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Ag+ driven antimicrobial activity of Ag+: ZnO nanowires immobilized on paper matrices
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Ag+ Driven Antimicrobial Activity of Ag+ :ZnO Nanowires Immobilized on Paper Matrices Sudiksha Aggrawal , Tapas Kumar Mandal , Paritosh Mohanty PII: DOI: Reference:
S2589-1529(19)30286-8 https://doi.org/10.1016/j.mtla.2019.100490 MTLA 100490
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Materialia
Received date: Accepted date:
12 July 2019 26 September 2019
Please cite this article as: Sudiksha Aggrawal , Tapas Kumar Mandal , Paritosh Mohanty , Ag+ Driven Antimicrobial Activity of Ag+ :ZnO Nanowires Immobilized on Paper Matrices, Materialia (2019), doi: https://doi.org/10.1016/j.mtla.2019.100490
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Ag+ Driven Antimicrobial Activity of Ag+:ZnO Nanowires Immobilized on Paper Matrices Sudiksha Aggrawal,a Tapas Kumar Mandal,a and Paritosh Mohanty*a a
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee - 247667,
Uttarakhand, India *E-mail: [email protected], [email protected] Tel: +91-1332-284859 Graphical abstract
Abstract The prevalence of infectious disease causing microbes in the environment is a worldwide critical concern. This is prevalent both in air and water bodies. Thus, fabricating cellulose based materials with superior antimicrobial activity is highly significant in day-to-day life. Herein, Ag/Ag+ doped zinc oxide nanowires have been immobilized on the cellulose fibers of the paper matrices using facile one step scalable autoclave technology. The incorporation of Ag+ in the ZnO lattice has indicated a synergy in the antimicrobial activity. The present work has demonstrated significant enhancement in antimicrobial activity with increased Ag+ 1
contents at the Zn sites of the wurtzite ZnO. Within a short time period of 2 h only, the Escherichia coli (E. coli) count of the order of 108 has been deactivated under the exposure of visible light. The synthesized paper matrices have also resisted the degradation even in the presence of Gloeophyllum trabeum (G. trabeum), a cellulose eating fungus. The paper remains intact even after the incubation for 7 days, which is unprecedented. The thorough characterization of the paper matrices along with their superior antimicrobial activities makes them a potential candidate for applications in healthcare and to produce pathogen free safe drinking water using a cheap point of use system. Keywords: G. trabeum, Ag/Ag+ doped ZnO, immobilization on cellulose, E. coli, antimicrobial activity
Statement of Significance The objectives of the current research are to demonstrate a methodology to immobilize Ag+ doped ZnO nanostructures on the surface of the cellulose fibers in the paper matrices without using linkers, binders or retention aid. Immobilization of metal oxides on the cellulose surface of paper matrices remains a challenge owing to the mismatch in the polarizability. Moreover, the role of Ag and Ag+ in the ZnO lattice and how these effect the antimicrobial activity are the central theme of the research. The synthesized paper matrices have deactivated E. coli (CFU~ 108) on 2h exposure of visible light. Moreover the specimen have also desisted the degradation of cellulose for 7 days even in the presence of G. trabeum.
1. Introduction The emergence of antibiotic resistant microbes commonly known as “superbugs” is most threatening in recent times [1]. As stated in the recent report in the journal “Nature”, an 2
estimated 700,000 people worldwide are being killed by the “superbugs” every year and the number could reach as high as 10 million by 2050 if proper steps are not taken [2]. These “superbugs” could further endangered human life to such an extent where the common medical procedures may not be performed because of the risk of the “superbugs” infection [3]. To add more into this problem, the activation of dormant microbes due to the melting of glacier owing to the global warming further pose a threatening scenario [4]. To add more to the worry, the research required to develop antibiotics for the “superbugs” is not very impressive because of the economic prospects [2]. One approach to overcome the spread of these bacteria is to deactivate them in the transmission medium such as water bodies [5]. The recent approach towards this is to use various metals and metal oxides to clean the waterbodies. Metal oxides such as TiO2 [6,7], CuO [8,9], Fe2O3 [10], Bi2O3 [11], ZnO [12,13], etc. and metals such as Ag [14-17], Au [18,19], Cu [20,21] etc. have been explored. Among these, ZnO have gained a lot of attention owing to its superior activity towards broader class of microbes, non-toxic to mammalian body, biocompatibility and costeffectiveness [12-14]. Moreover, control of microstructure in ZnO is comparatively easier that provides a pathway to further investigate the efficiency with respect to the microstructure [12]. Although, the antibacterial activity of ZnO has been investigated for various bacteria, but as documented in various research articles and even text books, the Escherichia coli (E. coli) as a model bacterium has mostly been studied [23]. Silver (Ag) is the most widely used metal for antibacterial studies since the ancient times. Even, in the 18th century Ag was used for treating the ulcers [24,25]. With the evolution of the nanotechnology, the focus on investigation of antibacterial activity of Ag nanostructures of various sizes and shapes have risen to a greater extent [24,25]. The Ag deactivates both Gram positive and Gram negative bacteria, however, is most effective against gram negative bacteria. This may be attributed to the larger thickness of peptidoglycan in the cell membrane in Gram positive bacteria [26].
3
Interestingly, due to the absence of peptidoglycan in mammalian cells, the Ag toxicity is restricted to the microbes and absent in mammals [26]. According to USEPA and WHO, the permissible limits of Ag in safe drinking water is 0.1 mg/L. On continuous exposure of Ag above this limit for a long time could cause argyria, a skin disorder [27,28]. Although, the fungi are not as threatening as the bacteria but few of them are pathogenic to the human [29]. However, they are more pathogenic towards plant. In this regard, G. trabeum, a fungus is considered as one of the main culprit to destroy the cellulose based products [30,31]. Paper, being prepared from cellulose, is highly prone to the G. trabeum attack [30]. This adversely affects its wide spread use in storing information, maps and ledgers, books in libraries, secure and long term documentation work etc. [32]. Thus, preserving paper and other cellulose based products is an important task, which needs a special attention. Various methods employed for the preservation and improvement of longevity of the cellulose materials includes but not limited to storing in sterile environments [33], gamma irradiation [34], and de-acidification etc. [32]. For these, metals and metal oxides such as Ag, Cu, ZnO, TiO2, CuO etc. have been incorporated into the paper matrices [35,36]. Among these, Ag and ZnO have shown better efficiency as compared to their peers. As discussed above, both Ag and ZnO have shown excellent antimicrobial activities and their combination have a synergy on the antimicrobial activity [37-39]. The synergic effect not only observed in the nanostructures but also in the bulk material [39]. Although, the exact mechanism for this observation is not known, however, one of the most acceptable reason is the annihilation of the native defects in ZnO on the doping Ag [37,38]. One of the major drawbacks of using the nanostructures for the antimicrobial activity is the recovery or separation of the active nanomaterials from the waterbodies [36]. On many occasions, ultra-small nanoparticles could show superior activity, but their separation becomes very difficult as neither filtering nor centrifugation could separate the suspended 4
nanoparticles from the solution [36]. In order to overcome these issues, nanostructures could be incorporated or immobilized in various substrates such as glass, ceramic, and paper etc. making nanocomposite type of materials that could be removed rather easily from the water bodies after the deactivation [36]. Paper, being a natural biopolymer, bio degradable, cheap, abundant is a very good alternative. Various metal and metal oxide nanostructures have been incorporated in the paper matrices for imparting their high end applications such as catalysis and photocatalysis, antimicrobial, and sensing etc. [36]. Although, the incorporation of the nanostructures in the paper matrices could circumvent a lot of issues with respect to the separation of the active materials, however, still the challenge exists to some extent as the retention of the ultra-small nanostructures in paper matrices needed linkers or binders [36]. The additional cost and accessibility of surface active sites of nanostructures hinder the two major drawbacks that limits their practical application. The recent invention by our research group on the development of the binder or linker free one step method for immobilization of the nanostructures on the cellulose surface of the paper matrices have opened up a new research avenue. The mechanism of the immobilization of the nanostructures through the formation of covalent bond between cellulose fibers and metal oxides have been documented [40-45]. Very importantly, this newly developed methodology do not allow any leaching of nanostructures from the cellulose surface during the experiments. Moreover, these can be stored in ambient condition for years. This leaching free methodology is very much beneficial as it has been recently reported that the microorganisms are sensitive to metal toxicity and almost 100 % pristine soil microbes are also killed by the leached metals to the soil [18]. In this article, the immobilization of Ag/Ag+ doped ZnO nanowires on the cellulose fiber surface of the paper matrices is reported. The paper matrices have shown a superior antimicrobial activity by deactivating the E. coli, a Gram negative bacterium in a short exposure time of 2 h and resisting the G. trabeum, a cellulose eating fungus to degrade the
5
paper matrices for one week. A mechanism is proposed for the observation of the extraordinary antimicrobial activity by using the information obtained from various analytical techniques. One of the very important observation of the reported method is the leaching free use of the nanostructures for the deactivation of bacteria and fungus. 2.
EXPERIMENTAL
2.1.
Materials
Silver nitrate (AgNO3, BDH Laboratories, India), Zinc chloride, (ZnCl2, Rankem RFCL Ltd., India), Sodium carbonate (Na2CO3, Rankem RFCL Ltd., India), ethanol (99.9 % pure, Merk), Luria Bertani (Himedia laboratories Pvt. Ltd., India), and bleached softwood pulp (Star Paper Mill Ltd., India) were procured. All the reagents were used as such without any purification. The E. coli (MTCC no. 1698) and G. trabeum (MTCC No. 3168) were procured from Microbial Type Culture Collection and Gene Bank, Chandigarh, India. 2.2.
Synthesis
Paper matrices immobilized with varying ZnO and Ag contents are prepared using a single step hydrothermal method followed by the paper making using the standard Handsheet making method following the TAPPI test method 205 sp-02 [40-45]. Typically, 1.2 g softwood pulp was dispersed in 40 mL deionized water. To it, different precursors were added as per the predetermined metal oxide content. The detailed precursor recipe required for the synthesis of individual specimen is given in Table 1. This was followed by a hydrothermal treatment at 120 °C using a 100 mL Teflon lined stainless steel autoclave for 12 or 18 h. The sample was cooled naturally to room temperature. The obtained cellulose matrices were washed several times alternatively with hot water and ethanol. The pulp was dried and further re-dispersed in water to prepare the paper matrices using standard
6
Handsheet making procedure in the British Sheet Former and dried in the MG drier for 3 h at 80 °C. Table 1. Composition of reagents added in the synthesis of nanostructures immobilized paper matrices.
2.3.
S.
Sample
AgNO3 ZnCl2 Na2CO3
Temp
Time
N.
ID's
(g)
(g)
(g)
(ᵒC)
(h)
1.
P
-
-
-
120
12
2.
AgP
0.0188
-
-
120
12
3.
ZnP
-
0.4
40
120
12
4.
AgZnP∙12
0.0188
0.4
40
120
12
5.
AgZnP∙18
0.0188
0.4
40
120
18
Characterization
The compositional investigation of metal and metal oxide present in the paper matrices has been carried out by standard combustion test [40-45] and TGA. Heating a paper matrix in air at a temperature over 500 °C would result in the decomposition followed by oxidation of the elements present in it, resulting in a complete mass loss. These experiments are carried out normally in furnaces. In the present work, a known weight of the paper matrix was kept in a crucible and placed in a muffle furnace. The furnace was ramped to a temperature of 520 °C with a heating rate of 10 °C per min and kept at that temperature for 4 h. It was then cooled down naturally below 50 °C and the remaining mass if any was collected. The difference in the weight of the sample used for the combustion test before and after heating could provide the amount of inorganic contents present in the specimen. The TGA experiments were carried out by heating the specimens in EXSTAR TG/DTA6300 with a heating rate of 5 °C per min in an argon flow of 200 sccm. We have tried to perform similar TGA experiments in air, but
7
at a particular temperature (~300 °C) the whole paper matrices get burnt with a short time period leading to an erroneous result. The phase analysis was performed in a Rigaku Ultima IV XRD using CuKα radiation (λ = 1.5405 Å) at a scanning speed of 4°/min in the range of 10 to 70° of the 2 scale. The obtained diffraction patterns were compared with the standard JCPDS (Joint Committee on Powder Diffraction Standards) files. Lattice parameters were refined by least-squares method using the PROSZKI program. The XPS analysis was carried on PHI-5000 VersaProbe III, ULVAC-PHI INC. The specimens were placed on the carbon tape and kept in ultra-high vacuum of 10-7 torr for 4 h in the sample introduction chamber. Then the samples were inserted into the main chamber and XPS data was recorded. The AlKα was used as X-ray source with energy of 1486.6 eV. The peaks were referenced with C 1s at binding energy of 284.8 eV. The microstructure is studied using FESEM (FEI operating at 10 kV) and TEM (TECNAI G2S-TWIN operating at 200 kV). Before FESEM investigation, the paper matrices owing to their non-conducting nature were coated with gold using a standard gold sputtering unit. Sample preparation for the TEM was carried out by collecting the nanowires from the surface of the paper matrices and dispersed in methanol using ultra-sonication. A drop of the dispersion was placed in the holey carbon coated copper grids and dried at room temperature. The leaching of metal and metal ions have been studied using ICP-OES (Teledyne Leeman Labs, Prodigy SPEC).
2.4.
Antimicrobial activity
The antibacterial activity in the presence of visible light was studied by inhibiting the growth of E. coli using the prepared ZnO, Ag and ZnO-Ag nanostructures immobilized paper matrices. The experiment was carried out in triplicate and standard deviation in log reduction 8
of E. coli is calculated. The experiment was carried out by following the standard protocol ISO 20743 with certain modifications. The paper matrices were first cut in the dimension of 8 cm × 1 cm. These were then put in individual test tubes filled with 10 mL of E. coli suspension. The test tubes were then incubated for 2 h at 37 °C under the exposure of compact fluorescent lamp (1.69 J/cm2). Serial dilutions of bacteria containing suspension was done and 100 μl of each dilution was spread on LB agar in petri-plates. The plates were incubated at 37 °C for 24 h. Colonies of the bacteria are counted and results are explained in form of % reduction and log reduction values. For the study of antifungal activity, prior grown fungal colony is picked using loop and transferred to potato dextrose agar plate. Paper matrices cut in the size of 2 cm × 2 cm and are sterilized using autoclave. These were then placed on the petri-plates containing the fungal colonies. The petri-plates were incubated at 28 °C for 7 days and investigated for the fungal growth. 3. RESULTS AND DISCUSSION The immobilization of ZnO nanowires (NWs) doped with Ag/Ag+ on the surface of the cellulose fibers has been carried out by a one-step hydrothermal method at 120 °C for 12 h using ZnCl2∙5H2O and AgNO3 as ZnO and Ag sources, and commercially available softwood pulp as cellulose source. In general, the extent of immobilization of the metal oxides in paper matrices is estimated by combustion test and thermogravimetric analysis (TGA) [40-43]. On heating the paper matrices in air above 520 °C, there is a complete decomposition and oxidation of organic matters leaving behind the inorganic components [40-43]. The paper specimens prepared in this research have been heated at 525 °C in air for 4 h in a muffle furnace. For comparison, blank paper matrix designated as specimen P has also been employed for an identical combustion. As expected, a complete mass loss (remaining ash content < 0.004 wt%) was observed in specimen P indicating the absence of any inorganic component. However, the combustion of paper matrices immobilized with only Ag/Ag+ 9
(designated as specimen AgP) and ZnO (designated as specimen ZnP) have remaining masses of 0.5 and 10.0 wt%, respectively. The combustion of Ag/Ag+ doped ZnO immobilized paper matrix (specimen AgZnP∙12) has shown to have leftover mass of 11.0 wt%. The paper matrix prepared by a prolonged hydrothermal treatment of 18 h (specimen AgZnP∙18) at 120 °C keeping all other experimental conditions same, was also having the same 11.0 wt% inorganic components as estimated by the combustion experiment. Table 2. Retention of inorganic content in paper matrices estimated by combustion test and TGA analysis of the specimens in argon atmosphere. Sample ID
Mass remaining (%) Combustion
TGA in Ar
P
0.004
12.0
AgP
0.5
12.0
ZnP
10.0
21.5
AgZnP∙12
11.0
21.2
AgZnP∙18
11.0
22.7
A similar conclusion can be drawn from the TGA investigations of the specimen heating up to 600 ºC in an argon atmosphere with a heating rate of 5 ºC/min as shown in Fig. 1. Around 12 % mass was left after heating the specimen P and AgP, which can be attributed to the leftover carbon. No appreciable mass difference can be observed in these two specimens similar to the combustion experiment. Moreover, remaining masses of 21.5, 21.2 and 22.7 wt% in ZnP, AgZnP∙12 and AgZnP∙18 specimens, respectively, corroborate the finding (Table 2).
10
100
P AgP ZnP AgZnP.12 · AgZnP.18
60
24 22 20
P AgP ZnP AgZnP.12 AgZnP.18
40
18 16
Weight (%)
Weight (%)
80
14 12
20
500
520
540
560
580
10 600
Temperature (C)
0
100
200
300
400
500
600
Temperature (C)
Fig. 1. TGA thermograms of ZnO and Ag+:ZnO NWs immobilized paper matrices in argon atmosphere. The phase analysis of the specimens was investigated by XRD (Fig. 2). Two broad peaks at d-values of 5.823 and 3.896 Å observed in all the samples are attributed to the (101) and (002) reflections of cellulose-I [40-45]. In addition, peaks at d-values of 2.832, 2.609, 2.488, 1.919, 1.632, 1.484 and 1.385 Å observed for the ZnP sample correspond to the (100), (002), (101), (102), (110), (103) and (112) reflections of hexagonal ZnO with the wurtzite structure. The refined lattice parameters of 3.258(1) and 5.209(5) Å are in agreement with the reported data for hexagonal ZnO (JCPDS PDF File No. 79-205). The XRD data for AgZnP∙12 indicates doublet feature for all the reflections corresponding to hexagonal ZnO structure, reflecting formation of two admixed ZnO phases. While the refined lattice parameters [3.246(1) and 5.199(2) Å] with one set of reflections resembled parent ZnO type phase, the other set indicated lattice expansion [3.311(1) and 5.306(2) Å]. This has been attributed to the incorporation of Ag+ in the ZnO lattice for the latter phase leading to lattice expansion due to larger size of Ag+ as compared to Zn2+ [46]. Interestingly, for the AgZnP.18 sample only one set of reflections corresponding to Ag+ incorporated hexagonal ZnO are observed and the value of refined lattice parameters [3.287(1) and 5.259(3) Å] are in between the two ZnO phases of the AgZnP∙12 sample. Moreover, the peaks due to fcc metallic Ag are 11
also evident in the XRD pattern of AgZnP∙18 and AgZnP∙12. It is to be noted that the intensity of the metallic Ag peaks increases on prolonging the reaction from 12 hours to 18 hours. The absence of metallic Ag peaks in the AgP sample is probably due to presence of very little amount of Ag as compared to cellulose content. However, the presence of Ag is clearly evident from the XPS data analysis. Interestingly, the enhancement in the metallic Ag peaks in AgZnP∙18 sample is attributed to better crystallization and growth of metallic Ag particles on prolonging the reaction. Subsequently, there is a decrease in the hexagonal lattice parameters for AgZnP∙18 to that of the AgZnP∙12. This appears to happen due to the diffusion of Ag+ from the Ag+ rich regions of ZnO (Zn1-xAgxO) to undoped ZnO (representing two admixed ZnO phases of AgZnP∙12) and finally resulting in a homogeneous solid solution of Ag+ doped ZnO (as in AgZnP∙18). Assuming linear Vegard’s law behaviour and using the experimental lattice volume expansion for AgZnP∙12 (5.2%) and AgZnP∙18 (2.6%), it is apparent that the lattice Ag+ incorporation is nearly double in AgZnP∙12 to that of AgZnP∙18.
.
AgZnP.18
AgZnP.12
AgZnP.18 AgZnP.12 ZnP AgP P
ZnP AgP P 10
20
30
40
50
60
70
Diffraction angle 2 (degree)
30
80
(101)
(002)
Intensity (a.u.)
(103)
(b)
(112)
. .
(110)
#
(102)
(100) (002) (101)
Intensity (a.u.)
# Cellulose-I
. Ag FCC
(100)
#
(a)
32
34
36
Diffraction angle 2 (degree)
Fig. 2. (a) XRD patterns of prepared paper matrices. (b) Selected region of the XRD patterns depicting the shift in the (100), (002), and (101) peak positions. In order to further understand the doping of the Ag+ in ZnO, all the specimens have been investigated by XPS. The survey scan of the specimens is given in Fig. 3. As expected, 12
only C1s and O1s peaks at 286.6 and 532.9 eV, respectively, were observed in the specimen P attributed to the cellulose [41]. In the sample AgP [Fig. 3a(ii)], in addition to the C1s and O1s peaks, a weak doublet (arrow marked) could be seen at the binding energy of 368 to 372 eV, which confirms the presence of silver in the specimen. Similarly, in addition to the C1s and O1s peaks, peaks originating from Zn were observed in the ZnP sample and are assigned to various levels as shown in Fig. 3a(iii). However, in specimens AgZnP∙12 and AgZnP∙18, peaks originating from Zn and Ag were observed in addition to the C1s and O1s peaks. To confirm the speciation of individual element present in these specimens, high resolution XPS spectra were recorded. For example, the high resolution spectra of C1s (Fig. 3b) has three peaks at 284.8, 286.5 and 287.7 eV attributed to carbon with one ether link, carbon linked to hydroxyl groups and carbon with two ether links, respectively as shown in Fig. 4. Similarly, two oxygen has been observed in high resolution O 1s XPS spectra of specimen P as shown in Fig. 3c at 533 and 534.8 eV. These could be attributed to the hydroxyl and glycosidic oxygen. A similar O1s spectrum was also observed in the AgP specimen. However, an additional O1s peak could be seen at 531.1 eV in the specimens ZnP, AgZnP∙12 and AgZnP∙18 attributed to the presence oxygen of zinc oxide. It is interesting to note that the shift in the C1s and O1s XPS spectra are consistent with the observation in XRD analysis. The high resolution Ag3d XPS spectra has provided critical information on the oxidation state of silver. As shown in Fig. 3d, all the three specimens AgP, AgZnP∙12 and AgZnP∙18 have two strong peaks assigned to Ag3d3/2 and Ag3d5/2, respectively, with a separation of 5.96 eV which is close to the standard value of separation of 6.00 eV. The Ag3d5/2 peak was observed at 367.87, 367.64 and 367.94 eV in the specimens AgP, AgZnP∙12 and AgZnP∙18, respectively, and are assigned to the Ag+. The presence of the metallic silver Ag0 in these specimens could be obtained on further deconvolution as shown 13
in Fig. 3d. It was observed at 369.15, 368.43 and 368.66 eV in the specimens AgP, AgZnP∙12 and AgZnP∙18, respectively [47]. The quantitative XPS analysis estimates the Ag content of
C1s
Zn3d
Zn3s Zn3p3
AgZnP.12
Ag3d3
AgZnP.18
Intensity (a.u.)
(a)
O1s ZnLMM
Zn2p1 Zn2p3
0.5, 0.5 and 0.52 at% in AgP, AgZnP∙12 and AgZnP∙18, respectively. It is interesting to note
(v) (iv)
ZnP
(iii)
AgP
(ii) P
(i) 1000
800
600
400
200
0
Binding energy (eV)
C1s
290
AgZnP.18
C1s
AgZnP.12
C1s
ZnP
C1s
AgP
C1s
P
288
286
284
Ag3d3/2
Ag3d3/2
AgP
O1s
ZnP
O1s
AgP
O1s
P
534
(e)
Ag3d5/2
AgZnP.18
AgZnP.12
AgZnP.12
532
530
Binding energy (eV)
AgZnP.12
Zn2p3/2
Zn2p1/2
AgZnP.12
Ag3d5/2
Ag3d5/2
Intensity (a.u.)
Intensity (a.u.)
Ag3d3/2
AgZnP.18
O1s
536
Binding energy (eV)
(d)
O1s
(c)
Intensity (a.u.)
Intensity (a.u.)
(b)
Zn2p3/2
Zn2p1/2
Zn2p3/2
ZnP Zn2p1/2
376
374
372
370
368
366
1050
Binding energy (eV)
1040
1030
1020
Binding energy (eV)
Fig. 3. (a) Full scan (Su1s) XPS spectra of the prepared paper matrices. The high resolution XPS spectra of paper matrices showing (b) C1s, (c) O1s, (d) Ag3d and (e) Zn2p3 peaks. 14
that the shifting in the peak position in the Ag3d XPS spectra are in consistent with the observation of XRD. Similarly, two peaks have been observed in high resolution Zn2p XPS spectra as shown in Fig. 3e, assigned to Zn2p3/2 at 1021.4 eV and Zn2p1/2 at 1044.6 eV. The difference between the two is 23.2 eV, which is matching well with the reported literature value of 23.0 eV [48]. A consistent shifting in Zn2p peaks further corroborate the results obtained in XRD investigation.
H
2 H2C
OH 2
2C
C
C H
H
3C
1 C
O OH
C H2C 2
O O
1
O 3C
H
OH
H H
O 1
H
H
1C
OH
2C H
OH
2
H C OH
Fig. 4. Structure of cellulose indicating three chemically different carbon. The microstructural analysis of the specimens was carried out by FESEM and TEM. The typical FESEM images of all the paper matrices are given in Fig. 5. As expected, fibers of diameter in the range of 20-100 m and length extended upto millimetre could be seen in all the specimens. Clean and smooth surface of the fibers was observed in high magnification FESEM image of the specimen P (Fig. 5b). A similar observation was derived in the AgP specimen that was made by immobilizing Ag in the paper matrices. On careful observation, it could be seen that small spherical nanoparticles of Ag with a dimension smaller than 200 nm are present on the surface of the cellulose fibers. The ZnO NWs of diameter 30-100 nm and length of 2-5 m could be seen in the high magnification FESEM images and uniform distribution of the NWs can be seen with the low magnification images of the specimens ZnP, AgZnP∙12 and AgZnP∙18 in Fig. 5. The observation of ZnO NWs immobilized on the surface of the
15
Fig. 5. FESEM images of specimen (a, b) P, (c, d) AgP, (e, f) ZnP, (g, h) AgZnP∙12 and (i, j) AgZnP∙18. 16
cellulose fibers is expected and consistent with our previous report [41]. There is no major difference that can be observed in the microstructures of these specimens.
Fig. 6. (a, c) TEM and (b, d) HRTEM images of AgZnP∙12 and AgZnP∙18, respectively. The corresponding SAED patterns are given in the inset of the TEM images. In order to further get a better insight on the size and nature of the NWs, TEM analysis was performed. Cellulose being too thick (of the order of 20 to 100 m thick) could easily restrict the passage of the electron beams in the TEM, thus, the analysis could not be performed under the available experimental conditions. Rather, the ZnO NWs were scratched and dispersed in the carbon coated copper grid for TEM investigation. ZnO NWs of uniform diameter in the range of 30 to 50 nm could be seen in AgZnP∙12 and AgZnP∙18 specimens, which is consistent with the observation of FESEM. The high resolution TEM images have indicated the single crystallinity nature of the ZnO NWs due to the observation of clear and uniform lattice fringes. The lattice spacing along the longer axis was measured to be 2.597 Å 17
which matches well with the (0002) plane indicating the growth of the NWs in the [0001] direction [48]. This lattice spacing also matches well with the value of 2.609 Å obtained from the XRD pattern. The single crystallinity of the NWs was further confirmed by the observation of regular arrays of the bright spots in SAED patterns of specimens AgZnP∙12 and AgZnP∙18 in Fig. 6. The antimicrobial activity of the prepared paper matrices has been carried out by choosing the most common and widely investigated Gram negative bacterium E. coli [23]. For the antibacterial studies, the colony count methodology is followed as shown in Fig. 7a. An initial bacterial colony counts of ~3.00 × 108 CFU (colony forming unit) per mL was taken for the investigation and the bacterial colonies are exposed to visible light for a short time period of 2 h only. However, in both the specimens AgP and ZnP, the reduction of the colonies is almost same i.e., ~53 % (1.4 × 108 CFU per mL) of the original counts. But in Ag doped ZnO NWs immobilized paper matrix, specimen AgZnP∙12, a reduction of 99.99999 % of the colonies from the original counts could be estimated. To our surprise, the specimen AgZnP∙18 could only achieve a ~74 % reduction of the bacterial colonies (8.0 × 107 CFU per mL) after 2 h of visible light exposure (Fig. 7a and Table 3). Log reduction values estimated for the E. coli growth with standard deviation in triplicate experiments has been carried out and depicted in Fig. 8, which clearly shows the efficiency of the AgZnP∙12 for the deactivation of E. coli. This high value of log reduction is in the higher side as compared to many of the recent reports [49,50]. The cellulose in paper matrices is vulnerable to microbial attack. Among the various microbes, G. trabeum, a cellulase producing wood decaying fungus, has proven to be the worst enemy of paper matrices [30,31]. The degradation mechanism involves the depolymerisation of cellulose and hemicellulose. The already grown G. trabeum fungal colony has been picked up and inoculated to potato dextrose agar (PDA) petri-plates and the 18
sterilized paper matrices have been introduced into it. The petri-plates have been incubated at 28 ºC for 7 days for the growth of the fungus which in turn could degrade the paper matrices. For understanding the extent of degradation, photographs of these petri-plates with various specimens have been compared (Fig. 7b). As expected, a similar trend in the antifungal activity of these paper matrices have been perceived. Neither degradation/damage nor growth of fungus near the paper specimen AgZnP∙12 could be seen even after such a long incubation time of seven days. In all other specimens the extent of damage or degradation of the paper matrices follows the same trend as was observed for the antibacterial activity of these specimens. It has been observed in case of the sample AgZnP∙12, no growth of fungus is present, whereas in all the other samples, fungus have completely attacked the paper matrices. The consistent with the observation of antibacterial and antifungal activity of these specimens with a superior activity realized for the AgZnP∙12 could further be explained in term of the doping and chemical environment of the Ag+ in the ZnO NWs immobilized in the paper matrices.
Fig. 7. Photographs depicting the inhibition of growth of (a) E. coli and (b) G. trabeum. As reported by our group, the metal ion doesn’t leach out from the paper matrices made by the present method even after sonication, have removed the possibility of Ag or Zn leaching during the antimicrobial activity [41]. The ICP-OES investigation of the DI water
19
where the paper matrices were treated confirms the absence of Ag and Zn corroborate our earliest observation. It is important to note that the paper matrices are highly stable as these are not degraded by even the paper eating fungus G. trabeum, was also confirmed when a three years old prepared paper matrices shown the same activity with the newly made paper matrices. Table 3. Percent reduction and log reduction values in growth of E. coli after 2 h of visible light exposure. S. No.
Sample Id
CFU
% Reduction
Log Reduction
1.
P
3.0 × 108
No reduction
0
2.
AgP
1.4 × 108
53
0.32
3.
ZnP
1.4 × 108
53
0.32
4.
AgZnP∙12
4 × 10
99.99999
6.81
5.
AgZnP∙18
8.0 × 107
74
0.55
6.86
7
Log reduction
6 5 4 3 2 1 0 -1
0
P
0.32
0.32
AgP
ZnP
0.56
AgZnP.12
AgZnP.18
Sample Id Fig. 8. Log reduction values in E. coli growth after 2h of visible light exposure including standard deviation. Although, the overall Ag/Ag+ content in all the three specimens AgP, AgZnP∙12 and AgZnP∙18 are very much close to each other as estimated from the XPS investigation, the superior activity of AgZnP∙12 could be explained based on two factors (i) synergy of ZnO 20
and Ag+ and (ii) concentration of Ag+ ion the ZnO lattice [35]. The antimicrobial activity of ZnO and Ag individually is well known and has been documented in large numbers of research articles [12-18,51]. The combination of Ag and ZnO has shown to produce a synergy and could show a much better activity as compared to only ZnO or Ag [52,53]. Until recently, it was commonly accepted that the activity was due to the formation of reactive oxygen species by the Ag and ZnO. It was reported by Duran et al. that the activity of Ag+ is superior as compared to the metallic Ag [18]. This was mainly due to a higher affinity of Ag+ for organic amines, phosphates and thiols [54]. Three steps have been suggested by Marambio-Jones et al. for this superior activity i.e., disruption of ATP (adenine triphosphate) production by silver ions, generation of reactive oxygen species followed by the damage of cell membrane [55]. In this investigation, as the AgZnP∙12 specimen has the maximum Ag+ content (almost double as compared to AgZnP∙18 estimated from the XRD investigation), hence, the observation of superior activity could be expected. Although, a clear antifungal mechanism is not elucidated so far, but it is accepted that the accumulation of excessive carbohydrates and nucleic acids due to self-protecting mechanism of fungal cells against metal and metal oxides which lead to deformation of the fungal structures. It is expected that the increased Ag+ could facilitates the above mentioned mechanism. 4. CONCLUSION In summary, doping of Ag+ in the lattice of ZnO NWs immobilized on cellulose fibers of the paper matrices have shown to improve the antimicrobial activity against E. coli and G. trabeum. In the AgZnP∙12 with Ag+ concentration double as compared to AgZnP∙18 has shown the best performance, although, total Ag0 and Ag+ concentration in these samples are almost same. The underlying chemistry of the immobilization of the metal oxide nanostructures on the surface of cellulose fibers of paper matrices and tuning their application
21
on doping have not only made these paper matrices imperative in academic research purpose but also could open up commercial avenues to disinfect water bodies from microbes. ACKNOWLEDGEMENT The work was financially supported by SERB, New Delhi, Govt. of India with Grant code: EMR/2016/001693. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Ag+ Driven Antimicrobial Activity of Ag+ :ZnO Nanowires Immobilized on Paper Matrices Sudiksha Aggrawal , Tapas Kumar Mandal , Paritosh Mohanty PII: DOI: Reference:
S2589-1529(19)30286-8 https://doi.org/10.1016/j.mtla.2019.100490 MTLA 100490
To appear in:
Materialia
Received date: Accepted date:
12 July 2019 26 September 2019
Please cite this article as: Sudiksha Aggrawal , Tapas Kumar Mandal , Paritosh Mohanty , Ag+ Driven Antimicrobial Activity of Ag+ :ZnO Nanowires Immobilized on Paper Matrices, Materialia (2019), doi: https://doi.org/10.1016/j.mtla.2019.100490
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Ag+ Driven Antimicrobial Activity of Ag+:ZnO Nanowires Immobilized on Paper Matrices Sudiksha Aggrawal,a Tapas Kumar Mandal,a and Paritosh Mohanty*a a
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee - 247667,
Uttarakhand, India *E-mail: [email protected], [email protected] Tel: +91-1332-284859 Graphical abstract
Abstract The prevalence of infectious disease causing microbes in the environment is a worldwide critical concern. This is prevalent both in air and water bodies. Thus, fabricating cellulose based materials with superior antimicrobial activity is highly significant in day-to-day life. Herein, Ag/Ag+ doped zinc oxide nanowires have been immobilized on the cellulose fibers of the paper matrices using facile one step scalable autoclave technology. The incorporation of Ag+ in the ZnO lattice has indicated a synergy in the antimicrobial activity. The present work has demonstrated significant enhancement in antimicrobial activity with increased Ag+ 1
contents at the Zn sites of the wurtzite ZnO. Within a short time period of 2 h only, the Escherichia coli (E. coli) count of the order of 108 has been deactivated under the exposure of visible light. The synthesized paper matrices have also resisted the degradation even in the presence of Gloeophyllum trabeum (G. trabeum), a cellulose eating fungus. The paper remains intact even after the incubation for 7 days, which is unprecedented. The thorough characterization of the paper matrices along with their superior antimicrobial activities makes them a potential candidate for applications in healthcare and to produce pathogen free safe drinking water using a cheap point of use system. Keywords: G. trabeum, Ag/Ag+ doped ZnO, immobilization on cellulose, E. coli, antimicrobial activity
Statement of Significance The objectives of the current research are to demonstrate a methodology to immobilize Ag+ doped ZnO nanostructures on the surface of the cellulose fibers in the paper matrices without using linkers, binders or retention aid. Immobilization of metal oxides on the cellulose surface of paper matrices remains a challenge owing to the mismatch in the polarizability. Moreover, the role of Ag and Ag+ in the ZnO lattice and how these effect the antimicrobial activity are the central theme of the research. The synthesized paper matrices have deactivated E. coli (CFU~ 108) on 2h exposure of visible light. Moreover the specimen have also desisted the degradation of cellulose for 7 days even in the presence of G. trabeum.
1. Introduction The emergence of antibiotic resistant microbes commonly known as “superbugs” is most threatening in recent times [1]. As stated in the recent report in the journal “Nature”, an 2
estimated 700,000 people worldwide are being killed by the “superbugs” every year and the number could reach as high as 10 million by 2050 if proper steps are not taken [2]. These “superbugs” could further endangered human life to such an extent where the common medical procedures may not be performed because of the risk of the “superbugs” infection [3]. To add more into this problem, the activation of dormant microbes due to the melting of glacier owing to the global warming further pose a threatening scenario [4]. To add more to the worry, the research required to develop antibiotics for the “superbugs” is not very impressive because of the economic prospects [2]. One approach to overcome the spread of these bacteria is to deactivate them in the transmission medium such as water bodies [5]. The recent approach towards this is to use various metals and metal oxides to clean the waterbodies. Metal oxides such as TiO2 [6,7], CuO [8,9], Fe2O3 [10], Bi2O3 [11], ZnO [12,13], etc. and metals such as Ag [14-17], Au [18,19], Cu [20,21] etc. have been explored. Among these, ZnO have gained a lot of attention owing to its superior activity towards broader class of microbes, non-toxic to mammalian body, biocompatibility and costeffectiveness [12-14]. Moreover, control of microstructure in ZnO is comparatively easier that provides a pathway to further investigate the efficiency with respect to the microstructure [12]. Although, the antibacterial activity of ZnO has been investigated for various bacteria, but as documented in various research articles and even text books, the Escherichia coli (E. coli) as a model bacterium has mostly been studied [23]. Silver (Ag) is the most widely used metal for antibacterial studies since the ancient times. Even, in the 18th century Ag was used for treating the ulcers [24,25]. With the evolution of the nanotechnology, the focus on investigation of antibacterial activity of Ag nanostructures of various sizes and shapes have risen to a greater extent [24,25]. The Ag deactivates both Gram positive and Gram negative bacteria, however, is most effective against gram negative bacteria. This may be attributed to the larger thickness of peptidoglycan in the cell membrane in Gram positive bacteria [26].
3
Interestingly, due to the absence of peptidoglycan in mammalian cells, the Ag toxicity is restricted to the microbes and absent in mammals [26]. According to USEPA and WHO, the permissible limits of Ag in safe drinking water is 0.1 mg/L. On continuous exposure of Ag above this limit for a long time could cause argyria, a skin disorder [27,28]. Although, the fungi are not as threatening as the bacteria but few of them are pathogenic to the human [29]. However, they are more pathogenic towards plant. In this regard, G. trabeum, a fungus is considered as one of the main culprit to destroy the cellulose based products [30,31]. Paper, being prepared from cellulose, is highly prone to the G. trabeum attack [30]. This adversely affects its wide spread use in storing information, maps and ledgers, books in libraries, secure and long term documentation work etc. [32]. Thus, preserving paper and other cellulose based products is an important task, which needs a special attention. Various methods employed for the preservation and improvement of longevity of the cellulose materials includes but not limited to storing in sterile environments [33], gamma irradiation [34], and de-acidification etc. [32]. For these, metals and metal oxides such as Ag, Cu, ZnO, TiO2, CuO etc. have been incorporated into the paper matrices [35,36]. Among these, Ag and ZnO have shown better efficiency as compared to their peers. As discussed above, both Ag and ZnO have shown excellent antimicrobial activities and their combination have a synergy on the antimicrobial activity [37-39]. The synergic effect not only observed in the nanostructures but also in the bulk material [39]. Although, the exact mechanism for this observation is not known, however, one of the most acceptable reason is the annihilation of the native defects in ZnO on the doping Ag [37,38]. One of the major drawbacks of using the nanostructures for the antimicrobial activity is the recovery or separation of the active nanomaterials from the waterbodies [36]. On many occasions, ultra-small nanoparticles could show superior activity, but their separation becomes very difficult as neither filtering nor centrifugation could separate the suspended 4
nanoparticles from the solution [36]. In order to overcome these issues, nanostructures could be incorporated or immobilized in various substrates such as glass, ceramic, and paper etc. making nanocomposite type of materials that could be removed rather easily from the water bodies after the deactivation [36]. Paper, being a natural biopolymer, bio degradable, cheap, abundant is a very good alternative. Various metal and metal oxide nanostructures have been incorporated in the paper matrices for imparting their high end applications such as catalysis and photocatalysis, antimicrobial, and sensing etc. [36]. Although, the incorporation of the nanostructures in the paper matrices could circumvent a lot of issues with respect to the separation of the active materials, however, still the challenge exists to some extent as the retention of the ultra-small nanostructures in paper matrices needed linkers or binders [36]. The additional cost and accessibility of surface active sites of nanostructures hinder the two major drawbacks that limits their practical application. The recent invention by our research group on the development of the binder or linker free one step method for immobilization of the nanostructures on the cellulose surface of the paper matrices have opened up a new research avenue. The mechanism of the immobilization of the nanostructures through the formation of covalent bond between cellulose fibers and metal oxides have been documented [40-45]. Very importantly, this newly developed methodology do not allow any leaching of nanostructures from the cellulose surface during the experiments. Moreover, these can be stored in ambient condition for years. This leaching free methodology is very much beneficial as it has been recently reported that the microorganisms are sensitive to metal toxicity and almost 100 % pristine soil microbes are also killed by the leached metals to the soil [18]. In this article, the immobilization of Ag/Ag+ doped ZnO nanowires on the cellulose fiber surface of the paper matrices is reported. The paper matrices have shown a superior antimicrobial activity by deactivating the E. coli, a Gram negative bacterium in a short exposure time of 2 h and resisting the G. trabeum, a cellulose eating fungus to degrade the
5
paper matrices for one week. A mechanism is proposed for the observation of the extraordinary antimicrobial activity by using the information obtained from various analytical techniques. One of the very important observation of the reported method is the leaching free use of the nanostructures for the deactivation of bacteria and fungus. 2.
EXPERIMENTAL
2.1.
Materials
Silver nitrate (AgNO3, BDH Laboratories, India), Zinc chloride, (ZnCl2, Rankem RFCL Ltd., India), Sodium carbonate (Na2CO3, Rankem RFCL Ltd., India), ethanol (99.9 % pure, Merk), Luria Bertani (Himedia laboratories Pvt. Ltd., India), and bleached softwood pulp (Star Paper Mill Ltd., India) were procured. All the reagents were used as such without any purification. The E. coli (MTCC no. 1698) and G. trabeum (MTCC No. 3168) were procured from Microbial Type Culture Collection and Gene Bank, Chandigarh, India. 2.2.
Synthesis
Paper matrices immobilized with varying ZnO and Ag contents are prepared using a single step hydrothermal method followed by the paper making using the standard Handsheet making method following the TAPPI test method 205 sp-02 [40-45]. Typically, 1.2 g softwood pulp was dispersed in 40 mL deionized water. To it, different precursors were added as per the predetermined metal oxide content. The detailed precursor recipe required for the synthesis of individual specimen is given in Table 1. This was followed by a hydrothermal treatment at 120 °C using a 100 mL Teflon lined stainless steel autoclave for 12 or 18 h. The sample was cooled naturally to room temperature. The obtained cellulose matrices were washed several times alternatively with hot water and ethanol. The pulp was dried and further re-dispersed in water to prepare the paper matrices using standard
6
Handsheet making procedure in the British Sheet Former and dried in the MG drier for 3 h at 80 °C. Table 1. Composition of reagents added in the synthesis of nanostructures immobilized paper matrices.
2.3.
S.
Sample
AgNO3 ZnCl2 Na2CO3
Temp
Time
N.
ID's
(g)
(g)
(g)
(ᵒC)
(h)
1.
P
-
-
-
120
12
2.
AgP
0.0188
-
-
120
12
3.
ZnP
-
0.4
40
120
12
4.
AgZnP∙12
0.0188
0.4
40
120
12
5.
AgZnP∙18
0.0188
0.4
40
120
18
Characterization
The compositional investigation of metal and metal oxide present in the paper matrices has been carried out by standard combustion test [40-45] and TGA. Heating a paper matrix in air at a temperature over 500 °C would result in the decomposition followed by oxidation of the elements present in it, resulting in a complete mass loss. These experiments are carried out normally in furnaces. In the present work, a known weight of the paper matrix was kept in a crucible and placed in a muffle furnace. The furnace was ramped to a temperature of 520 °C with a heating rate of 10 °C per min and kept at that temperature for 4 h. It was then cooled down naturally below 50 °C and the remaining mass if any was collected. The difference in the weight of the sample used for the combustion test before and after heating could provide the amount of inorganic contents present in the specimen. The TGA experiments were carried out by heating the specimens in EXSTAR TG/DTA6300 with a heating rate of 5 °C per min in an argon flow of 200 sccm. We have tried to perform similar TGA experiments in air, but
7
at a particular temperature (~300 °C) the whole paper matrices get burnt with a short time period leading to an erroneous result. The phase analysis was performed in a Rigaku Ultima IV XRD using CuKα radiation (λ = 1.5405 Å) at a scanning speed of 4°/min in the range of 10 to 70° of the 2 scale. The obtained diffraction patterns were compared with the standard JCPDS (Joint Committee on Powder Diffraction Standards) files. Lattice parameters were refined by least-squares method using the PROSZKI program. The XPS analysis was carried on PHI-5000 VersaProbe III, ULVAC-PHI INC. The specimens were placed on the carbon tape and kept in ultra-high vacuum of 10-7 torr for 4 h in the sample introduction chamber. Then the samples were inserted into the main chamber and XPS data was recorded. The AlKα was used as X-ray source with energy of 1486.6 eV. The peaks were referenced with C 1s at binding energy of 284.8 eV. The microstructure is studied using FESEM (FEI operating at 10 kV) and TEM (TECNAI G2S-TWIN operating at 200 kV). Before FESEM investigation, the paper matrices owing to their non-conducting nature were coated with gold using a standard gold sputtering unit. Sample preparation for the TEM was carried out by collecting the nanowires from the surface of the paper matrices and dispersed in methanol using ultra-sonication. A drop of the dispersion was placed in the holey carbon coated copper grids and dried at room temperature. The leaching of metal and metal ions have been studied using ICP-OES (Teledyne Leeman Labs, Prodigy SPEC).
2.4.
Antimicrobial activity
The antibacterial activity in the presence of visible light was studied by inhibiting the growth of E. coli using the prepared ZnO, Ag and ZnO-Ag nanostructures immobilized paper matrices. The experiment was carried out in triplicate and standard deviation in log reduction 8
of E. coli is calculated. The experiment was carried out by following the standard protocol ISO 20743 with certain modifications. The paper matrices were first cut in the dimension of 8 cm × 1 cm. These were then put in individual test tubes filled with 10 mL of E. coli suspension. The test tubes were then incubated for 2 h at 37 °C under the exposure of compact fluorescent lamp (1.69 J/cm2). Serial dilutions of bacteria containing suspension was done and 100 μl of each dilution was spread on LB agar in petri-plates. The plates were incubated at 37 °C for 24 h. Colonies of the bacteria are counted and results are explained in form of % reduction and log reduction values. For the study of antifungal activity, prior grown fungal colony is picked using loop and transferred to potato dextrose agar plate. Paper matrices cut in the size of 2 cm × 2 cm and are sterilized using autoclave. These were then placed on the petri-plates containing the fungal colonies. The petri-plates were incubated at 28 °C for 7 days and investigated for the fungal growth. 3. RESULTS AND DISCUSSION The immobilization of ZnO nanowires (NWs) doped with Ag/Ag+ on the surface of the cellulose fibers has been carried out by a one-step hydrothermal method at 120 °C for 12 h using ZnCl2∙5H2O and AgNO3 as ZnO and Ag sources, and commercially available softwood pulp as cellulose source. In general, the extent of immobilization of the metal oxides in paper matrices is estimated by combustion test and thermogravimetric analysis (TGA) [40-43]. On heating the paper matrices in air above 520 °C, there is a complete decomposition and oxidation of organic matters leaving behind the inorganic components [40-43]. The paper specimens prepared in this research have been heated at 525 °C in air for 4 h in a muffle furnace. For comparison, blank paper matrix designated as specimen P has also been employed for an identical combustion. As expected, a complete mass loss (remaining ash content < 0.004 wt%) was observed in specimen P indicating the absence of any inorganic component. However, the combustion of paper matrices immobilized with only Ag/Ag+ 9
(designated as specimen AgP) and ZnO (designated as specimen ZnP) have remaining masses of 0.5 and 10.0 wt%, respectively. The combustion of Ag/Ag+ doped ZnO immobilized paper matrix (specimen AgZnP∙12) has shown to have leftover mass of 11.0 wt%. The paper matrix prepared by a prolonged hydrothermal treatment of 18 h (specimen AgZnP∙18) at 120 °C keeping all other experimental conditions same, was also having the same 11.0 wt% inorganic components as estimated by the combustion experiment. Table 2. Retention of inorganic content in paper matrices estimated by combustion test and TGA analysis of the specimens in argon atmosphere. Sample ID
Mass remaining (%) Combustion
TGA in Ar
P
0.004
12.0
AgP
0.5
12.0
ZnP
10.0
21.5
AgZnP∙12
11.0
21.2
AgZnP∙18
11.0
22.7
A similar conclusion can be drawn from the TGA investigations of the specimen heating up to 600 ºC in an argon atmosphere with a heating rate of 5 ºC/min as shown in Fig. 1. Around 12 % mass was left after heating the specimen P and AgP, which can be attributed to the leftover carbon. No appreciable mass difference can be observed in these two specimens similar to the combustion experiment. Moreover, remaining masses of 21.5, 21.2 and 22.7 wt% in ZnP, AgZnP∙12 and AgZnP∙18 specimens, respectively, corroborate the finding (Table 2).
10
100
P AgP ZnP AgZnP.12 · AgZnP.18
60
24 22 20
P AgP ZnP AgZnP.12 AgZnP.18
40
18 16
Weight (%)
Weight (%)
80
14 12
20
500
520
540
560
580
10 600
Temperature (C)
0
100
200
300
400
500
600
Temperature (C)
Fig. 1. TGA thermograms of ZnO and Ag+:ZnO NWs immobilized paper matrices in argon atmosphere. The phase analysis of the specimens was investigated by XRD (Fig. 2). Two broad peaks at d-values of 5.823 and 3.896 Å observed in all the samples are attributed to the (101) and (002) reflections of cellulose-I [40-45]. In addition, peaks at d-values of 2.832, 2.609, 2.488, 1.919, 1.632, 1.484 and 1.385 Å observed for the ZnP sample correspond to the (100), (002), (101), (102), (110), (103) and (112) reflections of hexagonal ZnO with the wurtzite structure. The refined lattice parameters of 3.258(1) and 5.209(5) Å are in agreement with the reported data for hexagonal ZnO (JCPDS PDF File No. 79-205). The XRD data for AgZnP∙12 indicates doublet feature for all the reflections corresponding to hexagonal ZnO structure, reflecting formation of two admixed ZnO phases. While the refined lattice parameters [3.246(1) and 5.199(2) Å] with one set of reflections resembled parent ZnO type phase, the other set indicated lattice expansion [3.311(1) and 5.306(2) Å]. This has been attributed to the incorporation of Ag+ in the ZnO lattice for the latter phase leading to lattice expansion due to larger size of Ag+ as compared to Zn2+ [46]. Interestingly, for the AgZnP.18 sample only one set of reflections corresponding to Ag+ incorporated hexagonal ZnO are observed and the value of refined lattice parameters [3.287(1) and 5.259(3) Å] are in between the two ZnO phases of the AgZnP∙12 sample. Moreover, the peaks due to fcc metallic Ag are 11
also evident in the XRD pattern of AgZnP∙18 and AgZnP∙12. It is to be noted that the intensity of the metallic Ag peaks increases on prolonging the reaction from 12 hours to 18 hours. The absence of metallic Ag peaks in the AgP sample is probably due to presence of very little amount of Ag as compared to cellulose content. However, the presence of Ag is clearly evident from the XPS data analysis. Interestingly, the enhancement in the metallic Ag peaks in AgZnP∙18 sample is attributed to better crystallization and growth of metallic Ag particles on prolonging the reaction. Subsequently, there is a decrease in the hexagonal lattice parameters for AgZnP∙18 to that of the AgZnP∙12. This appears to happen due to the diffusion of Ag+ from the Ag+ rich regions of ZnO (Zn1-xAgxO) to undoped ZnO (representing two admixed ZnO phases of AgZnP∙12) and finally resulting in a homogeneous solid solution of Ag+ doped ZnO (as in AgZnP∙18). Assuming linear Vegard’s law behaviour and using the experimental lattice volume expansion for AgZnP∙12 (5.2%) and AgZnP∙18 (2.6%), it is apparent that the lattice Ag+ incorporation is nearly double in AgZnP∙12 to that of AgZnP∙18.
.
AgZnP.18
AgZnP.12
AgZnP.18 AgZnP.12 ZnP AgP P
ZnP AgP P 10
20
30
40
50
60
70
Diffraction angle 2 (degree)
30
80
(101)
(002)
Intensity (a.u.)
(103)
(b)
(112)
. .
(110)
#
(102)
(100) (002) (101)
Intensity (a.u.)
# Cellulose-I
. Ag FCC
(100)
#
(a)
32
34
36
Diffraction angle 2 (degree)
Fig. 2. (a) XRD patterns of prepared paper matrices. (b) Selected region of the XRD patterns depicting the shift in the (100), (002), and (101) peak positions. In order to further understand the doping of the Ag+ in ZnO, all the specimens have been investigated by XPS. The survey scan of the specimens is given in Fig. 3. As expected, 12
only C1s and O1s peaks at 286.6 and 532.9 eV, respectively, were observed in the specimen P attributed to the cellulose [41]. In the sample AgP [Fig. 3a(ii)], in addition to the C1s and O1s peaks, a weak doublet (arrow marked) could be seen at the binding energy of 368 to 372 eV, which confirms the presence of silver in the specimen. Similarly, in addition to the C1s and O1s peaks, peaks originating from Zn were observed in the ZnP sample and are assigned to various levels as shown in Fig. 3a(iii). However, in specimens AgZnP∙12 and AgZnP∙18, peaks originating from Zn and Ag were observed in addition to the C1s and O1s peaks. To confirm the speciation of individual element present in these specimens, high resolution XPS spectra were recorded. For example, the high resolution spectra of C1s (Fig. 3b) has three peaks at 284.8, 286.5 and 287.7 eV attributed to carbon with one ether link, carbon linked to hydroxyl groups and carbon with two ether links, respectively as shown in Fig. 4. Similarly, two oxygen has been observed in high resolution O 1s XPS spectra of specimen P as shown in Fig. 3c at 533 and 534.8 eV. These could be attributed to the hydroxyl and glycosidic oxygen. A similar O1s spectrum was also observed in the AgP specimen. However, an additional O1s peak could be seen at 531.1 eV in the specimens ZnP, AgZnP∙12 and AgZnP∙18 attributed to the presence oxygen of zinc oxide. It is interesting to note that the shift in the C1s and O1s XPS spectra are consistent with the observation in XRD analysis. The high resolution Ag3d XPS spectra has provided critical information on the oxidation state of silver. As shown in Fig. 3d, all the three specimens AgP, AgZnP∙12 and AgZnP∙18 have two strong peaks assigned to Ag3d3/2 and Ag3d5/2, respectively, with a separation of 5.96 eV which is close to the standard value of separation of 6.00 eV. The Ag3d5/2 peak was observed at 367.87, 367.64 and 367.94 eV in the specimens AgP, AgZnP∙12 and AgZnP∙18, respectively, and are assigned to the Ag+. The presence of the metallic silver Ag0 in these specimens could be obtained on further deconvolution as shown 13
in Fig. 3d. It was observed at 369.15, 368.43 and 368.66 eV in the specimens AgP, AgZnP∙12 and AgZnP∙18, respectively [47]. The quantitative XPS analysis estimates the Ag content of
C1s
Zn3d
Zn3s Zn3p3
AgZnP.12
Ag3d3
AgZnP.18
Intensity (a.u.)
(a)
O1s ZnLMM
Zn2p1 Zn2p3
0.5, 0.5 and 0.52 at% in AgP, AgZnP∙12 and AgZnP∙18, respectively. It is interesting to note
(v) (iv)
ZnP
(iii)
AgP
(ii) P
(i) 1000
800
600
400
200
0
Binding energy (eV)
C1s
290
AgZnP.18
C1s
AgZnP.12
C1s
ZnP
C1s
AgP
C1s
P
288
286
284
Ag3d3/2
Ag3d3/2
AgP
O1s
ZnP
O1s
AgP
O1s
P
534
(e)
Ag3d5/2
AgZnP.18
AgZnP.12
AgZnP.12
532
530
Binding energy (eV)
AgZnP.12
Zn2p3/2
Zn2p1/2
AgZnP.12
Ag3d5/2
Ag3d5/2
Intensity (a.u.)
Intensity (a.u.)
Ag3d3/2
AgZnP.18
O1s
536
Binding energy (eV)
(d)
O1s
(c)
Intensity (a.u.)
Intensity (a.u.)
(b)
Zn2p3/2
Zn2p1/2
Zn2p3/2
ZnP Zn2p1/2
376
374
372
370
368
366
1050
Binding energy (eV)
1040
1030
1020
Binding energy (eV)
Fig. 3. (a) Full scan (Su1s) XPS spectra of the prepared paper matrices. The high resolution XPS spectra of paper matrices showing (b) C1s, (c) O1s, (d) Ag3d and (e) Zn2p3 peaks. 14
that the shifting in the peak position in the Ag3d XPS spectra are in consistent with the observation of XRD. Similarly, two peaks have been observed in high resolution Zn2p XPS spectra as shown in Fig. 3e, assigned to Zn2p3/2 at 1021.4 eV and Zn2p1/2 at 1044.6 eV. The difference between the two is 23.2 eV, which is matching well with the reported literature value of 23.0 eV [48]. A consistent shifting in Zn2p peaks further corroborate the results obtained in XRD investigation.
H
2 H2C
OH 2
2C
C
C H
H
3C
1 C
O OH
C H2C 2
O O
1
O 3C
H
OH
H H
O 1
H
H
1C
OH
2C H
OH
2
H C OH
Fig. 4. Structure of cellulose indicating three chemically different carbon. The microstructural analysis of the specimens was carried out by FESEM and TEM. The typical FESEM images of all the paper matrices are given in Fig. 5. As expected, fibers of diameter in the range of 20-100 m and length extended upto millimetre could be seen in all the specimens. Clean and smooth surface of the fibers was observed in high magnification FESEM image of the specimen P (Fig. 5b). A similar observation was derived in the AgP specimen that was made by immobilizing Ag in the paper matrices. On careful observation, it could be seen that small spherical nanoparticles of Ag with a dimension smaller than 200 nm are present on the surface of the cellulose fibers. The ZnO NWs of diameter 30-100 nm and length of 2-5 m could be seen in the high magnification FESEM images and uniform distribution of the NWs can be seen with the low magnification images of the specimens ZnP, AgZnP∙12 and AgZnP∙18 in Fig. 5. The observation of ZnO NWs immobilized on the surface of the
15
Fig. 5. FESEM images of specimen (a, b) P, (c, d) AgP, (e, f) ZnP, (g, h) AgZnP∙12 and (i, j) AgZnP∙18. 16
cellulose fibers is expected and consistent with our previous report [41]. There is no major difference that can be observed in the microstructures of these specimens.
Fig. 6. (a, c) TEM and (b, d) HRTEM images of AgZnP∙12 and AgZnP∙18, respectively. The corresponding SAED patterns are given in the inset of the TEM images. In order to further get a better insight on the size and nature of the NWs, TEM analysis was performed. Cellulose being too thick (of the order of 20 to 100 m thick) could easily restrict the passage of the electron beams in the TEM, thus, the analysis could not be performed under the available experimental conditions. Rather, the ZnO NWs were scratched and dispersed in the carbon coated copper grid for TEM investigation. ZnO NWs of uniform diameter in the range of 30 to 50 nm could be seen in AgZnP∙12 and AgZnP∙18 specimens, which is consistent with the observation of FESEM. The high resolution TEM images have indicated the single crystallinity nature of the ZnO NWs due to the observation of clear and uniform lattice fringes. The lattice spacing along the longer axis was measured to be 2.597 Å 17
which matches well with the (0002) plane indicating the growth of the NWs in the [0001] direction [48]. This lattice spacing also matches well with the value of 2.609 Å obtained from the XRD pattern. The single crystallinity of the NWs was further confirmed by the observation of regular arrays of the bright spots in SAED patterns of specimens AgZnP∙12 and AgZnP∙18 in Fig. 6. The antimicrobial activity of the prepared paper matrices has been carried out by choosing the most common and widely investigated Gram negative bacterium E. coli [23]. For the antibacterial studies, the colony count methodology is followed as shown in Fig. 7a. An initial bacterial colony counts of ~3.00 × 108 CFU (colony forming unit) per mL was taken for the investigation and the bacterial colonies are exposed to visible light for a short time period of 2 h only. However, in both the specimens AgP and ZnP, the reduction of the colonies is almost same i.e., ~53 % (1.4 × 108 CFU per mL) of the original counts. But in Ag doped ZnO NWs immobilized paper matrix, specimen AgZnP∙12, a reduction of 99.99999 % of the colonies from the original counts could be estimated. To our surprise, the specimen AgZnP∙18 could only achieve a ~74 % reduction of the bacterial colonies (8.0 × 107 CFU per mL) after 2 h of visible light exposure (Fig. 7a and Table 3). Log reduction values estimated for the E. coli growth with standard deviation in triplicate experiments has been carried out and depicted in Fig. 8, which clearly shows the efficiency of the AgZnP∙12 for the deactivation of E. coli. This high value of log reduction is in the higher side as compared to many of the recent reports [49,50]. The cellulose in paper matrices is vulnerable to microbial attack. Among the various microbes, G. trabeum, a cellulase producing wood decaying fungus, has proven to be the worst enemy of paper matrices [30,31]. The degradation mechanism involves the depolymerisation of cellulose and hemicellulose. The already grown G. trabeum fungal colony has been picked up and inoculated to potato dextrose agar (PDA) petri-plates and the 18
sterilized paper matrices have been introduced into it. The petri-plates have been incubated at 28 ºC for 7 days for the growth of the fungus which in turn could degrade the paper matrices. For understanding the extent of degradation, photographs of these petri-plates with various specimens have been compared (Fig. 7b). As expected, a similar trend in the antifungal activity of these paper matrices have been perceived. Neither degradation/damage nor growth of fungus near the paper specimen AgZnP∙12 could be seen even after such a long incubation time of seven days. In all other specimens the extent of damage or degradation of the paper matrices follows the same trend as was observed for the antibacterial activity of these specimens. It has been observed in case of the sample AgZnP∙12, no growth of fungus is present, whereas in all the other samples, fungus have completely attacked the paper matrices. The consistent with the observation of antibacterial and antifungal activity of these specimens with a superior activity realized for the AgZnP∙12 could further be explained in term of the doping and chemical environment of the Ag+ in the ZnO NWs immobilized in the paper matrices.
Fig. 7. Photographs depicting the inhibition of growth of (a) E. coli and (b) G. trabeum. As reported by our group, the metal ion doesn’t leach out from the paper matrices made by the present method even after sonication, have removed the possibility of Ag or Zn leaching during the antimicrobial activity [41]. The ICP-OES investigation of the DI water
19
where the paper matrices were treated confirms the absence of Ag and Zn corroborate our earliest observation. It is important to note that the paper matrices are highly stable as these are not degraded by even the paper eating fungus G. trabeum, was also confirmed when a three years old prepared paper matrices shown the same activity with the newly made paper matrices. Table 3. Percent reduction and log reduction values in growth of E. coli after 2 h of visible light exposure. S. No.
Sample Id
CFU
% Reduction
Log Reduction
1.
P
3.0 × 108
No reduction
0
2.
AgP
1.4 × 108
53
0.32
3.
ZnP
1.4 × 108
53
0.32
4.
AgZnP∙12
4 × 10
99.99999
6.81
5.
AgZnP∙18
8.0 × 107
74
0.55
6.86
7
Log reduction
6 5 4 3 2 1 0 -1
0
P
0.32
0.32
AgP
ZnP
0.56
AgZnP.12
AgZnP.18
Sample Id Fig. 8. Log reduction values in E. coli growth after 2h of visible light exposure including standard deviation. Although, the overall Ag/Ag+ content in all the three specimens AgP, AgZnP∙12 and AgZnP∙18 are very much close to each other as estimated from the XPS investigation, the superior activity of AgZnP∙12 could be explained based on two factors (i) synergy of ZnO 20
and Ag+ and (ii) concentration of Ag+ ion the ZnO lattice [35]. The antimicrobial activity of ZnO and Ag individually is well known and has been documented in large numbers of research articles [12-18,51]. The combination of Ag and ZnO has shown to produce a synergy and could show a much better activity as compared to only ZnO or Ag [52,53]. Until recently, it was commonly accepted that the activity was due to the formation of reactive oxygen species by the Ag and ZnO. It was reported by Duran et al. that the activity of Ag+ is superior as compared to the metallic Ag [18]. This was mainly due to a higher affinity of Ag+ for organic amines, phosphates and thiols [54]. Three steps have been suggested by Marambio-Jones et al. for this superior activity i.e., disruption of ATP (adenine triphosphate) production by silver ions, generation of reactive oxygen species followed by the damage of cell membrane [55]. In this investigation, as the AgZnP∙12 specimen has the maximum Ag+ content (almost double as compared to AgZnP∙18 estimated from the XRD investigation), hence, the observation of superior activity could be expected. Although, a clear antifungal mechanism is not elucidated so far, but it is accepted that the accumulation of excessive carbohydrates and nucleic acids due to self-protecting mechanism of fungal cells against metal and metal oxides which lead to deformation of the fungal structures. It is expected that the increased Ag+ could facilitates the above mentioned mechanism. 4. CONCLUSION In summary, doping of Ag+ in the lattice of ZnO NWs immobilized on cellulose fibers of the paper matrices have shown to improve the antimicrobial activity against E. coli and G. trabeum. In the AgZnP∙12 with Ag+ concentration double as compared to AgZnP∙18 has shown the best performance, although, total Ag0 and Ag+ concentration in these samples are almost same. The underlying chemistry of the immobilization of the metal oxide nanostructures on the surface of cellulose fibers of paper matrices and tuning their application
21
on doping have not only made these paper matrices imperative in academic research purpose but also could open up commercial avenues to disinfect water bodies from microbes. ACKNOWLEDGEMENT The work was financially supported by SERB, New Delhi, Govt. of India with Grant code: EMR/2016/001693. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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