- Email: [email protected]
Photocatalytic action of AgCl nanoparticles and its antibacterial activity
Accepted Manuscript Photocatalytic action of AgCl nanoparticles and its antibacterial activity Deenadayalan Ashok Kumar, V. Palanichamy, Selvaraj Mohana Roopan PII: DOI: Reference:
S1011-1344(14)00203-6 http://dx.doi.org/10.1016/j.jphotobiol.2014.06.011 JPB 9776
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
9 May 2014 16 June 2014 18 June 2014
Please cite this article as: D. Ashok Kumar, V. Palanichamy, S.M. Roopan, Photocatalytic action of AgCl nanoparticles and its antibacterial activity, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.06.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Photocatalytic action of AgCl nanoparticles and its antibacterial activity a,
Deenadayalan Ashok Kumara, V. Palanichamy * Selvaraj Mohana Roopanb,* a
School of Bio Sciences & Technology, bChemistry Research Laboratory, Organic
Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India. Corresponding
Author:
*Email:
[email protected];
[email protected]; [email protected]; Tel: 09865610356; +91 04162 202352; fax: +91 416 224 5544/5766
Abstract The scientific community is searching for biosynthetic methods for the production of metallic nanoparticles. Biogenic pathway has now become a vast developing area of research. A novel route biological synthesis of silver chloride nanoparticles (AgCI-NPs) using aqueous leaf extract of M. citrifolia under ambient conditions were evaluated. Synthesized nanoparticles were confirmed by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and Transmission Electron Microscopy (TEM). The effects of pH on biosynthesis of AgCI-NPs were investigated using UV-vis spectroscopy. TEM images showed that the diameter of stable AgCI-NPs were approximately 12 nm. FTIR spectra provide the evidence for the presence of protein as possible biomolecules responsible for reduction and capping of nanoparticles. The synthesized AgCI-NPs were observed to have a good catalytic activity on the reduction of methylene blue (MB) dye by M. citrifolia extract which has been confirmed by decrease in absorbance maximum values of methylene blue with respect to time using UV-vis spectroscopy and was attributed to the electron relay effect. Keywords AgCl, Biosynthesis, Morinda citrifolia leaf, Electron microscopy, Catalytic activity
1. Introduction Nanoparticles synthesis is an evergreen research field of 21st century in which the connotation of the bio-mediated experimental process is highly important. Biosynthesis of nanoparticles has an emerging highlight of the intersection of nanotechnology and biotechnology. It has received increasing attention due to a growing need to develop rapid, clean, nontoxic, simple and environmentally friendly synthetic technologies. The 1
significance of such a synthetic protocol has been well demonstrated [1]. Biosynthesis of silver chloride nanoparticles using biological systems, such as plants extract has gained a great importance in recent years due to using of less harmful, biodegradable and cost effective reagents [2]. The biosynthesis of silver chloride nanoparticles has an emerging highlight due to the variety of application especially photochemical and biomedical applications. Also it has been used for the fabrication of photographic paper and photochromic lenses [3]. The important significance of biosynthetic protocol has been well demonstrated in our present work. Morinda citrifolia is one of the traditional folk medicinal plants in Southeast Asia, also known as noni [4]. Noni has been reported for a broad range of medical applications including antibacterial, antiviral, antifungal, antitumor, analgesic, hypotensive, antiinflammatory, and immune enhancing effects [5]. The primary usage of noni is to apply leaves as a traditional topical treatment thought to enhance wound healing. The leaf contains flavanol glycosides, β-carotene and iridoid glycosides. Noni leaf extract was capable of promoting wound healing in an animal model. In addition, the crude extract of noni leaf has been traditionally used in patients with bone fractures or dislocation to promote tissue repair and decrease inflammation. In addition, Joseph Betz a biochemist in FDA evaluated Morinda citrifolia had been analysis for a number of biological activities in animal models and antimicrobial studies. The fresh leaf is used for the treatment of wounds and also as a poultice for broken bones and sprains in most parts of India. However the detailed information regarding the wound healing activity of M. citrifolia is not scientifically documented [6]. In the present investigation an attempt was made to assess the effect of Morinda citrifolia leaf extract on silver chloride nanoparticles production. Followed by catalytic activity of the AgCI-NPs were evaluated with the help of methylene blue reduction using UV-vis spectroscopy. AgCI-NPs can act as a good catalyst due to the high Fermi potential. It can also catalyze much reduction reaction. The rate constant of the catalytic reaction is found to depend on the size of the nanoparticles.
2. Experimental section 2.1 Materials
2
The healthy leaves of Morinda citrifolia was collected in and around of Vellore town in Tamilnadu, India. Silver nitrate (AgNO3) were purchased form Merck (India). The reagents used in all the experiments were purely analytical grade. 2.2 Preparation of M. citrifolia aqueous extract The freshly leaves of M. citrifolia was washed several times with double distilled water. About 10 g of leaves were added to 100 mL of deionized water and boiled for 5-10 min in a water bath. The aqueous mixture was then filtered through Whatman filter paper No.1 to obtain aqueous extract (Fig. 1). The aqueous extract was stored in refrigerator at 4 ºC. The aqueous extracts were used as reducing as well as stabilizing agent [7]. 2.3 Biosynthesis of silver chloride nanoparticles using leaf extract In this study, the leaves extract acts as a capping agent for the biosynthesis of AgCI-NPs. In a typical biosynthesis of AgCI-NPs, 1 mL of aqueous leaves extract was added to 10 mL of 1 mM AgNO3 aqueous solution and kept in a dark place at room temperature. After few minutes, the colorless reaction mixture changed to dark brown color that indicates the formation of AgCI-NPs [8]. The dry powders of the silver chloride nanoparticles were obtained as follows; after the desired reaction period; aqueous mixture containing AgCI-NPs was centrifuged at 10,000 rpm for 10 min. The centrifugation and re-dispersion in double distilled deionized water were repeated three times [9]. 2.4 Effect of pH on phyto-synthesis of AgCI-NPs This pH parameter is an important factor for any phyto-synthesis reaction, especially for biochemical reaction. The effect of pH values of AgNO3 solution on the phyto-synthesis of AgCI-NPs were evaluated using various pH parameters (5.0, 6.0, 7.0, 8.0 and 9.0) with fixed aqueous concentration of AgNO3 (1 mM). The pH value was adjusted by 0.01 M HCl and 0.01 M NaOH solution. The effect of pH values were analyzed by UV-vis spectroscopy. 2.5 Effect of volume on phyto-synthesis of AgCI-NPs The concentration of precursor is also an important significant parameter for AgCI-NPs synthesis. The concentration of leaf extract was optimizing by varying the different volume of leaf aqueous extract (1 to 5 mL). The effect of leaf aqueous extract was evaluated using UV-vis spectroscopy. 2.6 Characterization of silver chloride nanoparticles The bio-reduced AgCI-NPs were monitored using a UV–vis spectrophotometer. The UV–vis spectrum was recorded on a Perkin-Elmer (Lambda 25 Model) UV–vis 3
spectrophotometer and the sample was measured in the wave- length region of 200–800 nm. The morphological studies of the phyto-synthesized AgCI-NPs were viewed by TEM (HITACHISU-6600 model) instrument [10]. The green phyto-synthesized AgCINPs were centrifuged at 10,000 rpm for 15 min to obtain the residue. The residual part of AgCI- NPs were washed with deionized water and dried in a hot air oven at 60 ºC for 24 h. The powdered AgCI-NPs were analyzed by a Bruker model D8 Advanced Powder Xray diffractometer. The intensity data for the nano silver powder were collected in the 2θ range 20–80º and the scanning speed at 0.02 min per degree. The green phyto-synthesis of AgCI-NPs were recorded by JASCO FTIR spectrophotometer using KBr pellet in the range of 400–4000cm-1 with the spectra resolution of 4 cm-1. 2.7 Photocatalytic activity of AgCI-NPs In order to assess the catalytic activity of phyto-synthesized AgCI-NPs, two reactions were carried out in a 3.5 mL capacity quartz cuvette and absorbance values were monitored using UV–visible spectrophotometer [11]. In the first reaction, 1 mL of methylene blue (1 ×10−4 M) was mixed with 0.2 mL of aqueous leaf extract and 1.8 mL of water, this reaction mixture was monitored for 30 min. In second reaction, 1 ml of methylene blue (1×10−4 M) was mixed with 0.2 mL leaf extract and 2 mL of synthesized silver nanoparticles (AgCI-NPs) and this reaction mixture was monitored at three different time intervals viz., 30, 45 and 60 min. In all the reactions total volume of the mixture was made up to 3 mL.
The values of absorption maxima (λ max) were
compared, with that of methylene blue. 2.8 Antimicrobial properties of AgCI-NPs The antibacterial activities of AgCI-NPs were evaluated by using the Kirby–Bauer method. Nutrient broth/Agar (1 g beef extract, 1 g peptone, 0.5 g NaCl dissolved in 100 ml of double distilled water) was used for culturing the bacteria. The Nutrient agar plates were inoculated with cultures of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa allowed for overnight growth at 30 °C. Sterile Whatman filter paper discs of 5 mm diameter was impregnated with 25 µL, 50 µL and 75 µL of AgCI-NPs and were placed on to the bacterial lawn in agar plates [12].
Standard
streptomycin antibiotic discs were used as a reference drug. The agar plates were incubated at 37 °C for 24 h. After 24 h of incubation, the zone of inhibition was measured. 4
3. Results and discussion 3.1 UV-vis spectroscopy analysis The biosynthesis of AgCI-NPs using leaf extract was monitored by UV-vis spectroscopy. It is well known that AgCI-NPs were exhibit maximum UV-vis absorption in the range of 400-500 nm [13]. The formations of AgCI-NPs were preliminarily confirmed by UVvisible spectral analysis of colored solutions that exhibit within 5 h. A strong small shift of absorbance toward the higher wavelength from 420 to 430 nm (Fig. 2a) was seen with increase of time. The inset in indicates the variation of absorbance at λmax with respect to time. In the present investigation, AgCI-NPs were rapidly formed after the addition of M. citrifolai leaf aqueous extract. The bioreduction reaction turned dark brown colour after 4-5 h. A wide absorption spectrum peak of UV-visible range indicates that the distribution of smaller sized nanoparticles will be higher in the reaction mixture [14]. From this current investigation it revealed that the formations of AgCI-NPs were depends on the pH parameter of the bioreduction reaction and the rate of AgCI-NPs were formed with high intensity in basic pH (7.0) than in acidic pH. The formations of AgCI-NPs were occurred quickly in neutral and basic pH which may be due to the ionization of the phytoconstituents present in the leaf aqueous extract of M. citrifolia. The absorbance spectrum peak was located at 424 nm in pH 7.0 and the rate of AgCI-NPs formation was slow reduction reaction at acidic pH condition. The present study was extended to evaluate the AgCI-NPs formation using M. citrifolia leaf extract at different pH level. In final conclude that leaf extract is responsible for the reduction in AgNO3 to form AgCINPs and suitable pH condition is 7.0. [15] Similarly we demonstrated different concentration ratios of leaf aqueous extract and silver nitrate solution were also optimized for high yield production of silver nanoparticles.
In this study different
volume of leaf extract from 1 to 5 mL added to 1 mM AgNO3 solution was turned to brown colour with in 30 min of incubation period, indicating rapid formation of AgCINPs. Thus the optimized leaf aqueous medium supported the maximum amount formation of silver chloride nanoparticles (AgCI-NPs) and the reaction was occurred very fast and rapidly.
3.2 Fourier transforms infrared spectroscopy (FTIR) The FTIR spectroscopy analysis was carried out to identify the biomolecules responsible +
for reduction of Ag into Ag0 and capping of the AgCI-NPs phyto-synthesized by leaf
5
extract (Fig.2b) [16]. The bands at 557 cm-1 is corresponds to C-CI stretch (alkyl halides). The band at 1068 cm-1 is assigned to be C-N stretch which is found to be aliphatic amines group. The absorption bands at 1384 cm-1 are characteristics of C-H alkyl halides group. The band at 1402 cm-1 is assigned to the aromatic group stretching. The band at 1598 cm-1 is occurred -C=C- stretch in alkenes group. The absorption spectrum peak at 2827 cm-1 is showed C-H stretching in aldehydes group. The peaks located at 3174cm-1, 3332 cm-1 and 3444cm-1 corresponds to stretching vibrations of =CH (alkene) and N-H stretching vibrations of amines, amides and O-H stretching vibrations in phenol groups. The present study well known that bio-constituents interact with silver ions salts and mediate reduction process with functional groups.
The current
investigation FTIR spectrum analysis the presence of phenol groups, amines, amides, alkene, aldehydes, aromatic, alkyl halides, and aliphatic amines groups in the leaf aqueous extract of M. citrifolia are responsible for capping agents in AgCI-NPs formation [17].
3.3 X-Ray diffraction analysis (XRD) Fig.2c shows the XRD pattern of AgCI-NPs evaluated in the present research study. After bioreduction reaction, the diffraction peaks at 2θ = 28.01, 32.08º, 38.16º, 46.17º, 54.91º, 57.40º, and 76.68º assigned to the (111), (200), (220), and (311) intensity planes of a faced centre cubic lattice of AgCI-NPs (Fig.2c) [18-20]. However, the diffraction peaks are formed which indicate that crystallite size obtained was matched with database of Joint Committee on Powder Diffraction Standard (JCPDS) file no. 99-101-1241. The minor peaks are due to the presence of silver nanoparticles [21]. The average size of the AgCI-NPs formed in the bioreduction reaction was evaluated using Debye-Scherrer formula, D=Kλ/βs cos θ where ‘D’ is particle diameter size, k is a constant equal 1, ‘λ’ is wavelength of X-ray source, ‘β’ is the full width half maximum (FWHM) and ‘θ’ is the diffraction angle corresponding to lattice plane.
3.4 Transmission electron microscopy (TEM) The biosynthesized AgNPs were analyzed by TEM to find the information of morphology and size of nanoparticles [22]. A drop of AgCI-NPs sample solution synthesized by leaf extract was deposited onto a carbon coated copper grid. The TEM images of the AgCI-NPs are depicted in Fig.3. The result was comparable with reported 6
data and the size of the AgNPs is controlled within 12 nm by capping and stabilizing agent [23].
3.5 Mechanism for the formation of AgCI-NPs Biosynthetic pathways responsible for the production of metal nanoparticles using plants are yet to be elucidated. Keeping in this mind we decided to propose the mechanism for the production of AgCl-NPs (Fig.4). The chlorine ion which is present in the leaf will react with Ag(0) to form AgCl. It has been confirmed through powder XRD. Hence, content of leaf extract contains chlorine could be most attributed source for the formation of AgCl-NPs [24-25].
3.6 Photocatalytic activity of AgCI-NPs The catalytic activities of phyto-synthesized AgCI-NPs were analyzed using methylene blue. In the electron transfer step, there can be the restriction of passage of electrons when there is a large redox potential difference between acceptor and donar. Electron transfer becomes easy when an effective catalyst has intermediate redox potential value between acceptor and donor. Here, we have reported the reduction of methylene blue by the aqueous extract of M. citrifolia containing AgCI-NPs (Fig.5). The absorbance value of methylene blue was recorded at 665 nm. After 30 min of mixing of plant aqueous extract into methylene blue, it was observed that the absorbance peak of methylene blue is decreased slowly and shifted to higher wavelength. But the reaction containing methylene blue, AgCI-NPs and extract, showed remarkable decrease in absorbance peak after 30 and 45 min. This decrease in absorbance peak indicates that the AgCI-NPs have potential to degrade methylene blue [26].
3.7 Antimicrobial activity of AgCl-NPs The antibacterial activity of P. longum fruit is also well established. To evaluate the antibacterial effect of AgCI-NPs, they were tested against four different bacterial species at different concentrations in this study. AgNPs were used at concentration against Bacillus cereus and Staphylococcus aureus and against Bacillus cereus, Pseudomonas aeruginosa and Bacillus subtilis. The antibiotic streptomycin was used as a standard in this assay. The zone of inhibition produced by AgCI-NPs in plates containing bacterial lawns was compared with plates treated with standard alone [27]. As could be observed 7
in Table 1, the antibacterial effect of AgCI-NPs was almost two fold higher than that of control alone. To confirm the potential antibacterial effect of AgCI-NPs, bacillus cereus culture was treated with AgCI-NPs at a high concentration 75 µL and its effect on the growth pattern of bacteria was analyzed. The AgCI-NPs caused disturbance in the growth behaviour of bacteria by increasing the lag phase time and by decreasing the number of viable cells in the log phase suggesting the toxic effect of AgCI-NPs against bacterial strains [28]. 4. Conclusion We have demonstrated an eco-friendly, easy and rapid method for biosynthesis of silver chloride nanoparticles using leaf extract of M. citrifolia. This leaf extract act as both reducing as well as capping agent and thus avoiding the use of harmful chemicals, which creates the serious environmental issue. TEM morphology confirmed that the size of nanoparticles is 12 nm with spherical morphology. The phyto-synthesized AgCI-NPs showed that they act as a redox catalyst and restrain the capability to degrade methylene blue dye. Acknowledgement We thank the management of VIT University for providing all research facilities to carry out this work mainly VIT-TBI, DST-FIST for providing XRD facility to VIT for characterization
of
the
nanoparticles
and
also
we
thankful
to
DBT
(No.BT/PR6891/GBT/27/491/2012) for providing financial support to carry out research process. References [1] [2] [3] [4] [5] [6] [7]
[8] [9]
S.M. Roopan, T.V. Surendra, G. Elango, S.H.S. Kumar, Appl. Microbiol. Biotechnol. 98 (2014) 5289–5300. S. Ashokkumar, S. Ravi, V. Kathiravan, S. Velmurugan, Spectrochim Acta A 121 (2014) 88–93. J.F. Hamilton, Adv. Phys. 37 (1988) 359–441. M.Y. Wang, B.J. West, C.J. Jensen, D. Nowicki, Acta. Pharmacol. Sin. 23 (2002) 1127-1141. A.D. Pawlus, D.A. Kinghorn, J. Pharm. Pharmacol. 59 (2007) 1587–609. S. Sang, X. Cheng, N. Zhu, M. Wang, J.W. Jhoo, R.E. Stark, V. Badmae, G. Ghai, R.T. Rosen, J. Nat. Prod. 64 (2001) 799-800. S.M. Roopan, A. Haritha, A. Prabhakarn, A.A. Rahuman, K. Velayutham, G. Rajakumar, R.D. Padmaja, M. Lekshmi, G. Madhumitha, Spectrochim. Acta A. 98 (2012) 86–90. B.N. Kannan, S. Natarajan, Mater. Res. Bull. 46 (2011) 1708–1713. R. Kumar, S.M.Roopan, A. Prabhakarn, V.G. Khanna, S. Chakroborty, Spectrochim. Acta A. 90 (2012) 173–176. 8
[10] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Colloid. Surface B. 76 (2010) 50–56 [11] R.M. Tripathia, K. Nishant, S. Archana, S. Priti, B.R. Shrivastav, J. Mol. Catal B. Enzyme. 96 (2013) 75– 80. [12] H. Bar, D.K Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Colloid Surface A 339 (2009) 134–139 [13] M.R. Bindhu, M. Umadevi, Spectrochim Acta A 121 (2014) 596–604. [14] T. Chandan, H. Moushumi, B. Manobjyoti, R. Manash, Mater Lett 102–103 (2013) 1–4. [15] C. Dipankar, S. Murugan, Colloid Surface B 98 (2012) 112–119. [16] S.P. Dubeya, M. Lahtinen, M. Sillanpaa, Colloid Surface A 364 (2010) 34–41 [17] K. Jeba, T.E. Immanuel, M.G. Sethuraman, Process. Biochem. 47 (2012) 1351– 1357. [18] S. Kaviya, J. Santhanalakshmi, B. Viswanathan, J. Muthumar, K. Srinivasan, Spectrochim. Acta A. 79 (2011) 594–598. [19] D.A. Kumar, V. Palanichamy, S.M. Roopan, Spectrochim. Acta A. 127 (2014) 168–171 [20] D. Mandal, M.E. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol. Biotechnol. 69 (2006) 485–492. [21] S.M. Roopan, G. Madhumitha, A.A. Rahuman, C. Kamaraj, A. Bharathi, T.V. Surendra, Ind. Crop. Prod.43 (2013) 631–635. [22] Y.S. Salprima, N. Doni, A. Eka, S. Totok, H. John, N. Yuta, Mater. Lett. 97 (2013) 181–183 [23] J.Y. Song, B.S .Kim, Bioprocess. Biosyst. Eng. 32 (2009) 79–84. [24] V.S. Suvith, P. Daizy, Spectrochim. Acta A. 118 (2014) 526–532. [25] F.Z. Mervat, W.H. Eisa, A.A. Shabaka, Spectrochim. Acta A. 98 (2012) 423–428. [26] T. Chandan, H. Moushumi, B. Manobjyoti, R. Manash Das, Mater Lett., 1-4 (2013)102–103. [27] K. Muthu, R.M. Rangasamy, Mater. Lett. 97 (2013) 141–143. [28] M. Thiruna, U. Balaji, S. Behera, P.K. Panda, B.K. Mishra, Spectrochim. Acta A. 116 (2013) 424–427.
9
Table 1 Inhibition zones of biosynthesized AgCI-NPs
Concentration Sample
(µL)
S. aureus
B. cereus
P. aeruginosa
B. subtilis
Diameter of zone (Cm)
Streptomycin 25 AgCl NPs 25 50 75
2.2 1.2 1.4 1.5
2.1 1.4 1.6 1.7
10
2.4 1.6 1.5 1.3
2.5 1.5 1.5 1.4
List of figures captions Fig. 1 Preparation of M.citrifolia leaf extract Fig. 2 AgCl-NPs (a) UV-visible absorption spectrum; (b) FT-IR spectra; (c) XRD pattern Fig. 3 TEM images of biosynthesized AgCl-NPs Fig. 4 Mechanism for the production of AgCl-NPs Fig. 5 Photocatalytic activity of biosynthesized AgCl-NPs using methylene blue dye
11
100 mL of double distilled H2O
10 – 15 min reflux
10 g leaf of M. citrifolia
M. citrifolia extract
Fig. 1
12
a
b
13
(200) 3000
c
2500
Intensity
2000
(220)
AgCl (111)
1500
1000
Ag (111) 500
(311) (222)
(200)
(220)
(400)
(420)
0 10
20
30
40
50
60
2 Theta
Fig. 2
14
70
80
Fig. 3
15
Ag+ +
AgNO3 (aq)
Cl
+ Ag+
NO3-
AgCl + RT
M. citrifolia aqueous leaf extract
M. citrifolia aqueous leaf extract
Fig. 4
16
Fig. 5
17
Research Highlights
AgNPs were prepared using M. citrifolia extract.
XRD pattern evidence for crystalline nature of AgNPs.
Biomolecules which is present in leaf extact act as capping agent.
AgNPs may provide potential applications as good catalytic activity.
18
S1011-1344(14)00203-6 http://dx.doi.org/10.1016/j.jphotobiol.2014.06.011 JPB 9776
To appear in:
Journal of Photochemistry and Photobiology B: Biology
Received Date: Revised Date: Accepted Date:
9 May 2014 16 June 2014 18 June 2014
Please cite this article as: D. Ashok Kumar, V. Palanichamy, S.M. Roopan, Photocatalytic action of AgCl nanoparticles and its antibacterial activity, Journal of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.06.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Photocatalytic action of AgCl nanoparticles and its antibacterial activity a,
Deenadayalan Ashok Kumara, V. Palanichamy * Selvaraj Mohana Roopanb,* a
School of Bio Sciences & Technology, bChemistry Research Laboratory, Organic
Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632 014, Tamil Nadu, India. Corresponding
Author:
*Email:
[email protected];
[email protected]; [email protected]; Tel: 09865610356; +91 04162 202352; fax: +91 416 224 5544/5766
Abstract The scientific community is searching for biosynthetic methods for the production of metallic nanoparticles. Biogenic pathway has now become a vast developing area of research. A novel route biological synthesis of silver chloride nanoparticles (AgCI-NPs) using aqueous leaf extract of M. citrifolia under ambient conditions were evaluated. Synthesized nanoparticles were confirmed by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and Transmission Electron Microscopy (TEM). The effects of pH on biosynthesis of AgCI-NPs were investigated using UV-vis spectroscopy. TEM images showed that the diameter of stable AgCI-NPs were approximately 12 nm. FTIR spectra provide the evidence for the presence of protein as possible biomolecules responsible for reduction and capping of nanoparticles. The synthesized AgCI-NPs were observed to have a good catalytic activity on the reduction of methylene blue (MB) dye by M. citrifolia extract which has been confirmed by decrease in absorbance maximum values of methylene blue with respect to time using UV-vis spectroscopy and was attributed to the electron relay effect. Keywords AgCl, Biosynthesis, Morinda citrifolia leaf, Electron microscopy, Catalytic activity
1. Introduction Nanoparticles synthesis is an evergreen research field of 21st century in which the connotation of the bio-mediated experimental process is highly important. Biosynthesis of nanoparticles has an emerging highlight of the intersection of nanotechnology and biotechnology. It has received increasing attention due to a growing need to develop rapid, clean, nontoxic, simple and environmentally friendly synthetic technologies. The 1
significance of such a synthetic protocol has been well demonstrated [1]. Biosynthesis of silver chloride nanoparticles using biological systems, such as plants extract has gained a great importance in recent years due to using of less harmful, biodegradable and cost effective reagents [2]. The biosynthesis of silver chloride nanoparticles has an emerging highlight due to the variety of application especially photochemical and biomedical applications. Also it has been used for the fabrication of photographic paper and photochromic lenses [3]. The important significance of biosynthetic protocol has been well demonstrated in our present work. Morinda citrifolia is one of the traditional folk medicinal plants in Southeast Asia, also known as noni [4]. Noni has been reported for a broad range of medical applications including antibacterial, antiviral, antifungal, antitumor, analgesic, hypotensive, antiinflammatory, and immune enhancing effects [5]. The primary usage of noni is to apply leaves as a traditional topical treatment thought to enhance wound healing. The leaf contains flavanol glycosides, β-carotene and iridoid glycosides. Noni leaf extract was capable of promoting wound healing in an animal model. In addition, the crude extract of noni leaf has been traditionally used in patients with bone fractures or dislocation to promote tissue repair and decrease inflammation. In addition, Joseph Betz a biochemist in FDA evaluated Morinda citrifolia had been analysis for a number of biological activities in animal models and antimicrobial studies. The fresh leaf is used for the treatment of wounds and also as a poultice for broken bones and sprains in most parts of India. However the detailed information regarding the wound healing activity of M. citrifolia is not scientifically documented [6]. In the present investigation an attempt was made to assess the effect of Morinda citrifolia leaf extract on silver chloride nanoparticles production. Followed by catalytic activity of the AgCI-NPs were evaluated with the help of methylene blue reduction using UV-vis spectroscopy. AgCI-NPs can act as a good catalyst due to the high Fermi potential. It can also catalyze much reduction reaction. The rate constant of the catalytic reaction is found to depend on the size of the nanoparticles.
2. Experimental section 2.1 Materials
2
The healthy leaves of Morinda citrifolia was collected in and around of Vellore town in Tamilnadu, India. Silver nitrate (AgNO3) were purchased form Merck (India). The reagents used in all the experiments were purely analytical grade. 2.2 Preparation of M. citrifolia aqueous extract The freshly leaves of M. citrifolia was washed several times with double distilled water. About 10 g of leaves were added to 100 mL of deionized water and boiled for 5-10 min in a water bath. The aqueous mixture was then filtered through Whatman filter paper No.1 to obtain aqueous extract (Fig. 1). The aqueous extract was stored in refrigerator at 4 ºC. The aqueous extracts were used as reducing as well as stabilizing agent [7]. 2.3 Biosynthesis of silver chloride nanoparticles using leaf extract In this study, the leaves extract acts as a capping agent for the biosynthesis of AgCI-NPs. In a typical biosynthesis of AgCI-NPs, 1 mL of aqueous leaves extract was added to 10 mL of 1 mM AgNO3 aqueous solution and kept in a dark place at room temperature. After few minutes, the colorless reaction mixture changed to dark brown color that indicates the formation of AgCI-NPs [8]. The dry powders of the silver chloride nanoparticles were obtained as follows; after the desired reaction period; aqueous mixture containing AgCI-NPs was centrifuged at 10,000 rpm for 10 min. The centrifugation and re-dispersion in double distilled deionized water were repeated three times [9]. 2.4 Effect of pH on phyto-synthesis of AgCI-NPs This pH parameter is an important factor for any phyto-synthesis reaction, especially for biochemical reaction. The effect of pH values of AgNO3 solution on the phyto-synthesis of AgCI-NPs were evaluated using various pH parameters (5.0, 6.0, 7.0, 8.0 and 9.0) with fixed aqueous concentration of AgNO3 (1 mM). The pH value was adjusted by 0.01 M HCl and 0.01 M NaOH solution. The effect of pH values were analyzed by UV-vis spectroscopy. 2.5 Effect of volume on phyto-synthesis of AgCI-NPs The concentration of precursor is also an important significant parameter for AgCI-NPs synthesis. The concentration of leaf extract was optimizing by varying the different volume of leaf aqueous extract (1 to 5 mL). The effect of leaf aqueous extract was evaluated using UV-vis spectroscopy. 2.6 Characterization of silver chloride nanoparticles The bio-reduced AgCI-NPs were monitored using a UV–vis spectrophotometer. The UV–vis spectrum was recorded on a Perkin-Elmer (Lambda 25 Model) UV–vis 3
spectrophotometer and the sample was measured in the wave- length region of 200–800 nm. The morphological studies of the phyto-synthesized AgCI-NPs were viewed by TEM (HITACHISU-6600 model) instrument [10]. The green phyto-synthesized AgCINPs were centrifuged at 10,000 rpm for 15 min to obtain the residue. The residual part of AgCI- NPs were washed with deionized water and dried in a hot air oven at 60 ºC for 24 h. The powdered AgCI-NPs were analyzed by a Bruker model D8 Advanced Powder Xray diffractometer. The intensity data for the nano silver powder were collected in the 2θ range 20–80º and the scanning speed at 0.02 min per degree. The green phyto-synthesis of AgCI-NPs were recorded by JASCO FTIR spectrophotometer using KBr pellet in the range of 400–4000cm-1 with the spectra resolution of 4 cm-1. 2.7 Photocatalytic activity of AgCI-NPs In order to assess the catalytic activity of phyto-synthesized AgCI-NPs, two reactions were carried out in a 3.5 mL capacity quartz cuvette and absorbance values were monitored using UV–visible spectrophotometer [11]. In the first reaction, 1 mL of methylene blue (1 ×10−4 M) was mixed with 0.2 mL of aqueous leaf extract and 1.8 mL of water, this reaction mixture was monitored for 30 min. In second reaction, 1 ml of methylene blue (1×10−4 M) was mixed with 0.2 mL leaf extract and 2 mL of synthesized silver nanoparticles (AgCI-NPs) and this reaction mixture was monitored at three different time intervals viz., 30, 45 and 60 min. In all the reactions total volume of the mixture was made up to 3 mL.
The values of absorption maxima (λ max) were
compared, with that of methylene blue. 2.8 Antimicrobial properties of AgCI-NPs The antibacterial activities of AgCI-NPs were evaluated by using the Kirby–Bauer method. Nutrient broth/Agar (1 g beef extract, 1 g peptone, 0.5 g NaCl dissolved in 100 ml of double distilled water) was used for culturing the bacteria. The Nutrient agar plates were inoculated with cultures of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa allowed for overnight growth at 30 °C. Sterile Whatman filter paper discs of 5 mm diameter was impregnated with 25 µL, 50 µL and 75 µL of AgCI-NPs and were placed on to the bacterial lawn in agar plates [12].
Standard
streptomycin antibiotic discs were used as a reference drug. The agar plates were incubated at 37 °C for 24 h. After 24 h of incubation, the zone of inhibition was measured. 4
3. Results and discussion 3.1 UV-vis spectroscopy analysis The biosynthesis of AgCI-NPs using leaf extract was monitored by UV-vis spectroscopy. It is well known that AgCI-NPs were exhibit maximum UV-vis absorption in the range of 400-500 nm [13]. The formations of AgCI-NPs were preliminarily confirmed by UVvisible spectral analysis of colored solutions that exhibit within 5 h. A strong small shift of absorbance toward the higher wavelength from 420 to 430 nm (Fig. 2a) was seen with increase of time. The inset in indicates the variation of absorbance at λmax with respect to time. In the present investigation, AgCI-NPs were rapidly formed after the addition of M. citrifolai leaf aqueous extract. The bioreduction reaction turned dark brown colour after 4-5 h. A wide absorption spectrum peak of UV-visible range indicates that the distribution of smaller sized nanoparticles will be higher in the reaction mixture [14]. From this current investigation it revealed that the formations of AgCI-NPs were depends on the pH parameter of the bioreduction reaction and the rate of AgCI-NPs were formed with high intensity in basic pH (7.0) than in acidic pH. The formations of AgCI-NPs were occurred quickly in neutral and basic pH which may be due to the ionization of the phytoconstituents present in the leaf aqueous extract of M. citrifolia. The absorbance spectrum peak was located at 424 nm in pH 7.0 and the rate of AgCI-NPs formation was slow reduction reaction at acidic pH condition. The present study was extended to evaluate the AgCI-NPs formation using M. citrifolia leaf extract at different pH level. In final conclude that leaf extract is responsible for the reduction in AgNO3 to form AgCINPs and suitable pH condition is 7.0. [15] Similarly we demonstrated different concentration ratios of leaf aqueous extract and silver nitrate solution were also optimized for high yield production of silver nanoparticles.
In this study different
volume of leaf extract from 1 to 5 mL added to 1 mM AgNO3 solution was turned to brown colour with in 30 min of incubation period, indicating rapid formation of AgCINPs. Thus the optimized leaf aqueous medium supported the maximum amount formation of silver chloride nanoparticles (AgCI-NPs) and the reaction was occurred very fast and rapidly.
3.2 Fourier transforms infrared spectroscopy (FTIR) The FTIR spectroscopy analysis was carried out to identify the biomolecules responsible +
for reduction of Ag into Ag0 and capping of the AgCI-NPs phyto-synthesized by leaf
5
extract (Fig.2b) [16]. The bands at 557 cm-1 is corresponds to C-CI stretch (alkyl halides). The band at 1068 cm-1 is assigned to be C-N stretch which is found to be aliphatic amines group. The absorption bands at 1384 cm-1 are characteristics of C-H alkyl halides group. The band at 1402 cm-1 is assigned to the aromatic group stretching. The band at 1598 cm-1 is occurred -C=C- stretch in alkenes group. The absorption spectrum peak at 2827 cm-1 is showed C-H stretching in aldehydes group. The peaks located at 3174cm-1, 3332 cm-1 and 3444cm-1 corresponds to stretching vibrations of =CH (alkene) and N-H stretching vibrations of amines, amides and O-H stretching vibrations in phenol groups. The present study well known that bio-constituents interact with silver ions salts and mediate reduction process with functional groups.
The current
investigation FTIR spectrum analysis the presence of phenol groups, amines, amides, alkene, aldehydes, aromatic, alkyl halides, and aliphatic amines groups in the leaf aqueous extract of M. citrifolia are responsible for capping agents in AgCI-NPs formation [17].
3.3 X-Ray diffraction analysis (XRD) Fig.2c shows the XRD pattern of AgCI-NPs evaluated in the present research study. After bioreduction reaction, the diffraction peaks at 2θ = 28.01, 32.08º, 38.16º, 46.17º, 54.91º, 57.40º, and 76.68º assigned to the (111), (200), (220), and (311) intensity planes of a faced centre cubic lattice of AgCI-NPs (Fig.2c) [18-20]. However, the diffraction peaks are formed which indicate that crystallite size obtained was matched with database of Joint Committee on Powder Diffraction Standard (JCPDS) file no. 99-101-1241. The minor peaks are due to the presence of silver nanoparticles [21]. The average size of the AgCI-NPs formed in the bioreduction reaction was evaluated using Debye-Scherrer formula, D=Kλ/βs cos θ where ‘D’ is particle diameter size, k is a constant equal 1, ‘λ’ is wavelength of X-ray source, ‘β’ is the full width half maximum (FWHM) and ‘θ’ is the diffraction angle corresponding to lattice plane.
3.4 Transmission electron microscopy (TEM) The biosynthesized AgNPs were analyzed by TEM to find the information of morphology and size of nanoparticles [22]. A drop of AgCI-NPs sample solution synthesized by leaf extract was deposited onto a carbon coated copper grid. The TEM images of the AgCI-NPs are depicted in Fig.3. The result was comparable with reported 6
data and the size of the AgNPs is controlled within 12 nm by capping and stabilizing agent [23].
3.5 Mechanism for the formation of AgCI-NPs Biosynthetic pathways responsible for the production of metal nanoparticles using plants are yet to be elucidated. Keeping in this mind we decided to propose the mechanism for the production of AgCl-NPs (Fig.4). The chlorine ion which is present in the leaf will react with Ag(0) to form AgCl. It has been confirmed through powder XRD. Hence, content of leaf extract contains chlorine could be most attributed source for the formation of AgCl-NPs [24-25].
3.6 Photocatalytic activity of AgCI-NPs The catalytic activities of phyto-synthesized AgCI-NPs were analyzed using methylene blue. In the electron transfer step, there can be the restriction of passage of electrons when there is a large redox potential difference between acceptor and donar. Electron transfer becomes easy when an effective catalyst has intermediate redox potential value between acceptor and donor. Here, we have reported the reduction of methylene blue by the aqueous extract of M. citrifolia containing AgCI-NPs (Fig.5). The absorbance value of methylene blue was recorded at 665 nm. After 30 min of mixing of plant aqueous extract into methylene blue, it was observed that the absorbance peak of methylene blue is decreased slowly and shifted to higher wavelength. But the reaction containing methylene blue, AgCI-NPs and extract, showed remarkable decrease in absorbance peak after 30 and 45 min. This decrease in absorbance peak indicates that the AgCI-NPs have potential to degrade methylene blue [26].
3.7 Antimicrobial activity of AgCl-NPs The antibacterial activity of P. longum fruit is also well established. To evaluate the antibacterial effect of AgCI-NPs, they were tested against four different bacterial species at different concentrations in this study. AgNPs were used at concentration against Bacillus cereus and Staphylococcus aureus and against Bacillus cereus, Pseudomonas aeruginosa and Bacillus subtilis. The antibiotic streptomycin was used as a standard in this assay. The zone of inhibition produced by AgCI-NPs in plates containing bacterial lawns was compared with plates treated with standard alone [27]. As could be observed 7
in Table 1, the antibacterial effect of AgCI-NPs was almost two fold higher than that of control alone. To confirm the potential antibacterial effect of AgCI-NPs, bacillus cereus culture was treated with AgCI-NPs at a high concentration 75 µL and its effect on the growth pattern of bacteria was analyzed. The AgCI-NPs caused disturbance in the growth behaviour of bacteria by increasing the lag phase time and by decreasing the number of viable cells in the log phase suggesting the toxic effect of AgCI-NPs against bacterial strains [28]. 4. Conclusion We have demonstrated an eco-friendly, easy and rapid method for biosynthesis of silver chloride nanoparticles using leaf extract of M. citrifolia. This leaf extract act as both reducing as well as capping agent and thus avoiding the use of harmful chemicals, which creates the serious environmental issue. TEM morphology confirmed that the size of nanoparticles is 12 nm with spherical morphology. The phyto-synthesized AgCI-NPs showed that they act as a redox catalyst and restrain the capability to degrade methylene blue dye. Acknowledgement We thank the management of VIT University for providing all research facilities to carry out this work mainly VIT-TBI, DST-FIST for providing XRD facility to VIT for characterization
of
the
nanoparticles
and
also
we
thankful
to
DBT
(No.BT/PR6891/GBT/27/491/2012) for providing financial support to carry out research process. References [1] [2] [3] [4] [5] [6] [7]
[8] [9]
S.M. Roopan, T.V. Surendra, G. Elango, S.H.S. Kumar, Appl. Microbiol. Biotechnol. 98 (2014) 5289–5300. S. Ashokkumar, S. Ravi, V. Kathiravan, S. Velmurugan, Spectrochim Acta A 121 (2014) 88–93. J.F. Hamilton, Adv. Phys. 37 (1988) 359–441. M.Y. Wang, B.J. West, C.J. Jensen, D. Nowicki, Acta. Pharmacol. Sin. 23 (2002) 1127-1141. A.D. Pawlus, D.A. Kinghorn, J. Pharm. Pharmacol. 59 (2007) 1587–609. S. Sang, X. Cheng, N. Zhu, M. Wang, J.W. Jhoo, R.E. Stark, V. Badmae, G. Ghai, R.T. Rosen, J. Nat. Prod. 64 (2001) 799-800. S.M. Roopan, A. Haritha, A. Prabhakarn, A.A. Rahuman, K. Velayutham, G. Rajakumar, R.D. Padmaja, M. Lekshmi, G. Madhumitha, Spectrochim. Acta A. 98 (2012) 86–90. B.N. Kannan, S. Natarajan, Mater. Res. Bull. 46 (2011) 1708–1713. R. Kumar, S.M.Roopan, A. Prabhakarn, V.G. Khanna, S. Chakroborty, Spectrochim. Acta A. 90 (2012) 173–176. 8
[10] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P.T. Kalaichelvan, N. Mohan, Colloid. Surface B. 76 (2010) 50–56 [11] R.M. Tripathia, K. Nishant, S. Archana, S. Priti, B.R. Shrivastav, J. Mol. Catal B. Enzyme. 96 (2013) 75– 80. [12] H. Bar, D.K Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Colloid Surface A 339 (2009) 134–139 [13] M.R. Bindhu, M. Umadevi, Spectrochim Acta A 121 (2014) 596–604. [14] T. Chandan, H. Moushumi, B. Manobjyoti, R. Manash, Mater Lett 102–103 (2013) 1–4. [15] C. Dipankar, S. Murugan, Colloid Surface B 98 (2012) 112–119. [16] S.P. Dubeya, M. Lahtinen, M. Sillanpaa, Colloid Surface A 364 (2010) 34–41 [17] K. Jeba, T.E. Immanuel, M.G. Sethuraman, Process. Biochem. 47 (2012) 1351– 1357. [18] S. Kaviya, J. Santhanalakshmi, B. Viswanathan, J. Muthumar, K. Srinivasan, Spectrochim. Acta A. 79 (2011) 594–598. [19] D.A. Kumar, V. Palanichamy, S.M. Roopan, Spectrochim. Acta A. 127 (2014) 168–171 [20] D. Mandal, M.E. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol. Biotechnol. 69 (2006) 485–492. [21] S.M. Roopan, G. Madhumitha, A.A. Rahuman, C. Kamaraj, A. Bharathi, T.V. Surendra, Ind. Crop. Prod.43 (2013) 631–635. [22] Y.S. Salprima, N. Doni, A. Eka, S. Totok, H. John, N. Yuta, Mater. Lett. 97 (2013) 181–183 [23] J.Y. Song, B.S .Kim, Bioprocess. Biosyst. Eng. 32 (2009) 79–84. [24] V.S. Suvith, P. Daizy, Spectrochim. Acta A. 118 (2014) 526–532. [25] F.Z. Mervat, W.H. Eisa, A.A. Shabaka, Spectrochim. Acta A. 98 (2012) 423–428. [26] T. Chandan, H. Moushumi, B. Manobjyoti, R. Manash Das, Mater Lett., 1-4 (2013)102–103. [27] K. Muthu, R.M. Rangasamy, Mater. Lett. 97 (2013) 141–143. [28] M. Thiruna, U. Balaji, S. Behera, P.K. Panda, B.K. Mishra, Spectrochim. Acta A. 116 (2013) 424–427.
9
Table 1 Inhibition zones of biosynthesized AgCI-NPs
Concentration Sample
(µL)
S. aureus
B. cereus
P. aeruginosa
B. subtilis
Diameter of zone (Cm)
Streptomycin 25 AgCl NPs 25 50 75
2.2 1.2 1.4 1.5
2.1 1.4 1.6 1.7
10
2.4 1.6 1.5 1.3
2.5 1.5 1.5 1.4
List of figures captions Fig. 1 Preparation of M.citrifolia leaf extract Fig. 2 AgCl-NPs (a) UV-visible absorption spectrum; (b) FT-IR spectra; (c) XRD pattern Fig. 3 TEM images of biosynthesized AgCl-NPs Fig. 4 Mechanism for the production of AgCl-NPs Fig. 5 Photocatalytic activity of biosynthesized AgCl-NPs using methylene blue dye
11
100 mL of double distilled H2O
10 – 15 min reflux
10 g leaf of M. citrifolia
M. citrifolia extract
Fig. 1
12
a
b
13
(200) 3000
c
2500
Intensity
2000
(220)
AgCl (111)
1500
1000
Ag (111) 500
(311) (222)
(200)
(220)
(400)
(420)
0 10
20
30
40
50
60
2 Theta
Fig. 2
14
70
80
Fig. 3
15
Ag+ +
AgNO3 (aq)
Cl
+ Ag+
NO3-
AgCl + RT
M. citrifolia aqueous leaf extract
M. citrifolia aqueous leaf extract
Fig. 4
16
Fig. 5
17
Research Highlights
AgNPs were prepared using M. citrifolia extract.
XRD pattern evidence for crystalline nature of AgNPs.
Biomolecules which is present in leaf extact act as capping agent.
AgNPs may provide potential applications as good catalytic activity.
18