Materials Science and Engineering C 50 (2015) 31–36

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Facile and green synthesis of silver nanoparticles using oxidized pectin Mythili Tummalapalli a, B.L. Deopura a, M.S. Alam b, Bhuvanesh Gupta a,⁎ a b

Bioengineering Lab, Department of Textile Technology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Department of Chemistry, Jamia Hamdard, New Delhi 110062, India

a r t i c l e

i n f o

Article history: Received 26 August 2014 Received in revised form 20 December 2014 Accepted 14 January 2015 Available online 17 January 2015 Keywords: Nanosilver Oxidized pectin Reduction Nanohydrogel Morphology

a b s t r a c t In the current work, an alternative route for facile synthesis of nanosilver is reported. Oxidized pectin has been used as the reducing agent as well as the stabilizing agent, resulting in the formation of oxidized pectinnanosilver (OP-NS) core sheath nanohydrogels. The effect of reaction parameters on the synthesized nanoparticles is investigated. The structural and morphological features have been analyzed using X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM) respectively. The crystal size of the synthesized nanosilver was calculated to be 28.76 nm. While the average size of the core sheath structure varied from 289 nm to 540 nm, the size of the silver nanoparticle entities at the core varied from 100 nm to 180 nm, with variation in reaction time. From the morphological examination, it could be seen that flower like nanostructures are formed with nanosilver in the core surrounded by a polymeric halo. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In recent years, nanotechnology has become a by-word in all walks of life. Nanocomposites constituting of polymers and metals, especially silver, have an important role to play in this aspect. Interest has veered towards the development of silver nanocomposites due to their diverse range of applications, e.g. in healthcare, sensors [1], catalysis [2], plasmonic devices [3], conducting materials [4,5], and food packaging [6, 7]. Nanosilver is particularly useful in preventing wound infection due to its excellent antimicrobial activity [8,9]. Indeed, silver and its compounds have traditionally been used through the millennia to treat wounds [10,11]. Polysaccharides are abundant in nature and have found application in a wide range of industries. Pectin is an anionic polysaccharide, poly(1,4-galacturonic acid), found in the cell walls of terrestrial plants. It is a hydrogel by nature and is therefore attractive for use as a gelling agent [12] and in wound care devices. The presence of groups like \OH, \COOH and \COOCH3 on the backbone of the pectin chain renders it readily susceptible to functionalization. Pectin and alkenyl succinic anhydride were used for the controlled deposition of nanoparticles that led to a high impact on the substrate surface properties of paper [13]. Pectin/chitosan/Eudragit® RS ternary films were developed for sigmoidal drug delivery [14]. Takei et al. oxidized citrus pectin and coupled it with an anticancer drug, doxorubicin [15]. It was suggested by Cipriani & co-workers that chemically sulfated citrus pectin possesses good antithrombogenic properties and could be used as wound ⁎ Corresponding author. Tel.: +91 11 26591416; fax: +91 11 26581061. E-mail address: [email protected] (B. Gupta). 0928-4931/© 2015 Elsevier B.V. All rights reserved.

dressings [16]. Coacervation between pectin and gelatin could be helpful for controlled drug delivery [17]. In our previous work, we reported the in-situ crosslinking between oxidized citrus pectin and gelatin and these matrices could potentially be used in wound dressings [18]. The synthesis of nanosilver has been a subject of enormous interest in recent years. Eid & Azzazy reported the controlled synthesis of hollow flower like silver nanostructures from AgNO3 with the aid of dextrose, trisodium citrate and sodium hydroxide [19]. Various reducing sugars like glucose, fructose and sucrose have been used to synthesize silver nanoparticles, both with and without stabilizing agents [20–22]. The fungus Trichoderma viride was used to reduce AgNO3 to AgNPs through an extracellular biosynthesis method [23]. Hydrazine hydrate was employed as the reducing agent by several researchers to synthesize AgNPs from AgNO3 [3,24]. Radiolytic synthesis of AgNPs has also been reported. Kristić et al. investigated the efficacy of chitosan/PVA blends as capping agents for AgNPs synthesized by gamma irradiation [25]. Carboxymethyl chitosan (CMCTS)/gelatin/nanosilver hydrogels were developed by radiation induced reduction and crosslinking [26]. These gels could potentially be used as wound dressings. CMCTS serves as a reducing agent as well as a stabilizing agent. Gamma radiation has been used to prepare nanosilver nanohydrogels of poly(methacrylic acid) where the radiation performs two functions — polymerization of the monomer and reduction of silver ions to nanosilver [27]. In our previous work, we reported the fabrication of OP and gelatin crosslinked matrices [18]. Incorporation of silver nanoparticles into these matrices can lead to the development of potential wound care devices. Obviating the ex situ reduction of silver salt to silver as reported in other methods, we aim to develop an in situ reduction approach wherein the silver nanoparticles would be synthesized within the system, thus reducing the cytotoxicity.


M. Tummalapalli et al. / Materials Science and Engineering C 50 (2015) 31–36





OP-NS powder

Fig. 1. Schematic representation of OP-NS nanohydrogel synthesis.

This sort of approach has not been reported earlier to the best of the authors' knowledge. In the present study, we report the synthesis and characterization of stable silver nanoparticles by reduction of silver nitrate with oxidized pectin. These nanoparticles are embedded in the oxidized pectin matrix and therefore can be termed as nanohydrogels. Core sheath structures with silver nanoparticles at the core and oxidized pectin as the sheath are produced. 2. Experimental

acid was added to a 2% solution of pectin in distilled water. The pH of the medium was adjusted to 3.5 using dilute hydrochloric acid and sodium bicarbonate solution. The reaction was allowed to take place at 40°C under constant stirring for 16 h. To prevent autooxidation due to light, the reaction vessel was wrapped in several layers of aluminum foil and the reaction was carried out in the dark. At the end of the reaction, oxidized pectin (OP) was precipitated out using excess isopropanol, separated by vacuum filtration and dried in vacuum at 60 °C. The aldehyde content of OP thus prepared is 2.1 mmol/g [9].

2.1. Materials 2.3. Reduction of silver nitrate by oxidized pectin Citrus pectin (Mw ∼ 30,000 g/mol, degree of esterification ∼ 72%) and isopropanol were purchased from CDH Fine Chemicals, India and Fisher Scientific, India, respectively. Silver nitrate and periodic acid were procured from Merck Chemicals, India. All other chemicals used were of analytical grade. Millipore water was used for all the experiments. 2.2. Oxidation of pectin by periodic acid The periodate oxidation of pectin was carried out according to the procedure reported in our earlier work [9]. 3 mL of 0.5 M periodic


5 min

1 wt.% of AgNO3 was added to a 2.8% solution of OP (initial aldehyde content 2.1 mmol/g). The reaction was allowed to continue for predetermined intervals of time under constant stirring. The temperature was maintained at 60 °C. The reaction vessel was wrapped in several layers of aluminum foil to prevent autooxidation of AgNO3 due to light. At the end of the reaction, oxidized pectin-nanosilver (OP-NS) was precipitated from the solution using excess isopropanol. Subsequently, it was separated out by vacuum filtration and dried in vacuum at 60 °C.

20 min

60 min

120 min

Reducon me Fig. 2. Appearance of OP-NS solutions with varying reaction time. (a) 5 min; (b) 20 min; (c) 60 min; (d) 120 min. Initial aldehyde content 2.1 mmol/g; reaction temperature 60 °C; pH 4.

M. Tummalapalli et al. / Materials Science and Engineering C 50 (2015) 31–36




OD [-CHO1734 cm-1/-CH2 2943 cm-1]

Aldehyde content (mmol/g)

1.60 2.09




2.01 0







1.55 1.50 1.45 1.40 1.35 1.30 1.25



Time (min)


Fig. 3. Variation of aldehyde content with reaction time. Initial aldehyde content 2.1 mmol/g; reaction temperature 60 °C; pH 4.








Time (min) Fig. 5. Variation in optical density with reaction time. Initial aldehyde content 2.1 mmol/g; reaction temperature 60 °C; pH 4.

2.4. Determination of the aldehyde content The amount of aldehyde consumption upon reduction of silver nitrate was calculated using a dinitrophenylhydrazine (DNPH) assay as per our previously reported work [9]. 100 μL of 0.3% OP-NS was added to 10 mL of freshly prepared DNPH solution. The reaction mixture was allowed to stand for 1 h and then centrifuged at 7000 rpm for 10 min. The absorbance of unreacted DNPH in the supernatant fluid was measured at λ = 326.4 nm using a Perkin Elmer Lambda 35 UV–vis spectrophotometer. The amount of aldehyde consumed was calculated according to:

2.5. Fourier transform infrared spectroscopy (FTIR) FTIR spectra of nanosilver immobilized oxidized pectin of varying reduction times were recorded using a KBr pellet on a Perkin Elmer Spectrum-BX FTIR system. The samples were scanned in the range of 4000–400 cm−1 at a resolution of 2 cm−1. 2.6. Ultraviolet–visible spectrophotometry (UV–vis)

2 6 Aldehyde conc: ðmmol=gÞ ¼ 6 4


Reacted DNPHðmmol=gÞ

3  10‐4


Nanohydrogel solutions of OP-NS with varying reduction times were analyzed in a spectral range of 300–600 nm using a Perkin Elmer Lambda 35 UV–vis spectrophotometer. The path length was maintained at 10 mm.

7 5

198:14 7

2.7. Energy dispersive X-ray analysis (EDX)

where, 198.14 is the molecular weight of DNPH.

Transmiance (%)

To identify the elements present in the OP-NS matrices, EDX analysis was conducted. The samples were placed on an aluminum stub and

Absorbance (a.u)

420 nm e

d c

e d c b

2943 cm-1



a 4000


1734 cm-1 3200






Wavenumber (cm-1) Fig. 4. FTIR spectra of OP-NS with varying reaction time. (a) Pure OP; (b) 10 min; (c) 40 min; (d) 60 min; (e) 120 min. Initial aldehyde content 2.1 mmol/g; reaction temperature 60 °C; pH 4.








Wavelength (nm) Fig. 6. UV–vis spectra of OP-NS solutions with varying reaction time. (a) Pure OP; (b) 5 min; (c) 20 min; (d) 60 min; (e) 120 min. Initial aldehyde content 2.1 mmol/g; reaction temperature 60 °C; pH 4.


M. Tummalapalli et al. / Materials Science and Engineering C 50 (2015) 31–36 cps/eV




Ag C O






0 0







Fig. 7. EDX spectra of OP-NS. Reaction time 120 min; reaction temperature 60 °C; pH 4.

coated with carbon using an Auto-Fine Coater JFC-1600 (Joel, USA Inc., USA). The images and the silver content of the samples were obtained with a Bruker-AXS Energy Dispersive X-ray system (Model QuanTax 200), SDD Technology, USA.

(L) of the synthesized nanosilver particles was calculated according to the Scherrer equation:

L¼ 2.8. Dynamic light scattering (DLS)

2.9. High resolution transmission electron microscopy (HRTEM) The HRTEM imaging of the OP-NS matrices was conducted on a TECNAI 200 kV TEM (Fei, Electron Optics) machine. A few drops of the OP-NS solutions were cast onto carbon coated copper mesh grid. The grids were subsequently stained with 0.1 M phosphotungstic acid and dried at 60 °C in a vacuum oven. These grids were then used for imaging purposes.

where β is the broadening of line diffraction measured at half of the maximum intensity (in radian), λ is the wavelength of the X-rays used (1.54 Ǻ) and θ is the Bragg's angle (in degree).

230 220 210

Parcle diameter (nm)

The nanoparticles were characterized by a dynamic light scattering (DLS) using a Beckmann Coulter (Delsa™ Nano) instrument. When light is scattered by a molecule or particle some of the incident light is scattered. If the molecule is stationary then the amount of light scattered would be a constant. However, since all molecules in solution diffuse with Brownian motion in relation to the detector there will be interference (constructive or destructive) which causes a change in light intensity. By measuring the time scale of light intensity fluctuations, DLS can provide information regarding the average size, size distribution, and polydispersity of molecules and particles in solution.

0:94λ β cosθ

200 190 180 170 160 150 140 0

2.10. X-ray diffraction (XRD)










Time (min) Wide angle X-ray diffraction patterns were obtained using a PANalytical X'Pert PRO instrument, operating at 40 kV and 40 mA using sealed-tube Cu Kα radiation (λ = 1.54 Ǻ). The crystallite size

Fig. 8. Effect of reaction time on the mean particle size of OP-NS nanohydrogels. Initial aldehyde content 2.1 mmol/g; reaction temperature 60 °C; pH 4.

M. Tummalapalli et al. / Materials Science and Engineering C 50 (2015) 31–36

3. Results & discussion Pectin, an anionic polysaccharide, with vicinal diols on its backbone chain can undergo Malaprade oxidation by periodic acid. Similar to other reducing sugars like fructose and glucose, the aldehyde moieties thus introduced into oxidized pectin can reduce silver nitrate to elemental silver according to the mechanism shown. −


AgNO3 →Ag þ NO3 þ




Ag þ H2 O þ OP‐CHO → Ag þ H þ OP‐COOH The schematic representation of the process is shown in Fig. 1. Furthermore, OP acts as a stabilizing agent for nanosilver particles by virtue of its long chain length. The nanosilver thus incorporated into the matrix by in-situ reduction is expected to enhance the performance of these systems in drug delivery and wound care applications [18]. The conversion of silver nitrate to nanosilver is almost instantaneous and can be crudely evidenced by the change in color as shown in Fig. 2. There is a gradual change in the color with an increase in the reaction time. This change is due to the different morphologies of the nanoparticles synthesized [28]. The amount of aldehyde consumption as a function of reaction time is presented in Fig. 3. The initial aldehyde content of OP was 2.101 mmol/g. Instantaneous reaction takes place as seen by the change in aldehyde content in 5 min. With an increase in reaction time from 5 min to 120 min, the aldehyde content reduced from 2.098 mmol/g to 2.04 mmol/g. A larger number of silver ions come under attack with an increase in the reaction time leading to a decrease in the leftover aldehyde content. However, it seems that a stable state is reached at around 60 min. In Fig. 4, we present the FTIR spectra of OP-NS nanohydrogels with varying reaction time. The peak at 1734 cm−1 is due to the carbonyl aldehyde stretching in oxidized pectin. Upon the reduction of silver nitrate, the intensity of this peak is reduced. The optical density (OD) of the aldehyde stretching peak was calculated with respect to the \CH2


stretching at 2943 cm−1, as depicted in Fig. 5. As the reaction time increases from 10 min to 120 min, the OD reduces from 1.596 to 1.231 lending credence to the statement that aldehyde groups reduce silver nitrate to nanosilver. The simplest way to observe the formation of nanosilver is through UV–vis assay. Fig. 6 displays the UV–vis spectra of OP-NS systems with different reaction times. A strong resonance centered around 420 nm was clearly observed, which is a characteristic peak of nanosilver [29]. The intensity of the resonance peak increased with an increase in reaction time, indicating the strong presence of nanosilver in the system. A representative EDX spectrum of OP-NS nanohydrogels (reaction time 120 min) is presented in Fig. 7. The peak at 3 keV corresponds to silver [27]. Since the unreacted AgNO3 dissolves in water and isopropanol during precipitation, it is reasonable to theorize that the silver detected by EDX is purely in the metallic form. The particle size analysis by DLS is presented in Fig. 8. The size of the OP-NS nanohydrogels varies from ~289 nm to ~540 nm when the reaction time increases from 10 min to 120 min. The samples were dispersed in water prior to the measurement. OP is not completely soluble in water due to the formation of hemiacetals between aldehyde groups and adjacent hydroxyl groups. This would lead to a swelling as OP is a hydrogel material and the results thus obtained pertain to the swollen nanohydrogels. Initially the rate of increase in size is rapid up to 40 min beyond which it is stabilized. The morphologies of the fabricated OP-NS nanohydrogels can be examined through HRTEM as shown in Fig. 9. Flower like structures are observed with a dark core and a cloudy formation surrounding it. We theorize that the dark core corresponds to NS while the cloud around it consists of polymeric petals. It has been theorized that the core-sheath structure can lead to better antimicrobial activity of the silver nanoparticles [30]. The average sizes of the core and core & shell of the OP-NS nanohydrogel with 5 min reaction time are 102 nm & 352 nm respectively. As the reaction time is increased, the OP halo undergoes fragmentation as shown in Fig. 9(b)–(d).





Fig. 9. HRTEM studies of OP-NS particles. (a) 5 min; (b) 20 min; (c) 60 min; (d) 120 min. Initial aldehyde content 2.1 mmol/g; reaction temperature 60 °C; pH 4.


M. Tummalapalli et al. / Materials Science and Engineering C 50 (2015) 31–36






Intensity (a.u.)




0 10








2θ Fig. 10. X-ray diffractogram of OP-NS. Initial aldehyde content 2.1 mmol/g; reaction time 40 min; reaction temperature 60 °C; pH 4.

The crystal structure of the synthesized OP-NS nanohydrogels was investigated using XRD. A representative X-ray diffractogram of OP-NS nanohydrogel (reaction time 10 min) is presented in Fig. 10. The peaks at 2θ values of 39.23°, 43.77° and 76.87° correspond to the (111), (200) and (311) crystal planes of silver respectively, signifying a face centered cubic (fcc) crystal structure [31,32]. The crystal size (L) calculated according to the Scherrer equation is 28.76 nm. The broad peak at 2θ of 22.31° is due to the crystal structure of pectin [33]. 4. Conclusions A facile mechanism for the synthesis of nanosilver is reported in the present work. Silver nitrate was reduced to nanosilver by the aldehyde groups present in oxidized pectin. Oxidized pectin also acts as a stabilizing agent due to its long chain length, thus resulting in the formation of OP-NS core-sheath nanohydrogels. An increase in the reaction time resulted in an increase in the average particle size of the nanohydrogel. Flower like structures of the OP-NS nanohydrogels were observed using HRTEM with a dense nanosilver core surrounded by a polymeric halo. From XRD, it was concluded that the synthesized nanosilver has a face centered cubic crystal structure with an average crystal size of 28.76 nm. Acknowledgments The authors duly acknowledge the facilities provided at SAIF, AIIMS, New Delhi to conduct the HRTEM analysis. References [1] Y. Sun, Y. Xia, Shape controlled synthesis of gold and silver nanoparticles, Science 298 (2002) 2176–2179. [2] J. Shen, W. Shan, Y. Zhang, J. Du, H. Xu, K. Fan, W. Shen, Y. Tang, Gas phase selective oxidation of alcohols: in situ electrolytic nano-silver/zeolite film/copper grid catalyst, J. Catal. 237 (2006) 94–101. [3] V. Germain, A. Brioude, D. Ingert, M.P. Pileni, Silver nanodisks: size selection via centrifugation and optical properties, J. Chem. Phys. 122 (2005) (124707-1-124707-8). [4] H.H. Lee, K.S. Chou, Z.W. Shih, Effect of nano-sized silver particles on the resistivity of polymeric conductive adhesives, Int. J. Adhes. Adhes. 25 (2005) 437–441. [5] W.T. Cheng, Y.W. Chih, W.T. Yeh, In situ fabrication of photocurable conductive adhesives with silver nano-particles in the absence of capping agent, Int. J. Adhes. Adhes. 27 (2007) 236–243.

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Facile and green synthesis of silver nanoparticles using oxidized pectin.

In the current work, an alternative route for facile synthesis of nanosilver is reported. Oxidized pectin has been used as the reducing agent as well ...
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