluorescence imaging in acidic conditions is appropriate for visualizing cellular compartments (Figure 8b). Thus, on the basis of the difference in autofluorescence, the cells incubated with 20 nm AuNPs can be clearly observed with a stronger fluorescent signal than those grown without AuNPs. Thus, successful application of tryptophan fluorescence suggests novel possibilities in the imaging of cells and tissues by using intrinsic fluorescence rather than external probes or labeling. Certainly, tryptophan-based fluorescence imaging can ensure the safety of biolocalization and deliver similar properties of the fluorescent probes or conjugated drugs.66 We introduced a novel imaging method using intrinsic fluorescence of the tryptophan without the interference from background autofluorescence and need of fluorescent dyes or tags.67 In the subsequent experiments, AuNPs were used as an imaging probe considering the possibility of changes in particle uptake efficiency and fluorescence intensities. The cells treated with AuNPs were illuminated in the ultraviolet region, the fluorescence of AuNPs on the cell membrane and inside cells was further observed. Figure 8c shows the corresponding fluorescence image of SH-SY5Y cells after treatment at pH 7.0. The intense fluorescence was homogeneously distributed in all parts of the cell indicating the increased uptake of the dispersed AuNPs, without any structural and morphological damage (Figure 8c). The SH-SY5Y cells exhibited stronger luminescence

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CONCLUSIONS

In summary, the temperature conditions in the current system seemed to influence the productivity and size distribution patterns of the resulting AuNPs. Depending on the tryptophan ratio and temperature, the AuNPs could be formed through either the classical nucleation and growth or aggregative growth route. All results in this report revealed that, in addition to being a reducing and stabilizing agent, tryptophan played a third role in the current system as a pH mediator. Through this understanding, it was possible to synthesize nearly disperse and highly stable AuNPs having sizes of about 20 nm by simply elevating the reaction temperature. Tryptophan has a few advantages: it is readily soluble in water, is biocompatible, and has excellent reducing and stabilizing properties. It was possible to distinguish the autofluorescence from SH-SY5Y and the fluorescent signal from the tryptophan present on the AuNPs. The luminescent signal intensity revealed that the AuNPs were increasingly distributed in the SH-SY5Y cells at the neutral and alkaline conditions. However, the formation of aggregates of the AuNPs restricts uptake and accumulation in acidic conditions; thus, imaging of the cell wall can be demonstrated in a facile, safer, and sustainable manner. This approach would be a safer alternative to the variety of fluorescent dyes and quantum dots used in biological imaging. The tryptophan-capped AuNPs had a small size and thus could be extended to image different type of cells (mammalian cells, fungi, and bacteria) despite different surface structures. I


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01101. Size distribution histograms of the corresponding HRTEM images. Temporal evolution of UVVis spectrum of the gold nanoparticles. Effect of diluted pH values and ionic strength on the UVVis spectrum of gold nanoparticles. Emission spectrum of tryptophan functionalized gold nanoparticles. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-31-961-5157. Fax: +82-31-961-5122. E-mail: [email protected]. ORCID

Gajanan Ghodake: 0000-0001-6527-3745 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by National Research Foundation South Korea under the Project No. 2017R1C1B-5017360. This work was partly supported by Dongguk University-Seoul, South Korea Research Fund 2016−2017.

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REFERENCES

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Temperature Dependent Synthesis of Tryptophan-Functionalized Gold

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