DSpace at VNU: Gelatin as an ecofriendly natural polymer for preparing colloidal silver gold nanobranches

*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 7000

*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

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

Espe-cially, 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

UV-visible 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

influ-enced 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.

1 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

appli-cations, such as catalysis, thermoelectrics,

microelectron-ics, sensing, and biodiagnostics Metal NPs, in particular,

possess specific physical and chemical properties due to

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 trian-gles, polybranched, and star-shaped NPs Interestingly, nonspherical rather than spherical NPs show strong cata-lytic 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 star-shaped Au NPs not only improves the EMF without the necessity for the aggregation of particles but also exhib-its plasmon resonances in either the visible or the near-infrared 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 prepa-ration 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 disper-sion stability Several agents, such as thiols, surfactants, polymers, and polyelectrolytes, have been used as protec-tive agents for preventing NPs from aggregation Particu-larly, polymers are commonly used as particle stabilizers, owing to their effective ability to avoid agglomeration and precipitation of particles, resulting in NPs with homoge-neous 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 tetrachloro-auric acid with sodium citrate [13] Besides, Ahmad et al synthesized Ag NPs in Cts, gelatin, and Cts/gelatin sus-pensions, 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 incor-porated into metal particles, which allow them to serve many biological applications (biosensors, diagnostics, and photothermal therapy)

In this study, we focused on the synthesis of

star-shaped 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

deter-mined by UV-visible spectroscopy (UV-Vis) and

trans-mission electron spectroscopy (TEM) for the purpose of

checking the plasmon adsorption maximum and its

corre-lation with different shapes The study is expected to offer

valuable information for stabilizing Ag@Au particles by

gelatin

2 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

pur-chased 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, 1 mm) was mixed

with the TSC solution (10 ml, 0.25 mm) Then, ice-cooled NaBH4

solu-tion (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

photodegrada-tion 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 deion-ized 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 tem-perature 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 1 mg/ml and equili-brated 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 dif-fractometer (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).

3 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 syn-thesis 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.

successfully synthesized by AgNO3, NaBH4, and TSC

[23] In this experiment, the results provided no

conclu-sive evidence for determining the stability of Ag seeds

because they were quickly used for the formation of

star-shaped 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

deter-mined by TEM (Figure 2A), and the seeds were spherical

in shape with a narrow size distribution ranging in

diam-eter from 6 to 10 nm

Our results were quite consistent with those of

previ-ous publications for the synthesis of Ag NPs via the

chemi-cal 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,

ascor-bic ions had a pronounced impact on the formation of

branched nanocrystals and the durability of NP

disper-sion plays a critical role in their application Importantly,

in this research, the effect of gelatin in the growth of

star-shaped 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

increas-ing absorption intensity compared to the

remain-ing 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

heterogeneous reactions, resulting in the lower absorp-tion intensity of NPs

In the present study, the stability of the samples was investigated at room temperature for 1 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 1 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 col-loidal protection at a relatively high concentration The results were completely consistent with the precipitated layer observed at the bottom region of the sample after 1 month

The TEM images of sample b (Figure 3C′) and sample f were examined after 1 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 aggre-gate 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 forma-tion of Ag@Au NPs All the Au has similar XRD patterns

1 0.8 0.6 0.4 0.2

0

300 400 500 600 700

Wavelength (nm)

Figure 2: TEM image (A), UV-Vis absorption spectrum (B), and digital photograph (C) of Ag seeds.

(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

1

0.8

0.6

0.4

0.2

0

400 450 500 550

Wavelength (nm)

1.2

1

a b c d e f

a b c d e f 0.6

0.8

0.4

0.2

0

600 650 700

40 30 20 10 0

400

15 20 25

30 35 40

60 45 30 15 0 15

10 20 25

Diameter (nm)

60 45 30 15 0

20 25 30 35 40 45 50

30 35 40

450 500 550 Wavelength (nm)

600 650 700

Figure 3: UV-Vis absorption spectrum of samples a to f (A) and after 1 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 1 month b (B′ and C′) and f (D′), respectively.

Table 1: Synthesis specifications of star-shaped Ag@Au NPs.

Sample   VHAuCl4 (ml)   VAA (ml)   VAg seeds (μl)   Vgelatin (ml)

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

asymmetri-cal 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

success-fully attached to the surface of Ag@Au NPs

4 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

star-shaped 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

parti-cles with high stability

Acknowledgments: This research was funded by the

Viet-nam National Foundation for Science and Technology

Development (NAFOSTED) grant no 104.02-2014.83

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Bionotes

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

associ-ate professor in 2011 She is also an invited lecturer at the Lac Hong

University and Ho Chi Minh City University of Natural Sciences.

Dai Hai Nguyen

Dai Hai Nguyen obtained his PhD in 2013 at Ajou University, Repub-lic of Korea Currently, he works as a researcher at the Institute of Applied Materials Science, Vietnam Academy of Science and Tech-nology He is also an invited lecturer at the TraVinh University and

Ho Chi Minh City University of Natural Sciences.