Foliar-mediated Ag:ZnO nanophotocatalysts: green synthesis, characterization, pollutants degradation, and in vitro biocidal activity

1 Introduction

On the basis of commendable size ranges particularly miniature ones, nanoparticle (NP) synthetic modes are occupying a significant dimension of nanotechnology. The evolution in this regard has been particularly rapid. These NPs of desirable shapes and sizes are marked with significant biocidal potential; thus, in contrast to the conventional antibiotics, these NPs are capable of exterminating around 650 cells. NPs have been synthesized through different physical and chemical routes enabling the production of NPs having alleviated sizes; however, these routes possess an inherent damage to the environment, economy, and needs manual effort. In contrary to these physicochemical routes, biological entities, i.e. bacteria, fungi, and plants, can be used as the biofactories [1], [2] for NP synthesis. Plants are heavily utilized for this purpose because of their abundant availability and inexpensiveness and there is no requirement for culture maintenance. Biogenic synthesis has been employed for the synthesis of metallic and metal oxide NPs with an aim to make use of environmental-friendly reducing chemicals. Utilization of plant extracts as a green alternative to reducing and stabilizing agents ensures effectiveness over the highly toxic and energy intensive physicochemical routes [3].

ZnO nanoscale materials in varied morphologies, i.e. NPs, nanoflowers, nanowires, nanorods, and thin films, have been known for their remarkable sensing and optical cum electrical properties and thus have been employed in biological and gas sensing applications [4]. ZnO, being a wide bandgap semiconductor of 3.37eV, has been proved as a significant photocatalyst, and it exceeds TiO₂ (3.20eV bandgap) in the case of hydrospheric remediation. Such higher photocatalysis can be attributed to the generation of reactive oxygen species, capacity to mineralize, and provisioning of multiple reactivity sites [5], [6], [7], [8]. Furthermore, ZnO is also advantageous because of economic viability and higher light absorption [5], [6], [7], [8], [9]. Addition of Ag in ZnO as a dopant entity acquires the interstitial sites and alternative Zn²⁺ sites [10], [11]. Such an addition is also reported to enhance the photocatalytic potential of ZnO via enhanced separation of charge and alleviation in the recombining capacity of electron hole [12] and has been utilized for a variety of organic pollutants’ degradation under ultraviolet (UV) light irradiance [13], [14], [15]. Photocatalytic potential improvement of Ag:ZnO can be attributed to the presence of dopant on metallic oxide that gives rise to the charge transfer between the semiconductor ZnO and dopant Ag.

The augmented photoresponse exhibited by the ZnO-based wider bandgap derived products is due to higher intrinsic gains through addition of impurity. For the recompense of n-type conductivity as a result of silver dopant addition, there is a dwindling concentration of donating defects. This in turn plays an important role in enhancing the performance of the Ag-doped ZnO-based devices [15]. Such parameters also enhance the scientific possibility of doped nanomaterials to be used in sensing devices, LEDS, photodetectors, and solar-based devices [16]. Photocatalytic technologies based on incorporation of semiconductors has gained momentum in terms of both application and advancements for environmental remediation of atmospheric and hydrospheric compartments [17]. Doping of different metals on ZnO has been done [10], [18] and found to be playing an influential role in altering the metal oxides’ properties [19]. Scientific knowledge regarding the lower dimensional Ag-doped ZnO materials has been scanty. Metals of Ib are found to be exceeding as diffusers in case of semiconductors [10], [16], [18].

Prunus cerasifera is an angiospermic plant belonging to Rosaceae, Prunoideae, and Prunus. This plant is commonly known as Cherry plum and distributed in European and Asian regions. In Pakistan, it is found in Parachinar valley. Fruits of P. cerasifera are particularly famous because of characteristic sweet sour flavor, and it is being utilized in making wine, jams, and marmalades in addition to its use as a remedy for jaundice. This rich resource has not been employed for its biological functions on a greater scale neither does it find a higher utilization rate. Prunus cerasifera leaves and branches are known for higher quantities of phenols and tannins with an elevated antioxidant action. This plant possesses agronomical significance because it emits various kinds of volatiles, i.e. (E)-3-hexen-1-ol, n-hexanol and n-hexanol, (Z)-3-hexenyl butyrate, and (E)-2-hexenyl butyrate benzaldehyde [20] from different organs that play a role in defense and relationships with surrounding plant community [21]. The current era dominated by urbanization has adopted various nonsustainable patterns triggering environmental degradation. Such patterns involve indiscriminate synthesis of materials and their reckless dumping into water bodies; e.g. textile industries are known for consuming highest dye levels up to 60%. Of the waste dye, 15% drains off to water in unmodified form [22]. Dyes are persistent in nature because of ringed structure, which is not easily degraded by UV/photoactivation mechanisms, thus posing higher toxicity levels. Thus, the photocatalytic potential of ZnO can further be enhanced via doping to combat this issue [23]. In addition to the vulnerability toward persistent pollutants, the planet Earth and its inhabitants are faced with serious issues of pathogenicity caused by various human and agricultural pathogens. The problem is aggravated when these pathogens exhibit multidrug resistance. There is a need to develop miniature materials that not only can be effective toward resistant strains but also are good in terms of the environment. Furthermore, engineered nanomaterials can be influential in biomedical applications because the occurrence of biological phenomenon including many cellular processes is also at nanoscale. Metallic oxides synthesized via physicochemical routes have been employed for antimicrobial activity [24], [25].

In the present investigation, the strong antioxidant potential and underutilization of P. cerasifera leaves has been taken into consideration. The biomimetic synthesis of foliar-mediated silver-doped zinc oxide NPs from P. cerasifera leaf extract (PCLE) as a reducing cum stabilizing agent has been reported for the first time by hydrothermal route. Foliar doped NPs (FDNPs) were characterized for different properties via X-ray diffraction (XRD), ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy. FDNPs were also assessed for their photocatalytic activity against brominated persistent dyes, i.e. bromocresol green (BG) and bromophenol blue (BB). Moreover, the in vitro antimicrobial activity of FDNPs against nine microbes, i.e. Xanthomonas citri, Psuedomonas syringae, Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus terreus, Penicillium chrysogenum, Fusarium solani, and Lasiodiplodia theobromae, was investigated by a standard disc diffusion assay in a dose-dependent manner and compared with the standard antibiotics.

2 Materials and methods

Pure silver nitrate (99%; AgNO₃), zinc acetate dihydrate (C₄H₆O₄Zn·2H₂O), BG (C₂₁H₁₄Br₄O₅S), and BB (C₁₉H₁₀Br₄O₅S) were purchased from Sigma Aldrich, St. Louis, MO, USA. Nutrient agar and potato dextrose agar (PDA) culture media were purchased from Merck, Darmstadt, Germany and Liofilchem, Roseto degli Abruzzi, Italy, respectively. All chemicals were of 99% purity and analytical grade and used without further purification. All experiments were done with deionized water (DI). All of the reagents used were purchased from Sigma Aldrich, St. Louis, MO, USA. Devices used in the experimental work were: hotplate (MSH 20D, Wisestir, Germany), centrifuge (C0060-230V, Labnet International, Inc., Edison, NJ, USA), incubator shaker (Irmeco GmbH, Geesthach, Germany), UV-Vis spectrophotometer (1602, Biomedical services, Madrid, Spain), Fourier transformer infra red (8400, Shimadzu, Tokyo, Japan), GCMS (QP5050, Shimadzu, Tokyo, Japan), XRD (Bruker AXS D-8, Shimadzu, Japan), Furnace (550, Ney Vulcan, Bridgeport, CT, USA), SEM (SEMSS-550 Superscan, Shimadzu, Japan), Laminar Flow (Streamline, Singapore), UV lamp (SN500712, Kohler, Hamburg, Germany) and oven (UN110, Memmert, Schwabach, Germany).

2.1 Reducing agent preparation

Healthy and fresh P. cerasifera leaves were sampled in May 2017 from Alizai, Parachinar, Pakistan (latitude: 33°53′1.29″N, longitude: 70°6′35.49″E). Leaves were twice tap water washed for removal of possible dust particles and shade dried until complete moisture removal. Leaves were then dried in an oven at 60°C for 30 min for achieving complete dried biomass and then ground to fine powder, sieved, and stored at room temperature. For aqueous extract, 30 g of leaves were dispersed into 30 ml of DI in a 250 ml Erlenmeyer flask at 30°C for 10 min. PCLE was then filtered with Whatman no. 1 filter paper (pore size: 11 μm) and refrigerated at 4°C for further use in synthesis of FDNPs (Figure 1). PCLE, before every experiment, was centrifuged at 4000 rpm to ensure complete mixing of extract.

Figure 1:

Prunus cerasifera leaves: (A) tree, (B) leaf powder, (C) leaf extract in deionized water, and (D) filtered leaf extract used as a reductant in foliar doped nanoparticles synthesis.

3 Results and discussion

FDNPs were synthesized with Ag as dopant and C₄H₆O₄Zn·2H₂O as host, and PCLE was used as a reducing cum stabilizing agent for its unique phytoconstituents, e.g. flavonoids, terpenes, tannins, phenols, and other substances of reducing nature. Formation of FDNPs was confirmed by conversion of color from greenish brown to white because of the richness of electron functional groups in leaf extract inducing the synthesis of FDNPs. As FDNPs were synthesized via hydrothermal route, instead of chemical reducing agents, e.g. NaOH and tannic acid meant for precipitating the mixture, the present work has alternatively used PCLE, and no reports are available on the angiospermic plant mediated Ag-doped ZnO NPs. There is a simultaneous nucleation of both the dopant and the host, i.e. Ag and ZnO; however, ZnO has elevated growth rates in comparison to Ag because of variation in heats of formation. Consequently, the hydrothermal green synthetic route enables the formation of Ag NPs on ZnO surface. The antioxidant potential of PCLE is involved in either ion exchange or giving rise to complexation between metallic ions and polyphenols consequently giving rise to reduction of metallic ions to metallic atoms by extract [26].

XRD pattern of Ag:ZnO NPs has exhibited hexagonal wurtzite geometry of crystals with an average crystallite size of 10.02 nm calculated by Scherrer’s equation:

The diffraction pattern found consistent with the Joint Committee on Powder Diffraction Standards card no. 36-1451 has expressed maximum diffraction from (101) crystal plane. Comparatively, smaller peak at 44.5° corresponds to Ag NP crystal planes, thus confirming the presence of Ag in the tested sample (Figure 2A) [27], [28]. Single Ag NP peak is evident because of smaller quantity and remarkable dispersion rates [29]. Furthermore, current results also confirm the production of Ag NPs and the second phase in the case of synthesis of Ag:ZnO NPs [30]. Ag:ZnO NPs were analyzed for the presence of functional groups by FTIR (Figure 2B and Table 1). The IR band at 3462 cm⁻¹ of the O–H stretch corresponding to alcohols and phenols signifies the reducing role of PCLE involved in the synthesis of FDNPs. The IR peaks at 2963, 1574, and 1261 cm⁻¹ were assigned to C-H stretch, N-O stretch, and C-N stretch of alkanes, asymmetric stretch of nitro compounds and aromatic amines, respectively [31]. Furthermore, the band at 1020 cm⁻¹ is assigned to the in-plane vibrational mode of (NO₃⁻) ions. Remarkable doping of Ag:ZnO NPs is expressed in the form of strong vibrational mode. For the calculation of direct energy bandgap of the as synthesized FDNPs, the Tauc plot was used:

Figure 2:

Foliar doped nanoparticles synthesis at highest doped percentage: (A) XRD diffraction spectrum, (B) FTIR spectra, and (C) plot between hν (eV) and (αhν)^1/2 for direct bandgap calculation.

In this equation,

α=absorption coefficient

h=Plank’s constant

ν=vibration frequency

E_g=bandgap.

Tauc plot has been obtained by taking hν on the horizontal and (αhν)² on the vertical axis. Figure 3C expresses the linear behavior and thus indicative of direct transition of the Ag:ZnO semiconducting NPs. The plot was extrapolated, and the conventional bandgap of ZnO, i.e. 3.37 eV, was found to reduce to 3.1 eV after being doped with Ag, thus expressive of p-type conductivity and revealing its effectiveness in optoelectronics. Achievement of reduction in bandgap ensures the potential of FDNPs as conspicuous candidate for nanophotocatalytic and solar cell based devices. The reduction in bandgap can be attributed to the creation of oxygen vacancy, which augments the electronic transference between valence and conduction bands. Scanning electron microscopy micrographs of FDNPs synthesized with different variations of PCLE are shown in Figure 3. It is quite evident from the micrographs that nanosized particles were obtained at all concentrations of PCLE. Size ranges of 60–101.98, 89.44–101.98, 63.25–120, and 60–126.49 nm were obtained for 30, 50, 70, and 90 ml, respectively. Such ranges signify that 30 and 50 ml is the ideal PLCE concentration for production of FDNPs ≤100 nm, while increasing the concentration beyond this produced FDNPs ≥100 nm. It can be because 30 and 50 ml are the optimum concentrations that can initiate the nucleation of Ag and ZnO in the hydrothermal route.

Figure 3:

Scanning electron microscopy micrographs of foliar doped nanoparticles at (A) 30, (B) 50, (C) 70, and (D) 90 ml P. cerasifera leaf extract concentration.

Human being and aquatic flora and fauna are adversely affected by the textile industry effluents being dumped into water bodies with any processing. Dyes of organic nature are one of the major contributors toward water pollution. Such dyes in most of the cases express higher degrees of recalcitrance and nonbiodegradability. Brominated dyes (Figure 4) in this regard are highly prevalent. Thus, efforts have been done with a variety of materials for remediation of BG [32], [33], [34], [35], [36] and BB [32], [37—40] for alleviating the level of harm caused by them to environmental compartments. BG is a brominated dye, which is the most commonly used triphenylmethane dye posing difficulty in breaking down because of the presence of three benzene rings [41]. For BG, photocatalytic and adsorptive processes have been devised to remove it [42], [43], but both of these processes are associated with incurring heavier capital costs and lower efficiencies. BB, also a brominated dye, is one of the most common effluent released from textile and chemical manufacturing plants. It not only interferes with the safety level of water but also affects the aesthetic outlook of water bodies. It is also used as an acid-base indicator and as a color marker for monitoring the process of electrophoretic processes. Excessive BB quantities are known for contamination of lithospheric and hydrospheric compartment because of its profound solubility in water. Thus, remediative technologies have been designed for its removal, e.g. separation mechanisms, precipitative and coagulative processes, oxidation, electrochemical treatment, and adsorptive removal for degradation of organic dyes to benign daughter products [44], [45], [46], [47]. Although efforts have been done for the removal of BG and BB with different materials (Table 2), studies involving foliar-mediated Ag:ZnO have never been reported. Thus, current research has attempted to photocatalyze the degradation of BG and BB with green synthesized FDNPs possessing lower bandgap. Upon observation of achromatization (Figure 5) when FDNPs’ treated dye solution was exposed to solar radiations, UV-Vis absorption spectra for BB and BG were recorded after every 2 min (Figure 6). Ag:ZnO nanophotocatalyst degraded 86% of BG in 12 min and 95% of BB in 14 min, thus signifying the remediative role of FDNPs (Figure 7).

Figure 4:

Chemical structures: (A) bromocresol green and (B) bromophenol blue.

Figure 5:

Foliar doped nanoparticles triggered achromatization in (A) bromocresol green and (B) bromophenol blue upon direct solar irradiation.

Figure 6:

Photocatalytic role of foliar doped nanoparticles and alleviating UV-Vis spectra with time: (A) bromocresol green and (B) bromophenol blue.

Figure 7:

Photocatalytic degradation percentages and reaction kinetics: (A, C) bromocresol green and (B, D) bromophenol blue.

The natural purification process for BG and BB is extremely slow and inflicts a heavier ecological difference. However, the use of FDNPs ensures quick removal as compared with other materials that have been proved comparatively slower (Table 2). FDNPs upon exposure to direct solar irradiance are marked with formation of charge carriers through light absorption [48], [49], [50], thus making FDNPs a unique nanophotocatalyst. Furthermore, the reaction kinetics for FDNPs was determined by plotting ln(A_t/A_o) vs. time (Figure 7). Photocatalytic degradation processes for both dyes, i.e. BG and BB, exhibited pseudo first-order kinetics with R²=0.83 and 0.95, respectively. FDNPs have exhibited a fast and an effective photocatalytic response toward both dyes because of surface plasmonic resonance when they were exposed to light; however, the exact mechanism of photolytic or dye-sensitized mechanism cannot be ruled out. FDNPs in contact with dye solutions are photoactivated because of incoming light, and there is an adherence between the FDNP surface and dye molecules. Consequently, the photoactivation of FDNPs by incoming light also influences and causes excitation of these dye molecules, which releases electron from the lowest unoccupied molecular orbital into the conduction band of semiconductor nanophotocatalyst. This process initiates the delivery of electrons to Ag NPs. Such an electronic transfer generates free radicals known as reactive oxygen species. Reactive oxygen species are remarkably strong oxidants and decompose the brominated dyes [51], [52], [53], [54]. Moreover, there is a promotion in the absorptivity of incident light and degradability of BG and BB into CO₂ and H₂O due to oxygen vacancies acting as impurity states [55]. Thus, it is evident that the photocatalytic degradation of BG and BB can be a cumulative effect of surface plasmonic resonance, photocatalysis, and photosensitization.

Heavier capital costs spent on antibiotic drugs on an annual basis have not only triggered the financial concerns but also have been proved useless because of the development of resistance in microbes. Thus, a large variety of microbes harmful toward human as well as agricultural crops is becoming stronger with passage of time. There is a need for the development of antimicrobial agents that not only kill those microbes but also are good in terms of environmental toxicity, cost benefit consideration, and drug resistance. Bacterial and fungal pathogens, i.e. X. citri, P. syringae, A. niger, A. flavus, A. fumigatus, A. terreus, P. chrysogenum, F. solani, and L. theobromae, are known for their higher toxicity rates and have been treated with a variety of antibiotic drugs but are found resistant. However, no studies have been reported on the inhibition of these microbes with foliar-mediated Ag:ZnO NPs. Therefore, the present study has attempted to inhibit the growth of these pathogenic microbes.

In the clinical field, antibacterial agents are of particular significance because these bactericidal agents are associated with fast and improved recovery from bacterial-induced infections and thus play a role in the minimization of drug resistance to some extent. Results confirmed that FDNPs were found effective against all tested microbes (Figure 8). FDNPs produced highly active zones of inhibition against all microbes up to 23.07 mm for X. citri followed by 22.02 mm for A. flavus. FDNP-induced zones of inhibition against A. terreus and L. theobromae were comparatively active up to 14.05 and 17.05 mm, respectively. Inhibitory mechanism incurred by metallic NPs is still to be investigated; however, FDNPs are expected to inhibit the bacterial growth either by production of reactive oxygen species on FDNP surface and generation of oxidative stress or by means of toxicity of free metallic ions when they are dissolved [56]. Ag component of FDNPs is known for the production of pits and gaps and ultimate fragmentation of cell of pathogenic microbes.

Figure 8:

In vitro biocidal activity of foliar doped nanoparticles against pathogenic strains exhibiting multidrug resistance (standard for bacterial strains ampicillin and standard for fungal strains amphotericin B).

FDNP-induced lytic mechanism is associated with the interaction of Ag ions with enzymatic disulfide or sulfhydryl groups and consequently annihilating the metabolic processes and cell death. In addition to Ag ions, the ZnO component of FDNPs is also known for its bactericidal activity against a variety of Gram-positive and Gram-negative bacteria in addition to its spores [57], [58]. Bacterial sustainability is affected by FDNPs through production of H₂O₂. Another governing factor in this regard is the agglomeration of FDNPs on bacterial surface due to the generation of electrostatic forces [59]. Bacterial cellular damage is also influenced by cellular membrane disruption and FDNP internalization. Earlier reports have supported the role of Ag and Zn in interrupting the transmembrane electronic transportation [60], [61], [62]. When zones of inhibitions were compared with standard antibiotic drugs, FDNPs were found to exceed the efficiency in the cases of X. citri, P. syringae, A. niger, and A. flavus for producing higher zones of inhibitions than that of standard bactericidal and fungicidal agent. However, the zones of inhibitions produced against A. flavus, A. terreus, and L. theobromae were comparable with standard antibiotic drugs but did not exceed. FDNP-induced inhibition of P. syringae is comparable with chemically synthesized Ag:ZnO NPs [63], and it can be attributed to the provisioning of larger surface area of FDNPs toward microbial contact. Studies have supported the effectiveness of combining NPs with antibiotic drugs, and their synergistic effects were commendable [64]. FDNP-driven antibacterial and antifungal activity was found to be enhancing as the FDNP dose was increased from 2 to 10 μl and was found highest for the highest dose; this finding is consistent with previous studies [65], [66], [67]. A remarkable inhibitory activity of FDNPs against X. citri is significant because this pathogenic strain is responsible for citrus cancer destroying citriculture on a global scale. Thus, FDNPs have very promising prospects to be developed into commercialized bactericidal products against X. citri [68]. FDNPs in addition to its development into bactericidal agent are equally effective as a fungicidal agent because of its enhanced antifungal activity [69]. In addition to bacterial action, even in the case of fungal inhibition, higher activity can be attributed to the fact that FDNPs were synthesized from P. cerasifera phytoconstituents, and these phytoconstituents have inherent antimicrobial activity, thus causing an augmentation into the antimicrobial action of FDNPs [70]. Antifungal activity of Ag and ZnO NPs against fungal species including P. chrysogenum have been demonstrated in a variety of reports [71], [72], [73], [74], but the results of present investigation have confirmed the effectiveness of FDNPs against nine microbes.

4 Conclusion

In conclusion, the reducing agents extracted from PCLE can be used for hydrothermal synthesis of silver-doped zinc oxide NPs. The synthesized NPs exhibited commendable crystalline hexagonal wurtzite structure. Ag doping in ZnO can induce an alleviation into direct energy bandgap signifying p-type conductivity and thus enhancing its photocatalytic potential. Moreover, synthesized NPs can also be used as an eco-friendly remediator of BG and BB in direct solar irradiance yielding ≥86% efficiency in less than 15 min. Foliar-mediated Ag:ZnO NPs are of special consideration for their use as an antimicrobial bullets against nine pathogenic microbes having drug resistance. The present study can be extended to the use of synthesized NPs in exploring the exact mechanism of photocatalysis and antibacterial and antifungal annihilation.