Green Process Synth 2016; 5: 467–472 Phuong Phong Nguyen Thi and Dai Hai Nguyen* Gelatin as an ecofriendly natural polymer for preparing colloidal silver@gold nanobranches DOI 10.1515/gps-2016-0036 Received March 7, 2016; accepted July 6, 2016; previously published online August 17, 2016 Abstract: We report star-shaped silver@gold (Ag@Au) nanoparticles (NPs) in gelatin suspensions for the purpose of enhancing the stability of Ag@Au NPs In this case, Ag NPs were designed as nucleating agents, whereas gelatin was used as a protecting agent for Au development Especially, variable gelatin concentrations were also prepared to explore its ability to increase the stability of Ag@Au NPs The obtained samples were then characterized by UVvisible spectroscopy, transmission electron spectroscopy (TEM), X-ray diffraction, and Fourier transform infrared spectroscopy The maximum absorption wavelength of all samples (566–580 nm) indicated that branched Ag@Au@ gelatin NPs were successfully synthesized In addition, our TEM results revealed that the size of branched Ag@Au@ gelatin NPs was found to be between 20 and 45 nm as influenced by the component ratio and the pH value These results can provide valuable insights into the improvement of Ag@Au NP stability in the presence of gelatin Keywords: gelatin; gold; silver; stabilizer; star-shaped Introduction Nanotechnology has been recognized as a revolutionary approach to materials science over the last decade [1–6] Furthermore, nanoparticles (NPs), one of the most active research areas of nanotechnology, have optimal clearance characteristics, including nanosize, distribution, and morphology, and many uses in the field of practical applications, such as catalysis, thermoelectrics, microelectronics, sensing, and biodiagnostics Metal NPs, in particular, possess specific physical and chemical properties due to *Corresponding author: Dai Hai Nguyen, Institute of Applied Materials Science, Vietnam Academy of Science and Technology, 01 Mac Dinh Chi, District 1, Ho Chi Minh City, Ho Chi Minh 70000, Vietnam, e-mail: nguyendaihai0511@gmail.com Phuong Phong Nguyen Thi: University of Science, National University of Ho Chi Minh City, 227 Nguyen Van Cu, District 5, Ho Chi Minh City, Ho Chi Minh 70000, Vietnam their small-size effect, surface effect, and quantum-size effect Among different kinds of metal materials, gold (Au) and silver (Ag) have advantages of creating various nanostructures in forms such as rods, cubes, flat triangles, polybranched, and star-shaped NPs Interestingly, nonspherical rather than spherical NPs show strong catalytic activity and surface-enhanced Raman spectroscopy (SERS) behavior caused by the anisotropic distribution of electromagnetic field (EMF) near the tips of the branched NPs [7, 8] Moreover, the unique morphology of starshaped Au NPs not only improves the EMF without the necessity for the aggregation of particles but also exhibits plasmon resonances in either the visible or the nearinfrared region Therefore, star-shaped Au NPs have been extensively used in many fields in recent years [9] Various methods, including physical, chemical, and biochemical techniques, have been studied for the preparation of branched Au and Ag NPs [1–3] However, the NPs produced by these methods are often unstable and tend to aggregate together; accordingly, stabilizers play a critical role in controlling the formation of NPs and their dispersion stability Several agents, such as thiols, surfactants, polymers, and polyelectrolytes, have been used as protective agents for preventing NPs from aggregation Particularly, polymers are commonly used as particle stabilizers, owing to their effective ability to avoid agglomeration and precipitation of particles, resulting in NPs with homogeneous distributions [10–12] For instance, Cheng et al reported a simple method to synthesize spiky star-shaped Au/Ag nanostructured materials using chitosan (Cts) as stabilizing reagents [9] Neupane et al also developed gelatin-stabilized Au NPs (Au NPs-gelatin) with variation of gelatin concentrations by reducing in situ tetrachloroauric acid with sodium citrate [13] Besides, Ahmad et al synthesized Ag NPs in Cts, gelatin, and Cts/gelatin suspensions, in which case Cts and gelatin were made up as natural stabilizers and solid supports [14] According to the TEM images, Ag NPs with the combination of both Cts and gelatin showed a good distribution compared to Ag NPs with Cts or gelatin alone This accounts for the potentially different properties of polymers that are incorporated into metal particles, which allow them to serve many biological applications (biosensors, diagnostics, and photothermal therapy) Brought to you by | University of Exeter Authenticated Download Date | 10/12/16 5:02 AM 468 P.P Nguyen Thi and D.H Nguyen: Gelatin for preparing silver@gold nanobranches In this study, we focused on the synthesis of starshaped Ag@Au@gelatin NPs via the seeded growth method In such system, gelatin was used as a stabilizer for Ag@Au NPs due to the presence of nonpolar amino acid content ( > 80%) of gelatin [15, 16] Initially, Ag seeds were prepared by the reduction of silver nitrate (AgNO3) using trisodium citrate (TSC) and sodium borohydride (NaBH4) Next, these preformed Ag seeds were used for the further preparation of Ag@Au particles with different gelatin concentrations These samples were mainly determined by UV-visible spectroscopy (UV-Vis) and transmission electron spectroscopy (TEM) for the purpose of checking the plasmon adsorption maximum and its correlation with different shapes The study is expected to offer valuable information for stabilizing Ag@Au particles by gelatin Materials and methods 2.1 Materials AgNO3 (99.8%), tetrachloroauric(III) acid trihydrate (HAuCl4·3H2O; 99.5%), and NaBH4 (Reagent Plus 98%) were purchased from Merck (Darmstadt, Germany) TSC and L(+)-ascorbic acid (AA) were purchased from Prolabo (Paris, France) Gelatin type A was obtained from Sigma-Aldrich (St Louis, MO, USA) All reagents and solvents were used without any further purification 2.2 Methods Preparation of Ag colloids: Ag seeds were synthesized using NaBH4 and sodium citrate as reducing agents according to a previous report [17] Briefly, the aqueous solution of AgNO3 (1.5 ml, mm) was mixed with the TSC solution (10 ml, 0.25 mm) Then, ice-cooled NaBH4 solution (20 ml, 10 mm) was immediately introduced into the mixture, leading to the formation of Ag NPs with greenish yellow color The Ag seed solution was dialyzed by a dialysis membrane (MWCO: 3500 Da; Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) against distilled water under static conditions for several hours Importantly, this sample should be protected against light to avoid photodegradation Lastly, the Ag seeds were then characterized by UV-Vis and TEM Preparation of Ag@Au@gelatin NPs: Ag@Au NPs were fabricated using the preformed Ag seeds (Figure 1) [18] In brief, the HAuCl4 solution (10 ml, 0.5 mm) containing 0.1% K2CO3, 200 μl of the Ag seeds, and different amounts of gelatin (0.04 mm) was placed in a 50 ml glass containing a magnetic stirring bar The AA solution (1.5 ml, 100 mm) was then added dropwise into the mixture, and deionized water was added to reach a final volume of 17 ml The color of the samples turned from transparent to pink, blue violet, and finally dark blue, which corresponds to the formation of Ag@Au@ gelatin NPs The mixture was stirred for another 5 h at room temperature to achieve complete reduction After that, samples were dialyzed for about 3 days against distilled water and stored at room temperature Lastly, Ag@Au@gelatin was then characterized by TEM, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy Characterization: To investigate the presence of Ag@Au NPs, the samples were examined by FTIR spectroscopy (a Magna-IR™ 550 spectrometer, Nicolet, USA) using KBr pellet UV-Vis spectra were obtained using a Cary 50 UV-Vis spectrometer from Varian (Palo Alto, CA, USA), and the spectra were recorded over a wavelength range of 300–800 nm The shape, particle size, and size distribution of NPs were imaged by TEM (300 kV; JEOL, Tokyo, Japan) The samples were prepared in distilled water at concentration of mg/ml and equilibrated at 37°C A drop of the solution was placed on a carbon-copper grid (300 mesh; Ted Pella, Inc., Redding, CA, USA) and air-dried for 10 min [19, 20] XRD was performed using a Rigaku DMAX 2000 diffractometer (Rigaku Americas Corp., Woodlands, TX, USA) equipped with Cu/Kα radiation at scanning rate of 4°/min in the 2θ range of 30°–70° (λ = 0.15405 nm, 40 kV, 40 mA) Results and discussion The Ag metal has the same lattice parameters and the face-centered crystal structure Moreover, the diversity of Ag properties depends on the variation in molar element ratio so that the use of Ag particles as seeds for the synthesis of star-shaped Au NPs is quite interesting In this case, NaBH4 was prepared as a reducing agent and TSC was used as a supporting, reducing agent as well as a stabilizer in the production of Ag seeds; the COO groups of TSC were advanced in stabilizing NPs and protecting them from clumping [21, 22] As a result, Ag seeds weere Figure 1: Schematic illustration of the preparation of star-shaped Ag@Au@gelatin NPs Brought to you by | University of Exeter Authenticated Download Date | 10/12/16 5:02 AM P.P Nguyen Thi and D.H Nguyen: Gelatin for preparing silver@gold nanobranches 469 successfully synthesized by AgNO3, NaBH4, and TSC [23] In this experiment, the results provided no conclusive evidence for determining the stability of Ag seeds because they were quickly used for the formation of starshaped Ag@Au NPs However, the information about Ag seeds was first indicated by the distinctive color in the colloidal solution with a plasmon absorption band near 394 nm, which means that Ag ions were reduced to Ag0 in the aqueous phase [23, 24] The Ag seeds were then determined by TEM (Figure 2A), and the seeds were spherical in shape with a narrow size distribution ranging in diameter from to 10 nm Our results were quite consistent with those of previous publications for the synthesis of Ag NPs via the chemical reduction method [23, 25] The mild reducing agent AA was carried out for reducing Au3+ ions because it not only favored the growth of Ag@Au core-shell but also limited the new generation of nuclei in the solution, the leading cause of a desirable size distribution In addition, ascorbic ions had a pronounced impact on the formation of branched nanocrystals and the durability of NP dispersion plays a critical role in their application Importantly, in this research, the effect of gelatin in the growth of starshaped Ag@Au NPs was discovered by adding different concentrations of gelatin solution to the reaction media (Table 1) [26] The results of UV-Vis showed that the maximum absorption wavelength (566–580 nm) of all samples has no significant difference The absorption peak of sample a was sharp and symmetrical with the increasing absorption intensity compared to the remaining samples, so more NPs with evenly distributed particle sizes could be formed As the amount of gelatin increases in the solution, NPs tend to clump together to provide highly specific aggregations, which caused A B heterogeneous reactions, resulting in the lower absorption intensity of NPs In the present study, the stability of the samples was investigated at room temperature for month The results indicated that there were no significant changes over the absorption wavelengths from 580 to 600 nm, whereas the intensive absorption band noticeably decreased by 62% corresponding to sample a After month, the intensive absorption of sample b was reduced to at least 9.3% with no precipitation; thus, that was the most appropriate amount of gelatin for the protection of star-shaped Ag@ Au NPs Besides, the continuous increase in the number of gelatin decreased the intensive absorption of NPs, and it may be because gelatin could no longer function in colloidal protection at a relatively high concentration The results were completely consistent with the precipitated layer observed at the bottom region of the sample after month The TEM images of sample b (Figure 3C′) and sample f were examined after month The results of both samples were significantly different Sample f induced structural change, whereas sample b just underwent a little change in structure Gelatin was, in particular, clumping at high concentration and was not performing its stabilization function; therefore, Ag@Au@gelatin NPs tended to aggregate together and then formed larger clusters Meanwhile, in sample b, gelatin showed excellent ability to protect Ag@Au NPs NPs were star in shape and possessed strong stability The results indicated that 3 ml of 0.04 mm gelatin was the appropriate amount for improving the stability of Ag@Au NPs Consequently, stable Ag@Au@gelatin NPs with the size of 20–45 nm have a great potential for passive targeting to cancer cells Powder XRD was further used to analyze the formation of Ag@Au NPs All the Au has similar XRD patterns C Absorbance (a.u.) 0.8 0.6 0.4 0.2 300 400 500 600 700 Wavelength (nm) Figure 2: TEM image (A), UV-Vis absorption spectrum (B), and digital photograph (C) of Ag seeds Brought to you by | University of Exeter Authenticated Download Date | 10/12/16 5:02 AM 470 P.P Nguyen Thi and D.H Nguyen: Gelatin for preparing silver@gold nanobranches Table 1: Synthesis specifications of star-shaped Ag@Au NPs Sample A B C D E F VHAuCl4 (ml) VAA (ml) VAg seeds (μl) Vgelatin (ml) 10 10 10 10 10 10 1.5 1.5 1.5 1.5 1.5 1.5 200 200 200 200 200 200 A A′ 1.2 (Figure 4A, i) The XRD peaks at about 38.3°, 44.2°, 65.1°, and 78° 2θ values corresponded to the 111, 200, 220, and 311 planes of the face-centered cubic crystal of Au, respectively For Ag@Au NPs, the XRD peaks appeared at 37.97°, 44.05°, 64.21°, and 77.21° related to Ag NPs Additionally, the existence of Ag@Au@gelatin NPs was confirmed as the final product (Figure 4A, ii) The (200), (220), and (311) Bragg reflection planes were weak and widened compared to the intense reflection at (111) 1.2 a a b b c d 0.8 Absorbance (a.u.) Absorbance (a.u.) c e f 0.6 0.4 0.2 0.2 400 450 500 550 600 650 400 700 B′ C 30 20 10 15 20 25 30 35 550 600 650 700 C′ 45 30 15 40 10 15 20 25 30 35 40 Diameter (nm) Diameter (nm) Frequently (%) 500 60 E 450 Frequently (%) Frequently (%) 40 D f Wavelength (nm) Wavelength (nm) B e 0.6 0.4 d 0.8 D′ 60 45 30 15 20 25 30 35 40 45 50 Diameter (nm) Figure 3: UV-Vis absorption spectrum of samples a to f (A) and after month (A′) and TEM image (scale bar, 50 nm) and size distribution of sample b (B and C) and sample f (D and E) and after month b (B′ and C′) and f (D′), respectively Brought to you by | University of Exeter Authenticated Download Date | 10/12/16 5:02 AM P.P Nguyen Thi and D.H Nguyen: Gelatin for preparing silver@gold nanobranches 471 A B Figure 4: (A) XRD of bare Ag@Au (i) and Ag@Au@gelatin (ii) and (B) FTIR spectroscopy of gelatin (i) and Ag@Au@gelatin (ii) These results indicated that Ag@Au@gelatin NPs were mainly oriented to the (111) plane and Ag@Au@gelatin NPs obtained from XRD were smaller in size compared to this NPs measured by TEM The vibration frequencies of gelatin and Ag@Au@ gelatin NPs were obtained by FTIR spectroscopy for comparing the secondary structure of the blank gelatin (Figure 4B, i) and the Ag@Au@gelatin NPs (Figure 4B, ii) The spectra of gelatin showed vibration bands at 3285 cm-1 (N-H stretch coupled with hydrogen bonding), 3085 cm-1 (alkenyl C-H stretch), 2956 cm-1 (CH2 asymmetrical stretching), 1631 cm-1 (C = O stretch/HB coupled with COO-), 1533 cm-1 (N-H bend coupled with CN stretch), 1444 cm-1 (CH2 bend), 1240 cm-1 (NH bend), and 1078 cm-1 (C-O stretch) These results implied that gelatin was successfully attached to the surface of Ag@Au NPs Conclusion Star-shaped Ag@Au@gelatin NPs were successfully fabricated and found to be stable for a long time with a suitable amount of gelatin The average diameter of starshaped Ag@Au@gelatin NPs was between 20 and 45 nm, with the maximum surface plasmon resonance peak at 566–580 nm In addition, star-shaped NPs tended to clump together at high gelatin concentrations The results suggest that star-shaped Ag@Au@gelatin NPs could serve as a foundation for developing a novel star-shaped particles with high stability Acknowledgments: This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) grant no 104.02-2014.83 References [1] Tetgure SR, Borse AU, Sankapal BR, Garole VJ, Garole DJ Amino Acids 2015, 47, 757–765 [2] Suarasan S, Focsan M, Soritau O, Maniu D, Astilean S Colloids Surf B 2015, 132, 122–131 [3] Ruan S, He Q, Gao H Nanoscale 2015, 7, 9487–9496 [4] Nguyen DH, Lee JS, Choi JH, Park KM, Lee Y, Park KD Acta Biomater 2016, 35, 109–117 [5] Nguyen DH, Lee JS, Choi JH, Lee Y, Son JY, Bae JW, Lee K, Park KD Macromol Res 2015, 23, 765–769 [6] Nguyen DH, Lee JS, Bae JW, Choi JH, Lee Y, Park KD Int J Pharm 2015, 495, 329–335 [7] Vigderman L, Zubarev ER Langmuir 2012, 28, 9034–9040 [8] Nguyen TPP, Nguyen MT, Nguyen DH J 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Hai Nguyen obtained his PhD in 2013 at Ajou University, Republic of Korea Currently, he works as a researcher at the Institute of Applied Materials Science, Vietnam Academy of Science and Technology He is also an invited lecturer at the TraVinh University and Ho Chi Minh City University of Natural Sciences Phuong Phong Nguyen Thi Phuong Phong Nguyen Thi received her PhD in 2003 at the Institute of Applied Materials Science, Vietnam Academy of Science and Technology She is head of the Nanochemistry Laboratory at the Ho Chi Minh City University of Natural Sciences She became an associate professor in 2011 She is also an invited lecturer at the Lac Hong University and Ho Chi Minh City University of Natural Sciences Brought to you by | University of Exeter Authenticated Download Date | 10/12/16 5:02 AM ... Authenticated Download Date | 10/12/16 5:02 AM P.P Nguyen Thi and D.H Nguyen: Gelatin for preparing silver@ gold nanobranches 471 A B Figure 4: (A) XRD of bare Ag@Au (i) and Ag@Au @gelatin (ii) and... structure Gelatin was, in particular, clumping at high concentration and was not performing its stabilization function; therefore, Ag@Au @gelatin NPs tended to aggregate together and then formed... Thi and D.H Nguyen: Gelatin for preparing silver@ gold nanobranches In this study, we focused on the synthesis of starshaped Ag@Au @gelatin NPs via the seeded growth method In such system, gelatin