Materials Science and Engineering C 52 (2015) 171–177
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Effect of Mg2 +, Ca2 +, Sr2 + and Ba2 + metal ions on the antifungal activity of ZnO nanoparticles tested against Candida albicans Abdulrahman Syedahamed Haja Hameed a,⁎, Chandrasekaran Karthikeyan a, Venugopal Senthil Kumar b, Subramanian Kumaresan b, Seemaisamy Sasikumar a a b
PG and Research Department of Physics, Jamal Mohamed College, Tiruchirappalli 620 020, Tamil Nadu, India Department of Plant Biology and Plant Biotechnology, R.K.M. Vivekananda College, Chennai 600 004, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 12 November 2014 Received in revised form 3 February 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: ZnO nanoparticles Metal doping Antifungal activity Candida albicans XRD SEM
a b s t r a c t The antifungal ability of pure and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO nanoparticles (NPs) prepared by the co-precipitation method was tested against the pathogenic yeast, Candida albicans (C. albicans), and the results showed that the Mg-doped ZnO NPs possessed greater effect than the other alkaline metal ion doped ZnO NPs. The impact of the concentration of Mg doped ZnO sample on the growth of C. albicans was also studied. The Minimal Fungicidal Concentration (MFC) of the Mg doped ZnO NPs was found to be 2000 μg/ml for which the growth of C. albicans was completely inhibited. The ZnO:Mg sample (1.5 mg/ml) with various concentrations of histidine reduced the fungicidal effect of the nanoparticles against C. albicans, which was deliberately explained by the role of ROS. The ZnO:Mg sample added with 5 mM of histidine scavenged the ample amount of generated ROS effectively. The binding of the NPs with fungi was observed by their FESEM images and their electrostatic attraction is confirmed by the zeta potential measurement. © 2015 Published by Elsevier B.V.
1. Introduction Zinc Oxide (ZnO) is an n-type semiconductor with a wide direct band gap of 3.37 eV and a high exciton binding energy of approximately 60 meV. It has also attracted an attention for use in electrical and optical applications such as light emitting diodes, piezoelectric transducers and photo-catalysts [1–3]. In recent years, the photocatalytic and antibacterial activities of the ZnO nanoparticles (Zn NPs) have attracted interest for investigation [4]. The ZnO NPs are believed to be nontoxic, safe and biocompatible. They have also been used as drug carriers, cosmetics, and fillings in medical materials [5]. Several reports have addressed the harmful impact of nanomaterials on living cells, but relatively low concentrations of ZnO are nontoxic to eukaryotic cells [6–11]. The ZnO NPs significantly inhibit the growth of a wide range of pathogenic bacteria under normal visible lighting conditions [12]. Several studies suggest that different morphologies (particle's size and shape) of ZnO NPs have different degrees of antimicrobial activities [12,13]. The antimicrobial activity of ZnO NPs generally depends on the presence of more reactive oxygen species (ROS), which is mainly attributed to the larger surface area of NPs, an increase in oxygen vacancies, the diffusion ability of the reactant molecules and the release of Zn2+ [14]. The superoxide radical, hydroxyl radical and hydrogen peroxide belonging to the ROS group can cause damage to DNA and cellular ⁎ Corresponding author. E-mail address: [email protected] (A.S. Haja Hameed).
http://dx.doi.org/10.1016/j.msec.2015.03.030 0928-4931/© 2015 Published by Elsevier B.V.
proteins, and may even lead to cell death [15]. Generally, nanoparticles with better photocatalytic activity have larger specific surface area and smaller crystal size which increase oxygen vacancies, resulting in more ROS [14]. In addition, the crystal growth direction is another important factor for the generation of ROS. Earlier studies have proved that the terminal polar Zn (0001) faces showed the highest photocatalytic activity for H2O2 generation [16]. Similarly, the Zn polar terminal has exhibited strong UV luminescence rather than the O nonpolar terminal [17]. The antimicrobial effect of the ZnO samples was found to be mainly due to the combination of ROS and the release of Zn2 +. In an earlier report [18], it showed that the Mg-doped ZnO NPs had the highest antimicrobial activity as compared to that of pure ZnO NPs. The genus Candida is a compilation of some 150 asporogenous yeast species. These Candida species are usually restricted among the fungi in the class deuteromycetes because of their inability to form a sexual stage [19]. Candida albicans is a dimorphic fungus existing both in the blastospore phase (synyeast phase, blastocomidial phase) and the hyphal or mycelial phase. C. albicans infections (candidiasis) may be divided into two stages; superficial (such as oral and vaginal thrush and chronic mucocutaneous candidiasis) and deep-seated (such as Candida due myocarditis and acute disseminated Candida septicemia). In superficial candidacies, the organism may exist in the blastospore phase and typically colonizes the mucocutaneous surfaces of the mouth and vagina, whereas in the deep-seated candidiasis, the hyphal phase can be a portal of entry into deeper tissues and able to cause a variety of infections when host defense systems are compromised [20].
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C. albicans is the most common opportunistic fungal pathogen of humans and animals, and moreover it is also a very frequent hospital-acquired infection, and is the fourth leading cause of nosocomial infection in the USA [21,22]. For several reasons (immunosuppressive treatments, long-term catheterization, use of broadspectrum antibiotics and longer survival of immunologically compromised individuals), Candida infections have increased dramatically over the last two decades. Hence C. albicans has drawn a major medical importance among the other species of its genus. Lipovsky et al. (2011) investigated the antifungal potential of ZnO nanoparticles against C. albicans [23]. However, to the best of our knowledge, the antifungal properties have not been reported for alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs against C. albicans. In the present investigation, pure and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+)-doped ZnO NPs are synthesized by the co-precipitation method. We have studied the structural and antifungal properties of the pure and alkaline metal ion doped ZnO NPs.
2. Material and methods 2.1. Preparation and characterization of the pure and alkaline metal ion doped ZnO NPs The following high purity chemicals such as Zinc (II) nitrate hexahydrate (Zn(NO3)2·6H2O), Magnesium nitrate hexahydrate (Mg(NO3)2·6H2O), Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), Strontium nitrate (Sr(NO3)2), Barium nitrate (Ba(NO3)2), Sodium hydroxide (NaOH) and Polyethylene glycol (PEG) were used as the precursors without further purification. The details of the experimental procedure for the preparation of pure ZnO and alkaline metal (Mg, Ca, Sr and Ba) doped ZnO samples have been reported in our previous paper [18]. The X-ray diffraction (XRD) patterns of the ZnO NP samples were collected using an X'PERT PRO PANalytical X-ray diffractometer with a CuKα (40 kV, 30 mA) radiation source. The ZnO NP samples were gently crushed before being smeared on a clean glass slide. The powder diffraction patterns were collected over the 2θ range between 10° and 80° with a scan speed and sampling width of 2 min−1 and 0.05° respectively. HRSEM was performed on the ZnO samples using a FEI-QUANDA 200F microscope operating at 30 kV. The microscope was equipped with a charge-coupled device (CCD) camera. The samples were prepared by 1 mg of ZnO NP samples coated with 1.2 nm gold particle separation on a carbon tape using the low vacuum. Energy dispersive X-ray (EDX) analysis was done using an EDX spectrometer (model: AMETEK) with a FEI-QUANDA 200F high resolution scanning electron microscope operated at 30 kV. The dry powdered samples were attached to the substrate using a double-sided carbon tape and mounted onto the sample holder.
2.3. The concentration dependent effect of Mg doped ZnO NPs on C. albicans The fungicidal activity of the Mg doped ZnO NPs was quantitatively ascertained against the C. albicans by the optical density (OD) method at 660 nm. Different concentrations of Mg doped ZnO NP samples were added to the C. albicans culture (0.5 McFarland standard) and subsequently the OD was measured at different time intervals. C. albicans culture without the addition of NPs served as the control. 2.4. Determination of Minimum Fungicidal Concentration The minimal quantity of the Mg doped ZnO NPs required for the fungicidal activity was resolved by adding different concentrations of the samples into the C. albicans culture and the OD was measured after 24 h. 2.5. The role of reactive oxygen species (ROS) in antifungal activity The ROS as the key factor in antifungal activity was assessed with the help of histidine, a well-known scavenger of ROS. C. albicans culture containing a definite quantity of Mg doped ZnO NP sample (1000 μg/ml) was treated with various amounts of histidine and the change in the OD was determined after 24 h. C. albicans culture without the addition of histidine served as the control. 2.6. Preparation of fungal samples for Field Emission Scanning Electron Microscope (FESEM) The control and Mg doped ZnO NP treated C. albicans cells were centrifuged at 6000 rpm for 10 min. at 4 °C. The pellets were gently and doubly washed with PBS (0.1 M) and fixed with 2.5% glutaraldehyde in PBS at 4 °C for 2 h. After fixation, the samples were dehydrated with 25, 50 and 75% ethanol series at one time each and then dehydrated with 100% ethanol by three times for 10 min. The dehydrated samples were kept overnight in a desiccator and thereafter gold coated by sputtering. The samples were then analyzed by FESEM (model: SUPRA: CARL ZEISS 55) with EDAX (model: ULTRA 55). 2.7. Zeta-potential measurement The zeta-potential measurement was conducted for the control and Mg doped ZnO NP treated C. albicans samples by a ZetaPlus Zeta Potential Analyzer (nano-ZS90). The minimum fungal concentration of the NPs was 2 mg/ml. For both samples, an appropriate amount of undiluted solution was placed into the cuvette, and the average zeta potential values were obtained for the control and treated samples as individual measurements. Water is the solution medium for all zeta potential measurements. 3. Results and discussion 3.1. Phase and crystal structure analysis
2.2. Antifungal assay Antifungal activity was determined by an agar disc diffusion method against the test fungi C. albicans (ATCC 10231) using potato dextrose agar [24]. The test strain was transferred into potato dextrose broth (PDB) and incubated at 30 °C until it achieved the turbidity of 0.5 McFarland standard. The media plates were inoculated with the test strain by streaking for 2–3 times by rotating the plate at a 60° angle for each streak to ensure uniform distribution of inoculum. Subsequently, sterile disc (6 mm) loaded with 1 mg of test samples (ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba) was placed onto the inoculated plates using sterile forceps and incubated at 30 °C for 24 h under visible light. The zone of inhibition formed around the disc was measured and recorded.
The phase and crystallinity of the as-synthesized pure and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs are studied by XRD and their patterns are shown in Fig. 1. The intense and sharp diffraction peaks suggest that the pure and alkaline metal ion doped ZnO NPs are highly crystalline in nature. The standard diffraction peaks show the hexagonal wurtzite structure for the pure ZnO NPs (space group: P63mc). This is also confirmed by the JCPDS data (card no. 361451). There is no impurity phase found in Magnesium and Calcium doped ZnO samples because of the ionic radii of Mg (0.66 Å) and Ca (0.99 Å). Furthermore, other dopants doped with ZnO have ionic radii above 1 Å (i.e., 1.13 Å for Sr2 + and 1.35 Å for Ba2 +) and they have two additional peaks located around 25.115° & 25.76° and 23.83° & 24.25° respectively, which correspond to the (111) and (021) planes. These two peaks indicate the presence of SrCO3 and BaCO3 respectively
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(e)
* BaCo3
* SrCo3
* * (c)
Intensity (a.u.)
3.2. Energy dispersive analysis of X-ray (EDAX) The compositional analysis of the pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs was carried out using EDAX. From the EDAX analysis, the amounts of alkaline metal ions present in the doped ZnO NPs are given in Table 1. In the doped samples, the concentrations of Mg, Ca, Sr and Ba are found to be 1.87%, 2.04%, 2.70% and 5.30% respectively. In the pure ZnO NPs, the chemical compositions of Zn and O are found to be 86.60% and 13.40% respectively. However, for the alkaline metal ion-doped ZnO NPs, the zinc percentage decreases whereas the oxygen percentage increases. In the ZnO NPs doped with magnesium, which has a smaller ionic radius, the compositions of Zn and O are estimated as 86.30% and 11.83% respectively. As compared to the pure ZnO NPs, a decrease in the oxygen percentage is observed for the Mg-doped ZnO NPs due to the effect of the smaller ionic radius Mg2+ metal ions.
** (d)
173
3.3. Antifungal studies
20
30
40
50
60
70
(202)
(103) (200) (112) (112) (004)
(102)
(110)
(100) (002)
(a)
(101)
(b)
80
2θ (deg) Fig. 1. X-ray powder diffraction patterns of (a) pure ZnO, (b) ZnO:Mg, (c) ZnO:Ca, (d) ZnO:Sr and (e) ZnO:Ba NPs [18].
for the ZnO:Sr and ZnO:Ba NPs. Thus, Sr(NO3) and Ba(NO3) are decomposed as SrO and BaO on the surface of ZnO NPs when calcined at higher temperature and then the formed SrO and BaO can absorb atmospheric CO2 leading to the formation of SrCO3 and BaCO3 [25,18]. The values of lattice constants ‘a’ and ‘c’ of pure ZnO NPs are 3.2564 Å and 5.2151 Å respectively. The values of lattice constants of the alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs are increased. But, interestingly, in the Ba2+ doped ZnO NPs, the values of lattice constants decrease as compared to that of pure ZnO NPs, because the barium atoms trapped in non-linear equilibrium position were shifted to a more equilibrium position. The values of lattice constants ‘a’ and ‘c’ are 3.2572 Å & 5.2107 Å, 3.2571 Å & 5.2142 Å, 3.2566 Å & 5.2157 Å and 3.2544 Å & 5.2106 Å for ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba respectively. The average crystallite sizes are found to be 38 nm, 31 nm, 37 nm, 23 nm and 28 nm for pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. The average crystallite size decreases in the Mg2 +, Ca2+, Sr2+ and Ba2+ doped ZnO NPs as compared to that of pure ZnO NPs. The reduction in the particle's size is mainly due to the distortion in the host ZnO lattice by the foreign impurities i.e., Mg2+, Ca2+, Sr2+ and Ba2+. From the HRSEM images (Fig. 2), the pure ZnO and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs exhibited a nanoflake like morphology. The aggregation of nanoflakes is observed in the Mg2+ doped ZnO NPs. The average thicknesses of the nanoflakes are 63 nm, 62 nm, 47 nm, 53 nm and 52 nm for the pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. The size of the nanoflakes in alkaline metal ion doped ZnO NPs decreases in comparison with that of the pure ZnO nanoflakes. The reduction of thickness is due to the distortion in the host metal by the foreign impurities that decrease the nucleation and subsequent growth rate of the ZnO NPs.
In the present investigation, pure ZnO and alkali metal ions Mg, Ca, Sr, Ba doped ZnO NPs were tested against C. albicans (ATCC 10231) using the disc diffusion method to analyze their ability as a potential candidate for an antifungal agent. The magnitude of the antifungal agent was determined, based on the size of the inhibition zone formed around each disc loaded with an appropriate test sample. The antimicrobial activity of ZnO NPs generally depends on the presence of higher ROS that generally comes from a better photocatalyst with larger surface area, crystallite size, increased oxygen vacancies, diffusion ability of the reactant molecules and the release of Zn2 + [14,26,18]. The oxidative stress induced by ROS generation, interaction of oxide NPs with the microbial cell wall and possible permeation of the NPs into the microbial cell are still not clearly understood [27]. Once inside the cell, nanomaterials induce intercellular oxidative stress by distributing the balance between oxidant and anti-oxidant processes. On the one hand, the oxidative stress induced by exposure to nanomaterials may stimulate an increase of the cytosolic calcium concentration or may cause the translocation of transcription factors to the nucleus, which regulate pro-inflammatory genes. Alternatively, exceeding oxidative stress may also modify protein, lipids and nucleic acids, which further stimulates the anti-oxidant defense system or even leads to cell death [28]. Generally, nanoparticles with better photocatalytic activity having a large specific surface area and a small crystallite size which increase oxygen vacancies, result in more ROS [14]. Fig. 3 shows the diameter of the inhibition zone formed around discs. Even though all of the samples exhibited antifungal activity, the Mg doped ZnO NPs showed the highest activity. The antifungal activity of pure ZnO and alkaline metal ion doped ZnO NPs is explained as follows. As mentioned earlier, the greater number of ROS is mainly attributed to the larger surface area, increase in oxygen vacancies and the diffusion ability of the reactant molecules. In the present investigation, the antifungal effect of the ZnO samples is mainly due to the combination of various factors such as ROS and the release of Zn2 +. The photo-catalysis seems to be the most important antifungal mechanism; ROS produced on the surface of these NPs in the presence of light cause oxidative stress in microbial cell and eventually lead to the death of the cell. ROS contain the most reactive hydroxyl radical (•OH), less toxic superoxide anion radical (•O− 2 ) and hydrogen peroxide with a weaker oxidizer (H2O2). The generation of H2O2 can penetrate into the cell membrane and kill the microbes [29]. The mechanism of light induced generation of ROS can be given as follows [30]. −
ZnO þ hυ⇁e þ h
þ
þ þ h þ H2 O⇁·OH þ H
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Fig. 2. HRSEM images of (a) pure ZnO, (b) ZnO:Mg, (c) ZnO:Ca, (d) ZnO:Sr and (e) ZnO:Ba NPs [18].
−
e þ O2 ⇁·O2
·O
2
−
−
þ
þ H ⇁HO2 · þ
−
HO2 · þ H þ e ⇁H2 O2 The (0001) Zn-polar faces and the increased number of Zn2+ ions enhance the diffusion ability of the Zn2+ ions, which is supported by the level of the intensity ratio (I(100)/I(002)) [26,18]. In previous report, the ratio is 1.1770 for ZnO NPs. The values of the intensity ratio are 1.232, 0.97, 1.1732 and 1.1006 for ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. From the results of the photoluminescence study for the ZnO and alkaline metal ion-doped ZnO NPs, the green and yellow emissions are due to the presence of single ionized oxygen vacancies and interstitial oxygen vacancies respectively [18]. The ZnO NPs exposing the [0001] Zn-polar surface carrying a positive charge are prone to contact with the C. albicans cell surface carrying a net negative charge via electrostatic force [31,26]. Such contact may not only inhibit fungal growth but also be killed by the generated ROS. In the Mg doped ZnO NPs, there is no release of Mg2 +. Thus, the release of Mg2+ may not be responsible for the biocidal activity of the sample. However, this increased activity in the Mg doped ZnO NPs is caused by replacement of Mg in Zn sites, which enhances the
photoactivity of the particles because of the similarity in ionic radii between Mg2+ (0.66 Å) and Zn2+ (0.74 Å). This can be explained as follows: the Mg doped ZnO has the highest value intensity ratio I(100)/I(002) (1.232) as compared to that of the pure ZnO, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs, possessing a greater proportion of (0001) Zn-polar faces which enhances the diffusion ability of Zn2+ ions [18]. The wavelengths of the green and yellow emissions are at 549 nm and 573 nm for the Mg doped ZnO NPs, whereas the wavelengths of the emissions are at 546 nm and 569 for the pure ZnO NPs. This shows the increased number of oxygen vacancies and interstitial oxygen vacancies in the Mg-doped ZnO NPs, leading to a higher number of ROS as compared to the ZnO NPs [18]. The NPs with uneven surfaces and rough edges have been found to adhere to the microbial wall and cause damage to the cell membrane [32]. From the HRSEM images, it is clear that the Mg-doped ZnO NPs have uneven ridges at their outer surfaces which lead to antifungal activity, whereas the other NPs have smooth surfaces.
Table 1 The elemental composition of the synthesized pure and doped ZnO NPs. Sample
ZnO ZnO:Mg ZnO:Ca ZnO:Sr ZnO:Ba
Weight % Zn
O
Doping amount
Total
86.60 86.30 81.27 79.64 73.18
13.40 11.83 16.69 17.65 21.52
– 1.87 (Mg) 2.04 (Ca) 2.70 (Sr) 5.30 (Ba)
100% 100% 100% 100% 100%
Fig. 3. The size of the zone of inhibition formed around each disc, loaded with test samples, indicating the antifungal activity towards C. albicans for the pure ZnO and doped ZnO NPs.
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3.4. Growth curves of C. albicans in the presence of Mg doped ZnO NPs The growth inhibitory potential of Mg doped ZnO NPs against C. albicans was determined at different concentrations of the Mg doped ZnO samples at different times by comparing it with control sample (without Mg doped ZnO NPs). Fig. 4 represents the optical density (OD) value at 660 nm for different concentrations (100–1000 μg/ml) at different times. From the growth curves, it is clearly evident that the presence of Mg doped ZnO NPs retarded the growth of C. albicans and moreover the rate of inhibition was strongly dependent on the concentration of the nanoparticles. In the growth curve, the considerable incline in the logarithmic phase of the control sample was not observed in the test sample, which indicated the delayed logarithmic phase in the Mg doped ZnO NP treated samples. This is because in photocatalyst, first order kinetics is more active and under visible light irradiation, the valence band holes are created by a photocatalytic effect, which is responsible for the formation of hydroxide and superoxide molecules [33]. 3.5. Minimal Fungicidal Concentration (MFC) The measurement of the Minimal Fungicidal Concentration (MFC) elucidated that a low concentration of Mg-doped ZnO NP sample was required for impairing the viability of C. albicans. Various concentrations of Mg-doped ZnO NP sample (100–2000 μg/ml) were added to potato dextrose broth (PDB) containing C. albicans and their survival capacity was analyzed (Fig. 5). It was found that the growth of C. albicans was reduced as the concentration of Mg doped sample increased. The MFC of the Mg doped ZnO sample was found to be 2000 μg/ml for which a complete inhibition in the growth of C. albicans was observed. 3.6. Impact of histidine on the growth curves of C. albicans for Mg doped ZnO NPs From Figs. 4 and 5, it is clear that the presence of Mg doped ZnO NPs inhibited the growth of C. albicans. The most likely mechanism contributing to the antifungal activity of the nanoparticles is the generation of ROS. ROS are probably best known in biology for their ability to cause oxygen species. They can damage DNA, cell membranes, and cellular proteins, and may even lead to cell death. Hydroxyl radical (•OH) is the most reactive oxygen radical known, and reacts very quickly with almost every type of molecule found in living cells [34]. Such reactions will probably dominate the recombination of two •OH radicals to form 2.5
Control 100µg/ml 200µg/ml 400µg/ml 600µg/ml 800µg/ml 1000µg/ml
OD (660 nm)
2.0
1.5
Fig. 5. Determination of Minimal Fungicidal Concentration (MFC) for Mg doped ZnO NPs.
hydrogen peroxide (H2O2). Superoxide anion radical (•O− 2 ), on the other hand, poorly permeates to cell membranes and is less toxic [35, 36]. Hydrogen peroxide is also considered to be a weaker oxidizer, but it can cause cell damage via hydroxyl radicals produced by the Fenton reaction [37,15]. In addition, hydrogen peroxide in the presence of ( • O− 2 ) can generate singlet oxygen, which is very toxic and has the greatest biological significance. ROS are produced continuously in all cells as metabolic byproducts of a number of intracellular systems. ·O
2
−
−
1
þ H2 O2 ⇁OH þ OH· þ O2
However, this process requires a metal or other catalysts [37]. Histidine plays an important role in the functioning of all biosystems. In biological systems, free radicals are generated due to interaction of biomolecules with molecular oxygen. These free radicals are responsible for degradation of biomolecules. Oxidation is also accountable for nutritional quality deterioration. Consumption of oxidized foods generates lipid peroxides and low molecular weight compounds which cause damage to the cell membrane. In the biological systems, histidine plays an important role for scavenging of these toxic free radicals. Histidine is a known scavenger of the hydroxyl radical and singlet oxygen [23,38]. In the present study, the effect of histidine on the fungicidal activity of the Mg doped ZnO NPs was studied by analyzing the optical density with different concentrations of histidine. The growth of C. albicans cultures containing Mg doped ZnO NPs (1000 μg/ml) with various amounts of histidine was estimated and the data are presented in Fig. 6. It is clear that the concentration of histidine influences the fungicidal activity of the Mg doped ZnO NPs. It is found that fungicidal activity is lower at higher histidine concentration. The sample containing Mg doped ZnO NPs with 5 mM of histidine showed a growth profile more or less matched that of control sample, indicating that the generated ROS is effectively scavenged by histidine.
1.0
0.5
0.0 0
10
20
30
40
50
Time (h) Fig. 4. Growth of C. albicans at different concentrations of Mg doped ZnO NPs.
Fig. 6. Growth of C. albicans in the Mg doped ZnO nanoparticle cultures containing different concentrations of histidine.
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The obtained results thus reveal that the antifungal activity of the Mg doped ZnO NPs is primarily related to the ROS mechanism. 3.7. Microscopic investigation of C. albicans The antifungal activity of the Mg doped ZnO NPs on the C. albicans was observed using SEM analysis to look for structural changes in the outer membrane of the cells (Fig. 7(a–b)). From their EDAX spectra (Fig. 7(c–d)), it is revealed that these NP aggregates contain Zn and Mg elements, showing a number of Mg doped ZnO NPs gathered on the surface of C. albicans (Fig. 7d) but there is no any other metal ions present in the control specimen (Fig. 7c). Fig. 7a shows that the untreated control cells of fungal species did not exhibit extensive injury to the cell membrane. In the case of Mg doped ZnO NPs treated with C. albicans, the cells are marked by the circles as shown in Fig. 7b. The Mg doped ZnO NPs are on the surface of C. albicans, which leads to disruption and disorganization of membranes. Thus, membrane damage may be due to one of the important mechanisms of antifungal activity of the Mg doped ZnO NPs against C. albicans. As explained earlier (Section 3.3), the highly active free radicals (hydroxyl radical (OH•), su• peroxide anion (O− 2 ) and perhydroxyl radical (HO2)) damage the cells of microorganism. But, the released Zn ions bind into proteins to deactivate them and the ions interact with microbial membrane to cause structural change and permeability. Moreover, since the Mg doped ZnO NPs are accumulated on the microbial membrane, they result in the membrane disorganization and microbial cellular internalization. 3.8. Zeta potential analysis The adsorption ability of the Mg doped ZnO NPs with C. albicans was studied by the measurement of their zeta potentials as shown in Fig. 8. Yin et al. have reported that the zeta potentials are positive for pure ZnO and transition metal doped ZnO NPs (pure ZnO NPs for 16.1 mV, Fe doped ZnO NPs for 12.2 mV and Mn doped ZnO NPs for 10.6 mV) [39]. In the present study, the control solution untreated with C. albicans
becomes negatively charged. After incubation, the potential of the Mg doped ZnO NPs treated with C. albicans is found to be positive. The doped ZnO NPs carrying a positive charge are prone to contact with the C. albicans cell surface carrying a net negative charge. The antifungal activity is observed when the Zn2+ released by ZnO comes into contact with the cell membranes of the microbe with a negative charge due to electrostatic attraction. Hence, Zn2+ penetrates into the cell membrane and reacts with sulfhydryl groups inside the cell membrane. This activity of synthetase in the microbe becomes so damaged that the cells lose the ability of growth through cell division, which leads to the death of the microbes. The binding of the Mg doped ZnO NPs with C. albicans driven by electrostatic interaction is shown in Fig. 8.
4. Conclusions In summary, undoped and alkaline metal ion doped ZnO NPs were prepared through the co-precipitation method. From the XRD analysis, average crystallite size was found to be 38 nm, 31 nm, 37 nm, 23 nm and 28 nm for pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. The size of ZnO NPs was decreased by the doping of alkaline metal ions. This reduction in the particle's size was mainly due to the distortion in the host ZnO lattice by the foreign impurities. From the HRSEM images, the particles exhibited a nanoflake like morphology. The antifungal studies performed against C. albicans strains showed that the Mg-doped ZnO NPs possessed a greater antifungal effect than the other alkaline metal ion doped ZnO NPs. The replacement of Mg in Zn sites enhanced the photoactivity of the particles as compared to the ZnO NPs. The increasing number of oxygen vacancies and interstitial oxygen vacancies in the Mg-doped ZnO NPs lead to a higher number of ROS as compared to the ZnO NPs. The growth of C. albicans was found to be reduced as the concentration of Mg doped ZnO sample increased. At a minimum concentration of 2000 μg/ml, the growth of C. albicans was completely inhibited. The generation of ROS as the key mechanism was confirmed by the observation that the fungicidal activity of the Mg doped ZnO NPs was considerably reduced by the addition of 5 mM
Fig. 7. FESEM images of (a) control and (b) C. albicans treated with 2 mg/ml of Mg doped ZnO NP slurries for 16 h. EDAX spectra of (c) control and (d) C. albicans treated Mg doped ZnO NPs.
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Fig. 8. Zeta potential of Mg doped ZnO before and after exposure with C. albicans.
of histidine. The binding of the NPs with the fungi was observed by FESEM analysis and the zeta potential measurement showed the electrostatic attraction between the Mg doped ZnO NPs and C. albicans. Acknowledgment One of the authors (A.S.H.) is grateful to the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India for sanctioning the financial assistance (F. No. SR/FTP/ PS-049/2013). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
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[16] E.S. Jang, J.H. Won, S.J. Hwang, J.H. Choy, Adv. Mater. 18 (2006) 3309–3312. [17] U.K. Gautam, M. Imura, C.S. Rout, Y. Bando, X. Fang, B. Dierre, L. Sakharov, A. Govindaraj, T. Sekiguchi, D. Golberg, C.N. Rao, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 13588–13592. [18] A.S. Haja Hameed, C. Karthikeyan, S. Sasikumar, V. Senthil Kumar, S. Kumaresan, G. Ravi, J. Mater. Chem. B 1 (2013) 5950–5962. [19] J. Lodder, The Yeasts: A Taxonomic Study, 2nd ed. North Holland, Amsterdam, 1970. [20] T.J. Rogers, E. Balish, Microbiol. Rev. 44 (1980) 660–682. [21] A. Bacci, C. Montagnoli, K. Perruccio, S. Bozza, R. Gaziano, L. Pitzurra, A. Velardi, C.F. d'Ostiani, J.E. Cutler, L. Romani, J. Immunol. 168 (2002) 2904–2913. [22] T.S. Richards, B.G. Oliver, T.G. White, J. Antimicrob. Chemother. 62 (2008) 349–355. [23] A. Lipovsky, Y. Nitzan, A. Gedanken, R. Lubart, Nanotechnology 22 (2011) 105101. [24] P. Suyana, S. Nishanth Kumar, B.S. Dileep Kumar, B.N. Nair, C.P. Suresh, A. Peer Mohamed, K.G.K. Warrier, U.S. Hareesh, RSC Adv. 4 (2014) 8439–8445. [25] J.P. Breen, M. Marella, C. Pistarino, J.R.H. Ross, Catal. Lett. 80 (2002) 123–128. [26] G.X. Tong, F.F. Du, Y. Liang, Q. Hu, R.N. Wu, J.G. Guan, X. Hu, J. Mater. Chem. B 1 (2013) 454–463. [27] W. Jiang, H. Mashayekhi, B. Xing, Environ. Pollut. 157 (2009) 1619–1625. [28] C. Karunakaran, P. Gomathisankar, G. Manikandan, Mater. Chem. Phys. 123 (2010) 585–594. [29] M. Fang, J.H. Chen, X.L. Xu, P.H. Yang, H.F. Hildebarnd, Int. J. Antimicrob. Agents 27 (2006) 513–517. [30] R.K. Dutta, B.P. Nenavathu, M.K. Gangishetty, A.V. Reddy, Colloids Surf. B Biointerfaces 94 (2012) 143–150. [31] W.W. Wilson, M.M. Wade, S.C. Holman, F.R. Champlin, J. Microbiol. Methods 43 (2001) 153–164. [32] X. Wang, F. Yang, W. Yang, X. Yang, Chem. Commun. (2007) 4419–4421. [33] N. Talebian, M.R. Nilforoushan, E.B. Zargar, Appl. Surf. Sci. 258 (2011) 547–555. [34] C.S. Moody, H.M. Hassa, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 2855–2859. [35] C.E. Schwartz, J. Krall, L. Norton, K. McKay, D. Kay, R.E. Lynch, J. Biol. Chem. 258 (1983) 6277–6281. [36] H.M. Hassan, I. Fridovich, J. Biol. Chem. 254 (1979) 10846–10852. [37] H. Rosen, S.J. Klebanoff, J. Exp. Med. 149 (1979) 27–39. [38] M. Shoeb, B.R. Singh, A.K. Javed, W. Khan, B.N. Singh, H.B. Singh, A.H. Naqvi, Adv. Nat. Sci. Nanosci. Nanotechnol. 4 (2013) 035015 (1–11). [39] H. Yin, P.S. Casey, RSC Adv. 4 (2014) 26149–26157.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Effect of Mg2 +, Ca2 +, Sr2 + and Ba2 + metal ions on the antifungal activity of ZnO nanoparticles tested against Candida albicans Abdulrahman Syedahamed Haja Hameed a,⁎, Chandrasekaran Karthikeyan a, Venugopal Senthil Kumar b, Subramanian Kumaresan b, Seemaisamy Sasikumar a a b
PG and Research Department of Physics, Jamal Mohamed College, Tiruchirappalli 620 020, Tamil Nadu, India Department of Plant Biology and Plant Biotechnology, R.K.M. Vivekananda College, Chennai 600 004, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 12 November 2014 Received in revised form 3 February 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: ZnO nanoparticles Metal doping Antifungal activity Candida albicans XRD SEM
a b s t r a c t The antifungal ability of pure and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO nanoparticles (NPs) prepared by the co-precipitation method was tested against the pathogenic yeast, Candida albicans (C. albicans), and the results showed that the Mg-doped ZnO NPs possessed greater effect than the other alkaline metal ion doped ZnO NPs. The impact of the concentration of Mg doped ZnO sample on the growth of C. albicans was also studied. The Minimal Fungicidal Concentration (MFC) of the Mg doped ZnO NPs was found to be 2000 μg/ml for which the growth of C. albicans was completely inhibited. The ZnO:Mg sample (1.5 mg/ml) with various concentrations of histidine reduced the fungicidal effect of the nanoparticles against C. albicans, which was deliberately explained by the role of ROS. The ZnO:Mg sample added with 5 mM of histidine scavenged the ample amount of generated ROS effectively. The binding of the NPs with fungi was observed by their FESEM images and their electrostatic attraction is confirmed by the zeta potential measurement. © 2015 Published by Elsevier B.V.
1. Introduction Zinc Oxide (ZnO) is an n-type semiconductor with a wide direct band gap of 3.37 eV and a high exciton binding energy of approximately 60 meV. It has also attracted an attention for use in electrical and optical applications such as light emitting diodes, piezoelectric transducers and photo-catalysts [1–3]. In recent years, the photocatalytic and antibacterial activities of the ZnO nanoparticles (Zn NPs) have attracted interest for investigation [4]. The ZnO NPs are believed to be nontoxic, safe and biocompatible. They have also been used as drug carriers, cosmetics, and fillings in medical materials [5]. Several reports have addressed the harmful impact of nanomaterials on living cells, but relatively low concentrations of ZnO are nontoxic to eukaryotic cells [6–11]. The ZnO NPs significantly inhibit the growth of a wide range of pathogenic bacteria under normal visible lighting conditions [12]. Several studies suggest that different morphologies (particle's size and shape) of ZnO NPs have different degrees of antimicrobial activities [12,13]. The antimicrobial activity of ZnO NPs generally depends on the presence of more reactive oxygen species (ROS), which is mainly attributed to the larger surface area of NPs, an increase in oxygen vacancies, the diffusion ability of the reactant molecules and the release of Zn2+ [14]. The superoxide radical, hydroxyl radical and hydrogen peroxide belonging to the ROS group can cause damage to DNA and cellular ⁎ Corresponding author. E-mail address: [email protected] (A.S. Haja Hameed).
http://dx.doi.org/10.1016/j.msec.2015.03.030 0928-4931/© 2015 Published by Elsevier B.V.
proteins, and may even lead to cell death [15]. Generally, nanoparticles with better photocatalytic activity have larger specific surface area and smaller crystal size which increase oxygen vacancies, resulting in more ROS [14]. In addition, the crystal growth direction is another important factor for the generation of ROS. Earlier studies have proved that the terminal polar Zn (0001) faces showed the highest photocatalytic activity for H2O2 generation [16]. Similarly, the Zn polar terminal has exhibited strong UV luminescence rather than the O nonpolar terminal [17]. The antimicrobial effect of the ZnO samples was found to be mainly due to the combination of ROS and the release of Zn2 +. In an earlier report [18], it showed that the Mg-doped ZnO NPs had the highest antimicrobial activity as compared to that of pure ZnO NPs. The genus Candida is a compilation of some 150 asporogenous yeast species. These Candida species are usually restricted among the fungi in the class deuteromycetes because of their inability to form a sexual stage [19]. Candida albicans is a dimorphic fungus existing both in the blastospore phase (synyeast phase, blastocomidial phase) and the hyphal or mycelial phase. C. albicans infections (candidiasis) may be divided into two stages; superficial (such as oral and vaginal thrush and chronic mucocutaneous candidiasis) and deep-seated (such as Candida due myocarditis and acute disseminated Candida septicemia). In superficial candidacies, the organism may exist in the blastospore phase and typically colonizes the mucocutaneous surfaces of the mouth and vagina, whereas in the deep-seated candidiasis, the hyphal phase can be a portal of entry into deeper tissues and able to cause a variety of infections when host defense systems are compromised [20].
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C. albicans is the most common opportunistic fungal pathogen of humans and animals, and moreover it is also a very frequent hospital-acquired infection, and is the fourth leading cause of nosocomial infection in the USA [21,22]. For several reasons (immunosuppressive treatments, long-term catheterization, use of broadspectrum antibiotics and longer survival of immunologically compromised individuals), Candida infections have increased dramatically over the last two decades. Hence C. albicans has drawn a major medical importance among the other species of its genus. Lipovsky et al. (2011) investigated the antifungal potential of ZnO nanoparticles against C. albicans [23]. However, to the best of our knowledge, the antifungal properties have not been reported for alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs against C. albicans. In the present investigation, pure and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+)-doped ZnO NPs are synthesized by the co-precipitation method. We have studied the structural and antifungal properties of the pure and alkaline metal ion doped ZnO NPs.
2. Material and methods 2.1. Preparation and characterization of the pure and alkaline metal ion doped ZnO NPs The following high purity chemicals such as Zinc (II) nitrate hexahydrate (Zn(NO3)2·6H2O), Magnesium nitrate hexahydrate (Mg(NO3)2·6H2O), Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), Strontium nitrate (Sr(NO3)2), Barium nitrate (Ba(NO3)2), Sodium hydroxide (NaOH) and Polyethylene glycol (PEG) were used as the precursors without further purification. The details of the experimental procedure for the preparation of pure ZnO and alkaline metal (Mg, Ca, Sr and Ba) doped ZnO samples have been reported in our previous paper [18]. The X-ray diffraction (XRD) patterns of the ZnO NP samples were collected using an X'PERT PRO PANalytical X-ray diffractometer with a CuKα (40 kV, 30 mA) radiation source. The ZnO NP samples were gently crushed before being smeared on a clean glass slide. The powder diffraction patterns were collected over the 2θ range between 10° and 80° with a scan speed and sampling width of 2 min−1 and 0.05° respectively. HRSEM was performed on the ZnO samples using a FEI-QUANDA 200F microscope operating at 30 kV. The microscope was equipped with a charge-coupled device (CCD) camera. The samples were prepared by 1 mg of ZnO NP samples coated with 1.2 nm gold particle separation on a carbon tape using the low vacuum. Energy dispersive X-ray (EDX) analysis was done using an EDX spectrometer (model: AMETEK) with a FEI-QUANDA 200F high resolution scanning electron microscope operated at 30 kV. The dry powdered samples were attached to the substrate using a double-sided carbon tape and mounted onto the sample holder.
2.3. The concentration dependent effect of Mg doped ZnO NPs on C. albicans The fungicidal activity of the Mg doped ZnO NPs was quantitatively ascertained against the C. albicans by the optical density (OD) method at 660 nm. Different concentrations of Mg doped ZnO NP samples were added to the C. albicans culture (0.5 McFarland standard) and subsequently the OD was measured at different time intervals. C. albicans culture without the addition of NPs served as the control. 2.4. Determination of Minimum Fungicidal Concentration The minimal quantity of the Mg doped ZnO NPs required for the fungicidal activity was resolved by adding different concentrations of the samples into the C. albicans culture and the OD was measured after 24 h. 2.5. The role of reactive oxygen species (ROS) in antifungal activity The ROS as the key factor in antifungal activity was assessed with the help of histidine, a well-known scavenger of ROS. C. albicans culture containing a definite quantity of Mg doped ZnO NP sample (1000 μg/ml) was treated with various amounts of histidine and the change in the OD was determined after 24 h. C. albicans culture without the addition of histidine served as the control. 2.6. Preparation of fungal samples for Field Emission Scanning Electron Microscope (FESEM) The control and Mg doped ZnO NP treated C. albicans cells were centrifuged at 6000 rpm for 10 min. at 4 °C. The pellets were gently and doubly washed with PBS (0.1 M) and fixed with 2.5% glutaraldehyde in PBS at 4 °C for 2 h. After fixation, the samples were dehydrated with 25, 50 and 75% ethanol series at one time each and then dehydrated with 100% ethanol by three times for 10 min. The dehydrated samples were kept overnight in a desiccator and thereafter gold coated by sputtering. The samples were then analyzed by FESEM (model: SUPRA: CARL ZEISS 55) with EDAX (model: ULTRA 55). 2.7. Zeta-potential measurement The zeta-potential measurement was conducted for the control and Mg doped ZnO NP treated C. albicans samples by a ZetaPlus Zeta Potential Analyzer (nano-ZS90). The minimum fungal concentration of the NPs was 2 mg/ml. For both samples, an appropriate amount of undiluted solution was placed into the cuvette, and the average zeta potential values were obtained for the control and treated samples as individual measurements. Water is the solution medium for all zeta potential measurements. 3. Results and discussion 3.1. Phase and crystal structure analysis
2.2. Antifungal assay Antifungal activity was determined by an agar disc diffusion method against the test fungi C. albicans (ATCC 10231) using potato dextrose agar [24]. The test strain was transferred into potato dextrose broth (PDB) and incubated at 30 °C until it achieved the turbidity of 0.5 McFarland standard. The media plates were inoculated with the test strain by streaking for 2–3 times by rotating the plate at a 60° angle for each streak to ensure uniform distribution of inoculum. Subsequently, sterile disc (6 mm) loaded with 1 mg of test samples (ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba) was placed onto the inoculated plates using sterile forceps and incubated at 30 °C for 24 h under visible light. The zone of inhibition formed around the disc was measured and recorded.
The phase and crystallinity of the as-synthesized pure and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs are studied by XRD and their patterns are shown in Fig. 1. The intense and sharp diffraction peaks suggest that the pure and alkaline metal ion doped ZnO NPs are highly crystalline in nature. The standard diffraction peaks show the hexagonal wurtzite structure for the pure ZnO NPs (space group: P63mc). This is also confirmed by the JCPDS data (card no. 361451). There is no impurity phase found in Magnesium and Calcium doped ZnO samples because of the ionic radii of Mg (0.66 Å) and Ca (0.99 Å). Furthermore, other dopants doped with ZnO have ionic radii above 1 Å (i.e., 1.13 Å for Sr2 + and 1.35 Å for Ba2 +) and they have two additional peaks located around 25.115° & 25.76° and 23.83° & 24.25° respectively, which correspond to the (111) and (021) planes. These two peaks indicate the presence of SrCO3 and BaCO3 respectively
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(e)
* BaCo3
* SrCo3
* * (c)
Intensity (a.u.)
3.2. Energy dispersive analysis of X-ray (EDAX) The compositional analysis of the pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs was carried out using EDAX. From the EDAX analysis, the amounts of alkaline metal ions present in the doped ZnO NPs are given in Table 1. In the doped samples, the concentrations of Mg, Ca, Sr and Ba are found to be 1.87%, 2.04%, 2.70% and 5.30% respectively. In the pure ZnO NPs, the chemical compositions of Zn and O are found to be 86.60% and 13.40% respectively. However, for the alkaline metal ion-doped ZnO NPs, the zinc percentage decreases whereas the oxygen percentage increases. In the ZnO NPs doped with magnesium, which has a smaller ionic radius, the compositions of Zn and O are estimated as 86.30% and 11.83% respectively. As compared to the pure ZnO NPs, a decrease in the oxygen percentage is observed for the Mg-doped ZnO NPs due to the effect of the smaller ionic radius Mg2+ metal ions.
** (d)
173
3.3. Antifungal studies
20
30
40
50
60
70
(202)
(103) (200) (112) (112) (004)
(102)
(110)
(100) (002)
(a)
(101)
(b)
80
2θ (deg) Fig. 1. X-ray powder diffraction patterns of (a) pure ZnO, (b) ZnO:Mg, (c) ZnO:Ca, (d) ZnO:Sr and (e) ZnO:Ba NPs [18].
for the ZnO:Sr and ZnO:Ba NPs. Thus, Sr(NO3) and Ba(NO3) are decomposed as SrO and BaO on the surface of ZnO NPs when calcined at higher temperature and then the formed SrO and BaO can absorb atmospheric CO2 leading to the formation of SrCO3 and BaCO3 [25,18]. The values of lattice constants ‘a’ and ‘c’ of pure ZnO NPs are 3.2564 Å and 5.2151 Å respectively. The values of lattice constants of the alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs are increased. But, interestingly, in the Ba2+ doped ZnO NPs, the values of lattice constants decrease as compared to that of pure ZnO NPs, because the barium atoms trapped in non-linear equilibrium position were shifted to a more equilibrium position. The values of lattice constants ‘a’ and ‘c’ are 3.2572 Å & 5.2107 Å, 3.2571 Å & 5.2142 Å, 3.2566 Å & 5.2157 Å and 3.2544 Å & 5.2106 Å for ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba respectively. The average crystallite sizes are found to be 38 nm, 31 nm, 37 nm, 23 nm and 28 nm for pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. The average crystallite size decreases in the Mg2 +, Ca2+, Sr2+ and Ba2+ doped ZnO NPs as compared to that of pure ZnO NPs. The reduction in the particle's size is mainly due to the distortion in the host ZnO lattice by the foreign impurities i.e., Mg2+, Ca2+, Sr2+ and Ba2+. From the HRSEM images (Fig. 2), the pure ZnO and alkaline metal ion (Mg2+, Ca2+, Sr2+ and Ba2+) doped ZnO NPs exhibited a nanoflake like morphology. The aggregation of nanoflakes is observed in the Mg2+ doped ZnO NPs. The average thicknesses of the nanoflakes are 63 nm, 62 nm, 47 nm, 53 nm and 52 nm for the pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. The size of the nanoflakes in alkaline metal ion doped ZnO NPs decreases in comparison with that of the pure ZnO nanoflakes. The reduction of thickness is due to the distortion in the host metal by the foreign impurities that decrease the nucleation and subsequent growth rate of the ZnO NPs.
In the present investigation, pure ZnO and alkali metal ions Mg, Ca, Sr, Ba doped ZnO NPs were tested against C. albicans (ATCC 10231) using the disc diffusion method to analyze their ability as a potential candidate for an antifungal agent. The magnitude of the antifungal agent was determined, based on the size of the inhibition zone formed around each disc loaded with an appropriate test sample. The antimicrobial activity of ZnO NPs generally depends on the presence of higher ROS that generally comes from a better photocatalyst with larger surface area, crystallite size, increased oxygen vacancies, diffusion ability of the reactant molecules and the release of Zn2 + [14,26,18]. The oxidative stress induced by ROS generation, interaction of oxide NPs with the microbial cell wall and possible permeation of the NPs into the microbial cell are still not clearly understood [27]. Once inside the cell, nanomaterials induce intercellular oxidative stress by distributing the balance between oxidant and anti-oxidant processes. On the one hand, the oxidative stress induced by exposure to nanomaterials may stimulate an increase of the cytosolic calcium concentration or may cause the translocation of transcription factors to the nucleus, which regulate pro-inflammatory genes. Alternatively, exceeding oxidative stress may also modify protein, lipids and nucleic acids, which further stimulates the anti-oxidant defense system or even leads to cell death [28]. Generally, nanoparticles with better photocatalytic activity having a large specific surface area and a small crystallite size which increase oxygen vacancies, result in more ROS [14]. Fig. 3 shows the diameter of the inhibition zone formed around discs. Even though all of the samples exhibited antifungal activity, the Mg doped ZnO NPs showed the highest activity. The antifungal activity of pure ZnO and alkaline metal ion doped ZnO NPs is explained as follows. As mentioned earlier, the greater number of ROS is mainly attributed to the larger surface area, increase in oxygen vacancies and the diffusion ability of the reactant molecules. In the present investigation, the antifungal effect of the ZnO samples is mainly due to the combination of various factors such as ROS and the release of Zn2 +. The photo-catalysis seems to be the most important antifungal mechanism; ROS produced on the surface of these NPs in the presence of light cause oxidative stress in microbial cell and eventually lead to the death of the cell. ROS contain the most reactive hydroxyl radical (•OH), less toxic superoxide anion radical (•O− 2 ) and hydrogen peroxide with a weaker oxidizer (H2O2). The generation of H2O2 can penetrate into the cell membrane and kill the microbes [29]. The mechanism of light induced generation of ROS can be given as follows [30]. −
ZnO þ hυ⇁e þ h
þ
þ þ h þ H2 O⇁·OH þ H
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Fig. 2. HRSEM images of (a) pure ZnO, (b) ZnO:Mg, (c) ZnO:Ca, (d) ZnO:Sr and (e) ZnO:Ba NPs [18].
−
e þ O2 ⇁·O2
·O
2
−
−
þ
þ H ⇁HO2 · þ
−
HO2 · þ H þ e ⇁H2 O2 The (0001) Zn-polar faces and the increased number of Zn2+ ions enhance the diffusion ability of the Zn2+ ions, which is supported by the level of the intensity ratio (I(100)/I(002)) [26,18]. In previous report, the ratio is 1.1770 for ZnO NPs. The values of the intensity ratio are 1.232, 0.97, 1.1732 and 1.1006 for ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. From the results of the photoluminescence study for the ZnO and alkaline metal ion-doped ZnO NPs, the green and yellow emissions are due to the presence of single ionized oxygen vacancies and interstitial oxygen vacancies respectively [18]. The ZnO NPs exposing the [0001] Zn-polar surface carrying a positive charge are prone to contact with the C. albicans cell surface carrying a net negative charge via electrostatic force [31,26]. Such contact may not only inhibit fungal growth but also be killed by the generated ROS. In the Mg doped ZnO NPs, there is no release of Mg2 +. Thus, the release of Mg2+ may not be responsible for the biocidal activity of the sample. However, this increased activity in the Mg doped ZnO NPs is caused by replacement of Mg in Zn sites, which enhances the
photoactivity of the particles because of the similarity in ionic radii between Mg2+ (0.66 Å) and Zn2+ (0.74 Å). This can be explained as follows: the Mg doped ZnO has the highest value intensity ratio I(100)/I(002) (1.232) as compared to that of the pure ZnO, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs, possessing a greater proportion of (0001) Zn-polar faces which enhances the diffusion ability of Zn2+ ions [18]. The wavelengths of the green and yellow emissions are at 549 nm and 573 nm for the Mg doped ZnO NPs, whereas the wavelengths of the emissions are at 546 nm and 569 for the pure ZnO NPs. This shows the increased number of oxygen vacancies and interstitial oxygen vacancies in the Mg-doped ZnO NPs, leading to a higher number of ROS as compared to the ZnO NPs [18]. The NPs with uneven surfaces and rough edges have been found to adhere to the microbial wall and cause damage to the cell membrane [32]. From the HRSEM images, it is clear that the Mg-doped ZnO NPs have uneven ridges at their outer surfaces which lead to antifungal activity, whereas the other NPs have smooth surfaces.
Table 1 The elemental composition of the synthesized pure and doped ZnO NPs. Sample
ZnO ZnO:Mg ZnO:Ca ZnO:Sr ZnO:Ba
Weight % Zn
O
Doping amount
Total
86.60 86.30 81.27 79.64 73.18
13.40 11.83 16.69 17.65 21.52
– 1.87 (Mg) 2.04 (Ca) 2.70 (Sr) 5.30 (Ba)
100% 100% 100% 100% 100%
Fig. 3. The size of the zone of inhibition formed around each disc, loaded with test samples, indicating the antifungal activity towards C. albicans for the pure ZnO and doped ZnO NPs.
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3.4. Growth curves of C. albicans in the presence of Mg doped ZnO NPs The growth inhibitory potential of Mg doped ZnO NPs against C. albicans was determined at different concentrations of the Mg doped ZnO samples at different times by comparing it with control sample (without Mg doped ZnO NPs). Fig. 4 represents the optical density (OD) value at 660 nm for different concentrations (100–1000 μg/ml) at different times. From the growth curves, it is clearly evident that the presence of Mg doped ZnO NPs retarded the growth of C. albicans and moreover the rate of inhibition was strongly dependent on the concentration of the nanoparticles. In the growth curve, the considerable incline in the logarithmic phase of the control sample was not observed in the test sample, which indicated the delayed logarithmic phase in the Mg doped ZnO NP treated samples. This is because in photocatalyst, first order kinetics is more active and under visible light irradiation, the valence band holes are created by a photocatalytic effect, which is responsible for the formation of hydroxide and superoxide molecules [33]. 3.5. Minimal Fungicidal Concentration (MFC) The measurement of the Minimal Fungicidal Concentration (MFC) elucidated that a low concentration of Mg-doped ZnO NP sample was required for impairing the viability of C. albicans. Various concentrations of Mg-doped ZnO NP sample (100–2000 μg/ml) were added to potato dextrose broth (PDB) containing C. albicans and their survival capacity was analyzed (Fig. 5). It was found that the growth of C. albicans was reduced as the concentration of Mg doped sample increased. The MFC of the Mg doped ZnO sample was found to be 2000 μg/ml for which a complete inhibition in the growth of C. albicans was observed. 3.6. Impact of histidine on the growth curves of C. albicans for Mg doped ZnO NPs From Figs. 4 and 5, it is clear that the presence of Mg doped ZnO NPs inhibited the growth of C. albicans. The most likely mechanism contributing to the antifungal activity of the nanoparticles is the generation of ROS. ROS are probably best known in biology for their ability to cause oxygen species. They can damage DNA, cell membranes, and cellular proteins, and may even lead to cell death. Hydroxyl radical (•OH) is the most reactive oxygen radical known, and reacts very quickly with almost every type of molecule found in living cells [34]. Such reactions will probably dominate the recombination of two •OH radicals to form 2.5
Control 100µg/ml 200µg/ml 400µg/ml 600µg/ml 800µg/ml 1000µg/ml
OD (660 nm)
2.0
1.5
Fig. 5. Determination of Minimal Fungicidal Concentration (MFC) for Mg doped ZnO NPs.
hydrogen peroxide (H2O2). Superoxide anion radical (•O− 2 ), on the other hand, poorly permeates to cell membranes and is less toxic [35, 36]. Hydrogen peroxide is also considered to be a weaker oxidizer, but it can cause cell damage via hydroxyl radicals produced by the Fenton reaction [37,15]. In addition, hydrogen peroxide in the presence of ( • O− 2 ) can generate singlet oxygen, which is very toxic and has the greatest biological significance. ROS are produced continuously in all cells as metabolic byproducts of a number of intracellular systems. ·O
2
−
−
1
þ H2 O2 ⇁OH þ OH· þ O2
However, this process requires a metal or other catalysts [37]. Histidine plays an important role in the functioning of all biosystems. In biological systems, free radicals are generated due to interaction of biomolecules with molecular oxygen. These free radicals are responsible for degradation of biomolecules. Oxidation is also accountable for nutritional quality deterioration. Consumption of oxidized foods generates lipid peroxides and low molecular weight compounds which cause damage to the cell membrane. In the biological systems, histidine plays an important role for scavenging of these toxic free radicals. Histidine is a known scavenger of the hydroxyl radical and singlet oxygen [23,38]. In the present study, the effect of histidine on the fungicidal activity of the Mg doped ZnO NPs was studied by analyzing the optical density with different concentrations of histidine. The growth of C. albicans cultures containing Mg doped ZnO NPs (1000 μg/ml) with various amounts of histidine was estimated and the data are presented in Fig. 6. It is clear that the concentration of histidine influences the fungicidal activity of the Mg doped ZnO NPs. It is found that fungicidal activity is lower at higher histidine concentration. The sample containing Mg doped ZnO NPs with 5 mM of histidine showed a growth profile more or less matched that of control sample, indicating that the generated ROS is effectively scavenged by histidine.
1.0
0.5
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10
20
30
40
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Time (h) Fig. 4. Growth of C. albicans at different concentrations of Mg doped ZnO NPs.
Fig. 6. Growth of C. albicans in the Mg doped ZnO nanoparticle cultures containing different concentrations of histidine.
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The obtained results thus reveal that the antifungal activity of the Mg doped ZnO NPs is primarily related to the ROS mechanism. 3.7. Microscopic investigation of C. albicans The antifungal activity of the Mg doped ZnO NPs on the C. albicans was observed using SEM analysis to look for structural changes in the outer membrane of the cells (Fig. 7(a–b)). From their EDAX spectra (Fig. 7(c–d)), it is revealed that these NP aggregates contain Zn and Mg elements, showing a number of Mg doped ZnO NPs gathered on the surface of C. albicans (Fig. 7d) but there is no any other metal ions present in the control specimen (Fig. 7c). Fig. 7a shows that the untreated control cells of fungal species did not exhibit extensive injury to the cell membrane. In the case of Mg doped ZnO NPs treated with C. albicans, the cells are marked by the circles as shown in Fig. 7b. The Mg doped ZnO NPs are on the surface of C. albicans, which leads to disruption and disorganization of membranes. Thus, membrane damage may be due to one of the important mechanisms of antifungal activity of the Mg doped ZnO NPs against C. albicans. As explained earlier (Section 3.3), the highly active free radicals (hydroxyl radical (OH•), su• peroxide anion (O− 2 ) and perhydroxyl radical (HO2)) damage the cells of microorganism. But, the released Zn ions bind into proteins to deactivate them and the ions interact with microbial membrane to cause structural change and permeability. Moreover, since the Mg doped ZnO NPs are accumulated on the microbial membrane, they result in the membrane disorganization and microbial cellular internalization. 3.8. Zeta potential analysis The adsorption ability of the Mg doped ZnO NPs with C. albicans was studied by the measurement of their zeta potentials as shown in Fig. 8. Yin et al. have reported that the zeta potentials are positive for pure ZnO and transition metal doped ZnO NPs (pure ZnO NPs for 16.1 mV, Fe doped ZnO NPs for 12.2 mV and Mn doped ZnO NPs for 10.6 mV) [39]. In the present study, the control solution untreated with C. albicans
becomes negatively charged. After incubation, the potential of the Mg doped ZnO NPs treated with C. albicans is found to be positive. The doped ZnO NPs carrying a positive charge are prone to contact with the C. albicans cell surface carrying a net negative charge. The antifungal activity is observed when the Zn2+ released by ZnO comes into contact with the cell membranes of the microbe with a negative charge due to electrostatic attraction. Hence, Zn2+ penetrates into the cell membrane and reacts with sulfhydryl groups inside the cell membrane. This activity of synthetase in the microbe becomes so damaged that the cells lose the ability of growth through cell division, which leads to the death of the microbes. The binding of the Mg doped ZnO NPs with C. albicans driven by electrostatic interaction is shown in Fig. 8.
4. Conclusions In summary, undoped and alkaline metal ion doped ZnO NPs were prepared through the co-precipitation method. From the XRD analysis, average crystallite size was found to be 38 nm, 31 nm, 37 nm, 23 nm and 28 nm for pure ZnO, ZnO:Mg, ZnO:Ca, ZnO:Sr and ZnO:Ba NPs respectively. The size of ZnO NPs was decreased by the doping of alkaline metal ions. This reduction in the particle's size was mainly due to the distortion in the host ZnO lattice by the foreign impurities. From the HRSEM images, the particles exhibited a nanoflake like morphology. The antifungal studies performed against C. albicans strains showed that the Mg-doped ZnO NPs possessed a greater antifungal effect than the other alkaline metal ion doped ZnO NPs. The replacement of Mg in Zn sites enhanced the photoactivity of the particles as compared to the ZnO NPs. The increasing number of oxygen vacancies and interstitial oxygen vacancies in the Mg-doped ZnO NPs lead to a higher number of ROS as compared to the ZnO NPs. The growth of C. albicans was found to be reduced as the concentration of Mg doped ZnO sample increased. At a minimum concentration of 2000 μg/ml, the growth of C. albicans was completely inhibited. The generation of ROS as the key mechanism was confirmed by the observation that the fungicidal activity of the Mg doped ZnO NPs was considerably reduced by the addition of 5 mM
Fig. 7. FESEM images of (a) control and (b) C. albicans treated with 2 mg/ml of Mg doped ZnO NP slurries for 16 h. EDAX spectra of (c) control and (d) C. albicans treated Mg doped ZnO NPs.
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Fig. 8. Zeta potential of Mg doped ZnO before and after exposure with C. albicans.
of histidine. The binding of the NPs with the fungi was observed by FESEM analysis and the zeta potential measurement showed the electrostatic attraction between the Mg doped ZnO NPs and C. albicans. Acknowledgment One of the authors (A.S.H.) is grateful to the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India for sanctioning the financial assistance (F. No. SR/FTP/ PS-049/2013). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
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