Antimicrobial Activity of UV-induced Chitosan Capped
Silver Nanoparticles
Ching-Wen Lou1, An-Pang Chen2, and Jia-Horng Lin2,3,4*
1
Institute of Biomedical Engineering and Material Science, Central Taiwan University of Science
and Technology, Taichung 40601, Taiwan. R.O.C.
*2Laboratory of Fiber Application and Manufacturing, Department of Fiber and Composite
Materials, Feng Chia University, Taichung City 40724, Taiwan, R.O.C.
*3School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan, R.O.C.
*4 Department of Fashion Design, Asia University, Taichung 41354, Taiwan, R.O.C.
Corresponding author: Jia-Horng Lin, Ph.D.
Email: jhlin@fcu.edu.tw
Affiliation: Laboratory of Fiber Application and Manufacturing
Department of Fiber and Composite Materials
Feng Chia University
Address: No. 100, Wenhwa Rd., Seatwen, Taichung 40724, Taiwan, R.O.C.
Tel: 886-4-24518661
Fax: 886-4-24510871
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Abstract. This study presents a green method to synthesize antibacterial silver
nanoparticles (Ag NPs) employing chitosan and silver nitrate. Chitosan acted
as dispersion and stabilizing agent. Ag NPs was radiated by UV ray to improve
their reaction efficiency. UV-Vis spectra showed that absorption peak of Ag
NPs occurred at 402 nm wavelength. TEM observed that the average size of Ag
NPs was 25-30 nm. XRD revealed that crystalline peak was Ag (1 1 1), Ag (2 0
0) and Ag (2 2 0). The synthesized Ag NPs had antibacterial activity against E.
coli.
Keywords: Nanoparticles, Synthesis, XRD, antibacterial.
Introduction
Microorganism infection is gradually received attention by more and more
people. Microorganism widely exists in the water, air, soil, animal body and
human body [1-3]. Recently, to prevent from harmful bacteria becomes an
important issue.
There has a big difference between nanomaterial and bulk metal material
[4]. Therein, nanomaterial usually employs in medicine, biology, biotechnology
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[5], chemistry [6], physics, catalysis [7], electronics [8] and opto-electronics
fields due to its superior properties such as good optics [9], excellent electricity
[10], good electromagnetism and chemical resistance. Among them,
nanoantibacterial material commonly comprises of antibacterial metal and
inorganic or organic carrier. Nanoparticle antibacterial metal is prepared
commonly using harmful chemicals, such as polar solvents and reduction agent.
Therefore, the current research goes toward to synthesize antibacterial metal
using green chemistry process. This approach reduces the generation of
contaminants and improves efficiency of energy. Guardia et al. replaced toxic
reagent based on green analytical chemistry theory [11]. Yun et al. introduced
series of rapid, clean, nontoxic, and eco-friendly manufacture procedures [12].
Meanwhile, Shankar et al. synthesized high-stability silver nanoparticles (16 −
40 nm) using extract of aqueous geranium leaf, and found that synthetic
duration of nanoparticles was as short as 60 min [13]. Lengke et al. described a
quick synthesis method of silver nanoparticles at high temperature, and
indicated that crystal growth at (1 1 1) faces depended on speediness of silver
atoms on cubic (1 0 0) faces [14]. Recently, more studies focused on formation of
gold or silver nanoparticles via surface plasmon resonance. UV irradiation
method was employed to produce strong electric field which was connected
3
with plasmonic of nanoparticles. Zapadinskii et al. utilized UV irradiation
technology to form gold nanoparticles after reducing Au3+ to Au0 in 3D
network polymer matrix due to surface plasmon resonance, and pointed out that
resonance intensity involved with synthesis condition and chemical structure of
oligomer among network polymer matrix [15].
This study presents a green method for silver nanoparticles (Ag NPs) using
chitosan and silver ion. Chitosan acted as dispersion and stabilizing agent. UV
radiation was induced to improve synthesis efficiency of Ag NPs. Lone pair
(-NH2) of nitrogen-atoms in chitosan has chelation effect with silver ion,
forming Ag NPs. Ag NPs was characterized by UV-Vis spectrometry (UV-Vis),
X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM).
Antimicrobial activity of Ag NPs was evaluated using E. coli.
Material and processing
Chitosan powder with 80 % deacetylation degree was provided from
Global Biological Technology Co., Ltd., Taiwan. Silver nitrate solution (extra
pure grade) was offered by Union Chemical Works Ltd., Taiwan.
Chitosan power was firstly dissolved in 1% acetic solution at 50 0C,
forming 2 wt% chitosan after magnetic stirring for 12 hours. 100 mM silver
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nitrate solution was blended with 2 wt% chitosan solution, forming Ag NPs
after stirring for 6 hour at 90 0C and radiation by UV light (Lamp Wattage:
20W, Lamp Voltage: 105V, wavelength: UV-C). Ag NPs were characterized by
UV–Vis spectra. The size of Ag NPs was observed by TEM. Crystalline
structure of Ag NPs was analyzed by XRD.
Measurements
The amount of Ag NPs was confirmed by UV-Vis spectra. The scanning
wavelength was 350-550 nm. The average size and distribution of Ag NPs was
analyzed by TEM (Hitachi H-7650, Hitachi High-Technologies Corporation,
Japan). Ag NPs solution was firstly deposited on the 200-mesh copper grid, and
then absorbed excessive agent after drying, which is used for TEM observation.
Crystalline structure of Ag NPs was characterized by XRD attached with
Rigaku Miniflex II analyzer (Rigaku Corporation, Japan). Cu was used as
target material. Kα was 1.542 Å, and scanning angle (2θ) was 5~70°.
Antibacterial activity of Ag NPs was evaluated against E. coli according to JIS
L1902. Sterilized agar was firstly injected into 90-mm-diameter culture dish,
forming solid medium. Then 0.1 ml E. coli bacterial suspension was evenly
coated on the solid medium, preparing bacterial medium. Then Ag NPs film
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was attached on the bacterial medium. Finally, the inhibition zone was
generated after co-culturing Ag NPs and bacteria at 37 ◦C for 24 hours.
Results and discussion
UV-Vis Spectrum
Figure 1 shows the UV-Vis spectrum of Ag NPs and Chitosan solution. As
silver nitrate adds, the reaction solution becomes from yellow to brown,
showing the presentation of Ag NPs which is confirmed by absorption peak at
405 nm [16]. The location of absorption peak correlated with surface plasmon
resonance of Ag NPs. The absorbance enhances after UV radiation, which
reflects that UV radiation is conductive to improve reaction efficiency. This is
because silver had strong absorption efficiency due to its surface plasmon
resonance (SPR) effect when silver electron produced polarization and
resonance with light wave after UV radiation [17]. SPR oscillations assisted to
produce impact between silver and chitosan, promote generation of chelating
bond and then improve absorbance of UV-visible light. Electrons generated
after UV radiation which induced a reduction of Ag+Ag0 [18], therefore, the
absorption peak moved toward to lower wavelength direction which was called
as blue shift. This blue shift was more prominent for small nanoparticles as
found by Palomba [19].
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Figure 1. UV-Vis spectra of Chitosan/Ag NPs solution before and after UV
radiation.
TEM Observation
Figure 2a shows that Ag NPs present uniform and spherical shape. Figure
2b displays that the nanoparticles are distributed as 5-60 nm. Figure 2c and 2d
show the particle size and distribution of Ag NPs after UV radiation. It is found
that a large number of Ag NPs had diameter of 10-45 nm. This is attributed to
incorporation of amidogen and silver ion. The increasing exponential
absorbance in Figure 1 indicated more Ag + was reduced into Ag0 nanoparticles
after UV radiation. This result agreed well with the earlier study about
chemically-produced Ag0 nanoparticles [20].
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Figure 2. TEM observation of chitosan/Ag NPs (a, b) and UV-induced
chitosan/Ag NPs (c, d) solution.
XRD Analysis
Figure 3(a) shows representative XRD of Chitosan/Ag NPs solution before and
after radiation. Chitosan had a crystalline peak at 2 θ=22.6°. This is due to fact
that chitosan had cellulose structure, and contained α-type and β-type
characteristic peaks [21]. Moreover, 2 θ of 38.08, 44.18, 64.48 displays
obviously silver crystalline peak, assigned to the (1 1 1), (2 0 0) and (2 2 0)
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planes of silver face-centered cubic [22]. These crystallization planes show that
fragment of chitosan encapsulated the silver ion to form the Ag NPS. Chitosan
has amino functional group and reacts with silver ion by the chelation effect as
in Figure 3(b). Chitosan contained lone pair of nitrogen atoms among NH2
group, which absorbed silver ions and provided excellent dispersive action. UV
induction promoted reaction efficiency of Ag NPs, which related to surface
plasmon resonance effect. Besides, Figure 3(a) also shows that 2 θ at (111),
(200) and (220) faces respectively changed from 38.08, 44.2, 64.48 to 38.18,
44.26 and 64.58. By Debye-Scherrer equation, the size of Ag NPs was shifted
from 29.1 nm to 25.4 nm after UV radiation. TEM observation displays that 40%
of Ag NPs had size of 25-30 nm via UV induction. Therefore, the size
calculated from Debye-Scherrer equation was close to that shown by TEM.
This shows that Debye-Scherrer Equation was appropriate to predict particle
size less than 100 nm [23, 24].
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Ag
(111)
X-ray diffraction
Ag
(200)
Ag
(220)
(a)
(b)
Figure 3 (a). XRD of chitosan, chitosan/Ag NPs, and UV-induced chitosan/Ag
NPs solution. (b) Chemical schematic of Ag NPs formed by chitosan and silver
nitrate.
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Antimicrobial Activity
Figure 4a shows that chitosan has low antimicrobial activity against E.
coli. With addition of silver nitrate, antimicrobial efficiency promotes as Ag
NPs generated. Therefore, an obvious inhibition zone is found in Figure 4b and
4c. Ag+ was released out in Ag NPs, behaving as the permanent source of the
cation [25]. Electropositive silver cation attracted with electronegative E. coli.
With smaller size of Ag NPs, intra-cellular protein thiol groups of
cytomembrane destroyed intensively, and then DNA replication of bacteria
weakened significantly [26, 27].
Figure 4. Antimicrobial activity of (a) chitosan, (b) chitosan/Ag NPs, (c)
UV-induced chitosan/Ag NPs against E. coli.
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Conclusion
This study prepared Ag NPs via green chemistry process. Ag NPs synthesized
using chitosan and silver nitrate. UV induction promoted reaction efficiency of
Ag NPs. The absorption peak occurred at 405 nm according to UV-Vis spectra,
however, it moved to 402 nm after UV induction. The average size of Ag NPs
was 27.1 nm by TEM observation, but mainly distributed around 25.7 nm after
UV induction. The crystalline peaks occurred at (1 1 1), (2 0 0) and (2 2 0)
planes of silver face-centered cubic, and UV-induced Ag NPs had the highest
diffraction intensity, according to XRD analysis. Ag NPs had strong
antimicrobial activity against E. coli. The resulting Ag NPs are expected to be
used as antibacterial nanomaterial in the future. Besides, nanoparticles
synthesis is accord with green chemistry and sustainable development.
References
[1] Jang J, Kim H, Han S. Sci Total Environ 2014;470–471:1558-1564
[2] Li A, Liu Z, Liu Y, Xu X, Pu Y. Energy Build 2012;47:497-505.
[3] Sabino R, Rodrigues R, Costa I, Carneiro C, Cunha M, Duarte A, Faria N,
Ferreira FC, Gargaté MJ, Júlio C, Martins ML, Nevers MB, Oleastro M,
12
Solo-Gabriele H, Veríssimo C, Viegas C, Whitman RL, Brandão J. J Sci Total
Environ 2014;472:1062-1069.
[4] Mazur M, Electro chem. Commun 2004; 6: 400.
[5] Kim B, Yalaz C, Pan D, Tetrahedron Lett 2012; 53: 4134-4137.
[6] Guo J, Ma L, Zhang X, Zhang Y, Lin T. Mater Lett 2014;118:142-145.
[7] Wang S, Li R, Zhang C, Li Y, Li B, Yang Y. Mater Lett 2013;106:71-74.
[8] Alena Řezníčková, Zdeňka Kolská, Petr Sajdl, Václav Švorčík. Mater Lett
2013;91:341-344.
[9] Xiang X, Xiang SB, Wang Z, Wang X, Hua G. Mater Lett 2012;88:27-29.
[10] Wiley BJ, Wang Z, Wei J, Yin Y, Cobden DH, Xia Y. Nano Lett 2006; 6(10):
2273–8.
[11] Armenta S, Garrigues S, Guardia MDL. Trac-Trends Anal Chem 2008; 27:
497-511.
[12] Akhtar PJ, Yun YG, ACS Sustainable Chemistry & Engineering 2013;1:591-602.
[13] Shankar SS, Ahmad A, Sastry M. J Biotechnol 2003;19:1627-1631.
[14] Lengke MF, Fleet ME, Southam G. Langmuir 2007; 23 (5):2691-2699.
[15] Zapadinskii BI, Kotova AV. Russ J Phys Chem B 2010;4:864-873.
[16] Chhatre P, Solasa S, Sakle R, Thaokar A, Mehra. Colloid Surf A-Physicochem
Eng Asp 2014;404:83-92.
13
[17] Huang X, El-Sayed IH, Qian W, El-Sayed MA. J Am Chem Soc
2006;128:2115-2120.
[18] Langlet M, Kim A, Audier M, Guillard C, Herrmann JM, Thin Solid Films
2003;429:13-21.
[19] Palomba S, Novotny L, Palmer RE. Opt Commun 2008;281:480-483.
[20] Naghavi K, Saion E, Rezaee K, Yunus WMM. Radiat Phys Chem
2010;79:1203-1208.
[21] Prashanth KVH, Kittur FS, Tharanathan RN. Carbohydr Polym 2002;50:27-33.
[22] Yin LY, Cheng YW. Energy Environ Sci 2011; 45: 2360.
[23] Debanath MK, Karmakar S. Mate Lett 2013;111(15):116-119.
[24] Gonçalves NS, Carvalho JA, Lima ZM, Sasaki JM. Mater Lett 2012;72:36-38.
[25] Liu X, Gurel V. Adv Mater 2007;19:2790.
[26] Kim SS, Park JE, Lee J. J Appl Polym Sci 2010;119: 2261-2267.
[27] Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO. J Biomed Res
2000;52:662-668.
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