diatomite nanocomposite by electron beam irradiation

Author’s Accepted Manuscript SYNTHESIS AND CHARACTERIZATION OF SILVER/DIATOMITE NANOCOMPOSITE BY ELECTRON BEAM IRRADIATION Truong Thi Hanh, Nguyen Thi Thu, Le Anh Quoc, Nguyen Quoc Hien www.elsevier.com/locate/radphyschem

PII: DOI: Reference:

S0969-806X(16)30406-6 http://dx.doi.org/10.1016/j.radphyschem.2017.04.004 RPC7514

To appear in: Radiation Physics and Chemistry Received date: 21 September 2016 Revised date: 7 April 2017 Accepted date: 12 April 2017 Cite this article as: Truong Thi Hanh, Nguyen Thi Thu, Le Anh Quoc and Nguyen Quoc Hien, SYNTHESIS AND CHARACTERIZATION OF SILVER/DIATOMITE NANOCOMPOSITE BY ELECTRON BEAM I R R A D I AT I O N , Radiation Physics and Chemistry, http://dx.doi.org/10.1016/j.radphyschem.2017.04.004 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 galley proof before it is published in its final citable 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.

SYNTHESIS AND CHARACTERIZATION OF SILVER/DIATOMITE NANOCOMPOSITE BY ELECTRON BEAM IRRADIATION Truong Thi Hanh*, Nguyen Thi Thu, Le Anh Quoc, Nguyen Quoc Hien Research and Development Center for Radiation Technology, Vietnam Atomic Energy Institute, 202A, Street 11, Linh Xuan Ward, Thu Duc District, Ho Chi Minh City, Viet Nam *

Corresponding author. Tel: +84 8 62829159; fax: +84 8 38975921. E-mail address:

[email protected] (Truong Thi Hanh)

ABSTRACT

Silver nanoparticles (AgNPs) with diameter about 9 nm were deposited on diatomite by irradiation under electron beam of diatomite suspension containing 10 mM AgNO3 in 1% chitosan solution, at the dose of 20.2 kGy. The AgNPs/diatomite nanocomposite was characterized by UV-Vis spectroscopy, TEM image and energy dispersive X-ray spectroscopy (EDX). The antibacterial activity of the AgNPs/diatomite against E. coli and S. aureus was evaluated by reduction of bacterial colonies on spread plates and inhibition zone diameter on diffusion disks. Keywords: Electron beam, silver nanoparticles, diatomite, nanocomposite, chitosan, antibacterial

1. Introduction Nanocomposites are composed by nanosized particles in a matrix such as polymer or inorganic substrate (Hussain et al., 2006; Pomogailo, 2005). These materials were improved in the characteristics of both constituents including nanoparticles and matrix. Nanoparticles received attention in the field of material as well as chemistry by high surface area reactivity and homogeneous physicochemical properties (He et al., 2014). Especially, AgNPs were concerned by the high antibacterial activity besides the other features. They inhibited the growth of bacteria, attacked and disrupted cell membranes of harmful bacteria such as Staphylococcus aureus (S. 1

aureus), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli)... (Jone & Hoek, 2010). Diatomite (DA) is an attractive material for industrial applications because of its low cost, well-defined porosity, small particles size, high thermal stability and high adsorption capacity (Lin & Lan, 2013). The present of diatomite in composition can provide the useful characteristics, so that it has been used as a filter aid, thermal insulator, absorbent and carrier (Reza et al., 2015). Modification of diatomaceous earth by agents containing Schiff base (N=CH-) and phenolic groups improved the adsorption of Ag+ ions on diatomite (Fatoni et al., 2010). Natural diatomite was also prepared for adsorption of radionuclides of Cs-137, Cs-134 and Co-60 from the liquid waste (Osmanlioglu, 2007). Furthermore, diatomaceous earth has been used as filter aids for removing impurities from beer without affecting the color, flavor or quality of beer (Martinovic et al., 2006). TiO2-diatomite photocatalysts were prepared for degradation of methyl orange dye under UV and visible-light irradiation (Xia et al., 2014). In recent years, the AgNPs have been incorporated into matrices such as ZnO, zeolite or silica in diatomite (SiO2.nH2O), ceramics induce the antibacterial activity or use for other purposes. The antibacterial effects of Ag/SiO2 nanocomposite against E. coli, S. aureus and Bacillus were reported by Singh et al. (2015). Lv (2014) proposed a new method for fixing AgNPs to the surface of porous ceramic by chemical bonding using an aminosilane coupling as a connecting bridge. The AgNPs remain tightly fixed to the surface of ceramic material so that they can contain a sufficient quantity of AgNPs for antibacterial treatment in drinking water purification. The nanocomposite of AgNPs/SiO2 core shell particles was fabricated by the Stöbe method in which silica was directly coated over the AgNPs core with addition of tetraethyl orthosilicate (TEOS). These nanocomposite particles of AgNPs core and silica shell were shown to be stable up to 1000oC and may be utilized as catalyst at high temperature (Chou & Chen, 2007). The advantage of radiolytic reduction method, over the other methods, is possibility to obtain a homogeneous distribution of synthesized AgNPs within polymer matrix as well as to control their size by changing the experimental conditions (Krstić et al., 2014). The obtained material is clean and sterilized at the same time, which is important to use nanocomposite as a decontaminant. In this study, AgNPs were immobilized on the diatomite by in situ synthesis. Silver ions were reduced to silver atoms by electron beam (EB) irradiation and simultaneously deposited on the 2

diatomite. The antibacterial activity of AgNPs/DA nanocomposite against E. coli and S. aureus was also investigated. 2. Experimental 2.1.Materials The diatomite powder was purchased from Phu Yen Diatomite Company (Vietnam) with over 63% SiO2, median particle size about 10-100 µm. Chitosan with a degree of deacetylation about 80% and Mw = 1.06 × 105 was prepared as reported previously (Hanh et al., 2014). All other chemicals, including silver nitrate (AgNO3), (S)-lactic acid (90%), sodium hydroxide (NaOH) were of reagent grade. Distilled water was used in all experiments. 2.2.Preparation of AgNPs/DA nanocomposite by EB-irradiation The diatomite samples were immersed in hydrochloric acid 3 % (v/v) at room temperature for 1 hour for demineralization and were washed with distilled water. Then samples were dried in an oven at 110oC. Next, an amount of 10 g DA was dispersed in 50 ml of 10 mM AgNO3 in 1% chitosan solution providing sufficient stabilizer with an acceptable solution viscosity at 30oC (Hanh et al., 2014; He et al., 2014). The mixture was stirred vigorously for 1 hour, and then suspension containing AgNO3/DA/chitosan was packed in polyethylene bag with thickness about 1 cm. Then samples were irradiated in air under an electron beam of UELR-10-15S2 linear accelerator (Russia), 10 MeV energy with beam current of 1.5 mA at absorbed doses of 6.5, 13.4, 20.2 and 25.9 kGy (dose rate of 5 kGy/s) measured by the Radiochromic film dosimeter (ASTM International, 2004). After that, the resultant powders of AgNPs/DA were rinsed with distilled water to remove all unbound particles from the surface, and then dried at 80oC for further experiments. 2.3. Characteristics of AgNPs/DA nanocomposite The UV-Vis spectra of the colloidal AgNPs were recorded on a Jasco V-630 spectrophotometer in the range from 200-600 nm after the solution was diluted 20 times with distilled water. EDX spectra were carried out on a SEM-EDX instrument – SEM S4800 (Hitachi, Japan) combined with a H7593 (Horiba, England) X-ray analyzer. Transmission electron microscopy (TEM) images were performed with a JEOL (Japan), JEM-1400 electron microscope at an accelerated voltage of 100 kV. The content of silver nano in AgNPs/diatomite nanocomposite was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Perkin Elmer Optima 5300 DV, USA). 3

2.4. Antibacterial efficacy of AgNPs/DA nanocomposite The antibacterial activity of the prepared AgNPs/DA sample against E. coli (ATCC 6538) and S. aureus (ATCC 25923) was evaluated using spread-plate method on LB agar Petri plate with some modifications (Ma et al., 2015). The tested bacterial suspension was prepared with 1×108 CFU/ml (CFU: colony forming units) of E. coli and added to 99 ml LB medium in each conical flask. Then 1 gram of AgNPs/DA nanocomposite was introduced into aqueous suspension of bacteria with the content of AgNPs about 10 mg/L. Flasks were shaken vigorously for 30 min, serial dilutions were made. From each of three suitable dilutions, 0.1 ml liquid was spread on LB agar plates, then incubated at 37oC for 24 h. Percent reduction of bacteria was evaluated by calculating the number of bacterial colonies on agar plates of sterilized water (control) and AgNPs/diatomite. The disk diffusion experiment was performed by placing spherical paper disks saturated with control, blank DA and AgNPs/DA nanocomposite on LB agar plates cultured E. coli or S. aureus. And then, the antibacterial effects are evaluated by measuring the inhibition zone diameter after plates were incubated at 37oC for 24 h. 3. Results and discussion 3.1. Preparation of AgNPs/DA nanocomposite Firstly, diatomite was treated with 1% hydrochloric acid (v/v) in order to reduce mineral then washed and dried at 110oC before stirring with AgNO3/chitosan solution. The aqueous ammonia was used as base to adjust the pH of suspension to 5-6. Reyad et al. (2003) and Caliskan et al (2011) reported that the pHzpc (isoelectric point) of diatomite was ~ 4 so the diatomite surface may be negatively charged above pHzpc. The electrophilic silver ions (Ag+) were adsorbed on the surface and embedded in amorphous silicon dioxide (SiO2) with the nucleophilic oxide groups (SiO─) by stirring formation of suspension before irradiation (Lawless et al., 1994). The •

•

solvated electrons (e-aq) with Eo (H2O/e-aq = - 2.87 VNHE and H radicals with Eo (H+/ H) = - 2.3 VNHE that were generated by water radiolysis can reduce adsorbed Ag+ ions to Ag atoms by irradiation effects in suspension solution (Eo(Ag+/Ag) = - 1.8 V). Continuous reduction of the Ag+ ions causes the aggregation into AgNPs cluster (Eo (Ag+n/Agn) = 0.79 VNHE) (Linnert et al., •

1990). Moreover, in chitosan solution, the radiolytic OH groups from irradiated water coordinate with chitosan by hydrogen abstraction to form macromolecule free radicals (R•1). At a slight acid medium pH 5-6, hydronium ion H3O+ represents an important scavenger for solvated •

electrons e-aq which are converted to H radicals (k = 2.3 × 107 m3.mol-1.s-1). These radicals also 4

contribute to reduction of Ag+ in chitosan solution to Ag0, so the value of total reduction yield (Gred) is unchanged and close to maximal reduction yield (Gred.max) in theory (Hanh et al. 2016). In this study, the reaction mechanism for the reduction of adsorbed Ag+ on silica (SiO2) in suspension of AgNO3/chitosan/DA by electron irradiation can be proposed as follows: Ag+

+

(SiO─)

H2O (EB) e-aq

+

•

→ +

Si-O•••Ag+

+

R(chitosan)

+

Si-O•••Ag+

+

nSi-O•••Ag0R1

•

H

+ H2 O

e-aq (•H) •

OH (•H)

→ →

R•1 →

(1)

OH + e-aq + •H + H2O2 + H2 + …

→

H3O

Si-O•••Ag+

→

Si-O•••Ag2R2

7

3

(2) -1 -1

k = 2.3 × 10 m .mol .s

(3)

Si-O•••Ag0

(4)

R•1 (H abstraction) +

H2O (H2)

(5)

→

Si-O •••Ag0R1

(6)

→

Si-O•••AgnRn

(7)

The binding of silver clusters with chitosan as well as with silica surface of diatomite are achieved through the Ag─O─Si bonds. Besides, the protonated amine groups (R─NH3+) of chitosan in acidic medium can stabilize silver clusters by electrostatics repulsions (Chen et al., 2007). The absorbed dose plays an important role in formation and growth of AgNPs by EBirradiation in situ synthesis in which Ag+ ions in AgNO3/chitosan/DA suspension were reduced to Ag0 and simultaneously immobilized on diatomite. Fig. 1 clearly indicated that the content of AgNPs in nanocomposite gradually increased with dose from 6.5 to 20.2 kGy. Above 20.2 kGy (25.9 kGy), the AgNPs content coordinated with DA changed insignificantly and obtained an approximate efficiency about 0.11% from ICP-AES analysis. This data is similar to the results of EDX analysis for elemental components in AgNPs/DA nanocomposite (Table 1). An amount of unbound Ag onto DA was removed by washing samples after irradiation. 3.2. Characterizations of AgNPs/DA nanocomposite Fig. 2 shows SEM images of DA and AgNPs/DA samples. The image revealed the blank porous structure of DA in Fig. 2a. On the other hand, the morphology of AgNPs/DA indicated that the particles were loaded into pores of DA which could be attributed to the presence of silver nano (Fig. 2c). The elemental compositions of DA are determined by EDX spectrum in Fig. 2b including peaks of O, Na, Al and Si at 0.5, 1.1, 1.5 and 1.8 keV, respectively. The EDX spectrum of AgNPs/DA nanocomposite depicted a new peak of Ag at 3 keV besides similar elements to 5

component of DA (Fig. 2d). Results in Table 1 also showed the weight and atomic percentages of Ag in AgNPs/DA nanocomposite in comparison with DA. The UV-vis spectra of the AgNPs colloidal solution in the AgNO3/DA suspensions after EBirradiation were shown in Fig. 3. The AgNPs colloidal solutions depicted peak maxima (

max)

in

the range from 417-422 nm with respective increase in absorbance with EB doses from 6.5 to 20.2 kGy. Above 20.2 kGy (25.9 kGy) the absorbance changed insignificantly. Therefore, the dose of 20.2 kGy was optimal for complete conversion of the 10 mM AgNO3 to AgNPs in DA suspension with 1% chitosan solution at ambient temperature. Chitosan was not only stabilizer of AgNPs but also binder of AgNPs on diatomite through the intermolecular hydrogen bonds. According to previous studies on diatomaceous structure, the hydroxyl groups are primary reactive sites on the surface of diatomite (Yuan et al., 2004; Ibrahim et al., 2012). These functional groups can bind with hydroxyl (─OH) or amino (─NH2) groups of chitosan which are able to form the intermediate connection between Ag0 and DA. The shape and dispersion of AgNPs on the surface of DA are shown by TEM image of the AgNPs. This nanocomposite was prepared by EB irradiation of 10mM AgNO3/diatomite in 1% chitosan solution at the dose of 20.2 kGy. The TEM image in Fig. 4a shows the AgNPs with spherical morphology that are well dispersed without aggregation on the surface of DA. In addition, a narrow size distribution of AgNPs/chitosan also indicated that AgNPs are homogeneous at experimental conditions (Fig. 4b). The size, shape and the formation rate of the metal nanoparticles which have been synthesized by irradiation depend on the irradiation dosage, the concentration of initial component and other conditions (Pattabi et al., 2009). The presence of AgNPs in SiO2 matrix demonstrated that the nanocomposite of AgNPs/DA can be synthesized by in situ reduction of AgNO3 in DA suspension containing SiO2 under EB irradiation. The average diameter of the AgNPs prepared at the dose of 20.2 kGy was determined from TEM image to be 9.3 nm. The advantage of embedding AgNPs in DA matrix was dispersion of AgNPs through silica avoiding their aggregration, so small sized AgNPs with a typical diameter between 1-10 nm were formed (Egger et al., 2009). 3.3. Antibacterial efficacy of AgNPs/DA nanocomposite Fig. 5 shows the test results for antibacterial efficacy against E. coli and S. aureus of samples, namely the control, the blank DA and the AgNPs/DA nanocomposite. The control specimens show the growth of bacteria on the LB nutrient substrate in Fig. 5Aa and 5Ad for E. coli and S. 6

aureus, respectively. The bacterial counts decreased after a contact time of 30 min with all samples containing DA which adsorbed bacteria into porous DA (Fig. 5Ab, 5Ac, 5Ae and 5Af). Especially, the nanocomposite of AgNPs/DA inhibited remarkably the growth of E. coli (Fig. 5Ac) and S. aureus (Fig. 5Af). The survival percentages of E.coli and S. aureus on LB agar for the specimens of AgNPs/DA in comparison with control were about 40 and 43 % respectively. Antibacterial efficacy can be enhanced when improvement in the reaction conditions, for example, prolongation of contact time between sample and bacterial suspension because of deep adsorption into DA of AgNPs. The bacterial reduction is clearly visible from observation of inhibition zone diameter in Fig. 5B for the AgNPs/DA samples both E. coli and S. aureus. The mechanism for bacterial reduction of AgNPs was proposed by Jone and Hoek (2010) that the AgNPs interact with the bacterial membrane and are able to penetrate inside the cell leading to increasing permeability and disturbing respiration. In this work, E. coli and S. aureus were selected as the objects of research with above experimental conditions. The next work, we are going to observe the inhibitory potential of AgNPs/DA nanocomposite against major fish pathogens such as bacteria of Edwardsiella ictaluri, Aeromonas hydrophila that threaten to the development of aquaculture. According to expert reports, nanotechnology plays an important role in providing new materials for water filtration and purification, and approaches to reduce the pathogeneous bacteria for fish in aquaculture system (Handy & Shaw, 2007; Muhling et al., 2009). Therefore, using nano materials for water disinfection in fishpond increased yields and survivals of fish (Huang et al., 2015). The porous silica particles can be used as a drug delivery matrix, for example, controlling release of substances as fish vaccines in aquaculture environment (Stromme et al., 2009). In this study, diatomite containing about 60% silica will be a suitable substrate bearing AgNPs to reduce contaminative bacteria for further testings in fishponds. Example data on the lethal concentrations (LC50) values for 48 - 96 h of NPs in fishes and invertebrates are mostly in the mg or tens of mg l−1 range (Handy, 2012). These values depicted the low acute toxicity for many materials NPs as Ag, Cu, Zn, so immediate threats to aquaculture systems and fisheries of NPs may be very small. Arora et al. (2009) investigated interactions of AgNPs with mammalian cells, the results showed that cell morphology of primary fibroblasts and liver cells remained unaltered in the presence of AgNPs with concentrations of 25 µg/ml and 100 µg/ml, respectively. Similar studies were done by Gopinath et al. (2008) on the baby 7

hamster kidney and human colon adenocarcinoma cell lines where necrosis starts at silver nanoparticles concentrations > 44 μg/ml. Oral administration of AgNPs at a limited dose of 5,000 mg/kg was neither mortality nor acute toxic signs for mice, substances with LD50 values greater than 5,000 mg/kg body weight are considered as low toxicity (Maneewattanapinyo et al., 2011) Controlling a suitable concentration of AgNPs for the treatment of pathogeneous bacteria in fishponds is based on the ecotoxicity data for aquaculture environment and concentration for safety to mammalian cells and higher level for safety warrants of consumer health. In this study, the AgNPs concentration was tested against cultured bacteria at a high density (108 CFU/ml), but in practice, bacterial contamination concentration in fish ponds is much lower compared with in vitro test. Thus, priority concentration should be selected for in vivo testing to be lower than 1 mg/L, a safety limitation for ecosystem and for mammalian and human cells. In vitro experiments for antibacterial effect of Edwardsiella ictaluri, Aeromonas hydrophila as well as in vivo testings in catfish ponds in Vietnam will be carried out. Moreover, the release of silver from AgNPs/DA nanocomposite should be considered. In the near future, we hope that AgNPs/DA material will be used for treatment of the pathogenous bacteria in fishponds of culture fish in Vietnam. 4. Conclusion The silver ions were reduced to atoms by EB irradiation and simultaneously deposited on the diatomite by in situ synthesis. The AgNPs content in diatomite was about 0.11% when irradiation of suspension containing diatomite in 10mM AgNO3/1% chitosan at the dose of 20.2 kGy. The AgNPs in diatomite were confirmed by UV-Vis spectra, SEM and TEM images. The average diameter of AgNPs on DA was determined to be about 9 nm. Antibacterial efficacy of the AgNPs/DA nanocomposite against E. coli and S. aureus was clearly demonstrated by reduction of bacterial growth in comparison with control. The other bacteria which are serious pathogens for fishes in aquaculture will be tested by using AgNPs/DA nanocomposite for further assays.

8

Acknowledgments The authors acknowledge funding in part for this work by Research and Development Center for

Radiation Technology, Vietnam Atomic Energy Institute.

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Captions- Figures Fig. 1. Effect of absorbed dose on the content of AgNPs in nanocomposite after removal of unbound silver on diatomite Fig. 2. SEM images and EDX spectra respectively for the diatomite (a, b) and AgNPs/DA nanocomposite (c, d) at the dose of 20.2 kGy Fig. 3. UV-Vis spectra of colloidal AgNPs from 10mM AgNO3/DA suspension diluted 20 times corresponding to absorbed doses of: (a) 6.5 kGy, (b) 13.4 kGy, (c) 20.2 kGy and (d) 25.9 kGy Fig. 4. TEM image (a) and particle size distributions (b) of AgNPs at the dose of 20.2 kGy Fig. 5. Photographs of antibacterial test results against E. coli and S. aureus: A) The survival colonies of E. coli (a, b, c) and S. aureus (d, e, f) on LB agar: Sterilized water- Control (a, d), Blank diatomite (b, e) and AgNPs/DA nanocomposite (c, f). B) inhibition zone E. coli and S. aureus for the samples LB (Sterilized water - Control), DA and AgNPs/DA

12

Table 1. Elemental percentages of the AgNPs/DA nanocomposite Material

Element

Diatomite

AgNPs/diatomite

O

Na

Al

Si

Ag

Total (%)

Weight %

57.63

1.76

1.63

38.98

100

Atomic %

70.25

1.49

1.18

27.08

100

Weight%

57.70

1.64

1.95

38.60

0.11

100

Atomic %

71.07

1.38

1.38

25.87

0.3

100

Highlights 

AgNPs/DA was synthesized in situ by EB irradiation of Ag+/chitosan/DA suspension.



The AgNPs/DA was characterized by TEM image, UV-Vis spectra and EDX analysis



The AgNPs/DA inhibited remarkably the growth of E. coli and S. aureus

13

AgNPs content in nanocomposite (%)

0.14

0.12 0.1

0.08 0.06 0.04 0.02 0 0

10

20 Dose (kGy)

Fig. 1

14

30

40

Fig. 2

Fig. 3

50

b Frequency, %

40

30 20

10 0

6

8

10

12 d, nm

Fig. 4 15

14

16

18

Fig. 5

16