Cytotoxicity, antifungal, antioxidant, antibacterial and photodegradation potential of silver nanoparticles mediated via Medicago sativa extract

2.1 Materials and equipment

Silver nitrate (AgNO₃; USA) and methanol (CH₃OH; Germany) were purchased from Sigma and Merck companies, respectively. In all experiments, deionized distilled water was used as a solvent for silver nitrate and plant extract. Standard strains of three species of Gram-positive bacteria, including Staphylococcus aureus (ATCC 29213; S. aureus), Staphylococcus epidermidis (ATCC 12228; S. epidermidis), and Enterococcus faecalis (ATCC 29212; E. faecalis) and three species of Gram-negative bacteria, including Escherichia coli (ATCC 25922; E. coli), Pseudomonas aeruginosa (ATCC27853; P. aeruginosa), and Klebsiella pneumoniae (ATCC 9997; K. pneumoniae) as well as one standard strain of Candida albicans (ATCC 10231; C. albicans) were used for antibacterial and antifungal studies. All were purchased from the Pasteur Institute of Iran. Moreover, ceftriaxone and amphotericin B were employed to compare the antibacterial and antifungal effects, respectively.

2.2 Preparation of Medicago sativa extract

Medicago sativa was collected and approved by the Herbarium Department of Birjand University, washed with distilled water, dried in the shade, and ground to yield powder. Subsequently, 25 g of the plant powder was stirred with 500 mL of methanol for 72 h at room temperature. The resultant suspension was filtered using a Buchner funnel and a Whatman filter paper, grade 41, before it was placed in a rotary vacuum evaporator. The obtained dried powder was refrigerated at 4 °C until used.

2.3 Green synthesis of silver nanoparticles

Biogenic AgNPs were synthesized according to a previous report (Hashemi et al., 2021). Hence, 16 mg of silver nitrate (AgNO₃) was dissolved in 10 mL of deionized distilled water under vigorous stirring at ambient temperature. Afterward, 10 mL of the M. sativa extract solution with a 10 mg/ml concentration and pH = 12 (using sodium hydroxide) was added drop-wise to the silver nitrate solution. At this stage, Ag⁺ ions were reduced to Ag⁰ nanoparticles. This conversion was accompanied by a change in the solution’s color from yellow to dark brown. The solution was stirred for 60 min. Moreover, the adsorption of the resultant solution was measured at 300 to 500 nm using UV–Vis for further study. Next, the silver solution was centrifuged at 6000 rpm for 5 min. Finally the product was washed with water to remove extracts and excess ions (three times) and dried. In sum, the bio-reduction of Ag(NO₃) into AgNPs using M. sativa extract is as follows: $$\mathrm{A}\mathrm{g}(\mathrm{N}\mathrm{O}3)\to \mathrm{A}\mathrm{g}++\mathrm{N}\mathrm{O}3$$

2.4 Measurements

Optimal conditions of silver nanoparticles using plant extracts:

To ensure optimal conditions for AgNPs synthesis, we evaluated the three parameters of silver nitrate concentration, reaction time, and reaction temperature using UV–Vis analysis (Table.1). Optimization of silver nitrate concentration: For optimal concentration, different concentrations of silver nitrate, including 3, 5, 10, and 15 mM, were used to synthesize AgNPs. The silver nitrate concentration with the highest absorption at a wavelength of 300 to 500 nm was selected as the optimal concentration for synthesis. Optimization of reaction time: The study built on the optimal concentration of silver nitrate to optimize the reaction time and performed the reaction at 5, 10, 30, 60, and 90 min. Moreover, the adsorption of the solution was investigated. Reaction temperature optimization: After optimal concentration and reaction time were calculated, the synthesis reaction of AgNPs was performed using extracts at different temperatures (room temperature, 65 °C, and 85 °C). Afterward, the optimal time for the formation of AgNPs was obtained by analyzing the obtained absorption diagrams.

2.5 Evaluation of antibacterial and antifungal activities

This study investigated the antibacterial effects of three species of Gram-negative bacteria, including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Klebsiella pneumoniae (K. pneumoniae), and three species of gram-positive bacteria, namely, Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), and Enterococcus faecalis (E. faecalis). Candida albicans (C. albicans) were also used to evaluate the antifungal activity. Ceftriaxone and amphotericin B were used as broad-spectrum antibiotics and antifungal agents, respectively, to evaluate their efficacy to that of synthesized MSE-AgNPs. The microdilution broth technique was used to explore the effects of nanoparticles on bacteria and fungi. For this purpose, 100 μL of different concentrations of MSE-AgNPs, M. sativa extract, ceftriaxone, and amphotericin B were poured into sterile microplate wells. Afterward, 100 μL of Mueller Hinton broth culture medium (MHB, Merck, Germany) containing the desired microbial suspension (equivalent to a dilution of 1:150 to the 0.5 McFarland) was added. Moreover, one well was considered a positive control containing 100 μL of desired microbial suspension in MHB medium (equivalent to a dilution of 1:150 to the 0.5 McFarland) and 100 μL of sterile physiology serum. Another well was regarded as a negative control containing only 200 μL of MHB medium. After 24 h, the turbidity of the wells was examined to evaluate the minimum inhibitory concentration (MIC). To evaluate the minimum bactericidal/fungicidal concentration (MBC, MFC), we cultured 10 μL of wells with MIC and higher concentrations on a blood agar medium (Merck, Germany) and incubated for 24 h. Subsequently, the concentration at which no growth was observed on the blood agar medium was selected as the minimum bactericidal/fungicidal concentration. All steps were reiterated three times to reach more robust results.

2.6 Evaluation of the antioxidant activity of MSE-AgNPs by DPPH assay

In this study, the free radical scavenging capacity was investigated through the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical method using a Zantox kit (Kavosh Arian Azma Co., Iran) (Brand-Williams et al., 1995). In this method, 10 μL of M. sativa extract samples (in concentrations of 5, 2, and 1 mg/mL), MSE-AgNPs (in concentrations of 43, 17.2, and 8.6 μg/mL), control sample (available in the kit), distilled water (blank sample), and various standards (in concentrations of 62.5, 125, 250, 500, and 1000 μM) were poured into micro-wells. Afterward, 250 μL of the prepared 1,1-Diphenyl-2-picrylhydrazyl solution was added to each of the micro-wells, and the plate was shaken for 30 s. Eventually, the plate was incubated for 20 min at room temperature. Typically, When DPPH radicals are dissolved in an organic solvent, they undergo a color change into purple that may be detected using a spectrophotometer at 517 nm. As the concentration of reducing compounds increases, the purple color fades more rapidly. The results of this test were reported as the percentage of DPPH radical inhibition. The following equation was used to compute the radical scavenging capacity of DPPH:

where A₀ is the absorbance of the DPPH solution, and A₁ is the absorbance of the sample.

2.7 Evaluation of cytotoxicity MSE-AgNPs

Human fibroblast cells were obtained from the cell bank of the cell bank in Pasteur Institute of Iran. The cells were grown to 80–90% confluence in a culture plate with the DMEM medium including streptomycin (100 μg/mL), penicillin (100 U/mL) and fetal bovine serum (FBS; 10%) at 37 °C. Then, the impact of MSE-AgNPs at different concentrations (3, 5, 12.5, 25, 50, 100 µg/mL) on fibroblast cell viability was determined for 24 h. MTT (Sigma, USA) staining was used to assess the cytotoxicity of the synthesized AgNPs on human fibroblasts. At first, the cell suspension was prepared from normal cells, and 200 µL of this suspension was added to each well from 96 house plates. Ten thousand cells were implanted in cell culture plates and treated with AgNPs concentrations of 3, 5, 12.5, 25, 50, 100 µg/mL. After 24 h, 20 μL of MTT dye (with a final concentration of 5 mg/ml) was added to the wells and incubated for 5 h at 37° C with 5% CO₂. The supernatant solution was discarded, and the formed formazan crystals were dissolved in 150 μL of DMSO. Additionally, the absorbance of the wells at 570 nm was read using an ELISA reader (Epoch, Biotek USA). The cell viability percentage was calculated as follows: $$\mathit{Cellviability}(\%)=(sampleabsorption/controlabsorption)\times 100$$

2.8 Investigation of the catalytic performance of MSE-AgNPs

The photocatalytic activity of the synthesized MSE-AgNPs occurred in sunlight and under UV irradiation. Different amounts of dyes as contaminants (5 ppm) were diluted in distilled water and poured into a beaker. A certain amount of biosynthesized MSE-AgNPs (35 mg) was added to the solution containing the dye. Before irradiation, the solution was aerated with oxygen (for equilibrium adsorption and desorption) under dark conditions for 30 min. The solution in the beaker was sampled after some time, and a UV–Vis optical spectrometer was employed to measure its adsorption (at specific times). The percentage of pollutant degradation was determined: Degradation (%) = (A₀-A_t)/A_t*100 (5), where A₀ is the initial adsorption and A_t is the adsorption at time t.

2.9 Characterization

The FT-IR (PerkinElmer Spectrum TwoTM IR spectrometer; Model L160000U) test was adopted to evaluate the functional groups of samples. Moreover, XRD analysis was used at an angle 2θ = 10–80° (Philips PW1800 using CuKα radiation) to determine the synthesized particles’ crystal structure and size. The formation of AgNPs with UV–Vis spectrum was confirmed using a Nanodrop device (BioTek model Epoch; USA). Particle size and surface charge of synthesized nanoparticles were confirmed by DLS and zeta potential (NanoBrook 90Plus-Brookhaven Instruments, model 18051; USA). FESEM (TESCAN BRNO-Mira3 LMU) and TEM (Zeiss-EM10C-100 KV) analyses evaluated the synthesized nanoparticles’ morphology and size.

2.10 Statistical analysis

At the very least, each experiment was performed in triplicates. Microsoft Excel and the Origin Pro 9 software program were used to determine the whole data. The data were analyzed using a one-way analysis of variance (ANOVA) and the Duncan post-hoc test (P-value < 0.05).

3.1 Optimization of synthesized nanoparticles

In this study, the UV–Vis spectrum at a wavelength of 300–500 nm was employed to validate the formation of AgNPs and analyze the impact of different parameters on the formation of nanoparticles. The intensification of the surface plasmon resonance of silver nanoparticles during the reaction leads to a change in the solution’s color to dark brown. This intensification of the surface plasmon creates an absorption band at a wavelength of 300–500 nm. Past results have shown that increasing the absorption rate of nanoparticles is directly related to an increased formation of synthesized nanoparticles (Naghizadeh et al., 2021). Thus, this absorption can be built on to explore the impact of different parameters on the formation of nanoparticles. Effect of silver ion concentration: Silver nitrate concentrations of 3, 5, 10, and 15 mM were examined to identify the optimal concentration for AgNP biosynthesis. Previous research indicates that more nanoparticles are formed with the increase in silver salt (Anand et al., 2017). As shown in Fig. 1a, the intensity of the absorption band increases as the concentration of silver nitrate rises to 10 mM, whereas it decreases after 10 mM. This decline in adsorption intensity can be due to the adhesion of nanoparticles to each other. Hence, the optimal concentration of silver nitrate for further experiments was considered to be 10 mM. Effect of reaction time: Fig. 1b illustrates the UV–Vis absorption spectra of the nanoparticle solution at different times (5, 10, 30, 60, and 90 min). As can be seen, the adsorption intensity increases with reaction time, reaching its maximum at 60 min. The band intensity dropped when the contact duration increased to 90 min. This reduction might be explained by sample aggregation. The optimal response time was 60 min based on the spectroscopy results. The reaction was investigated at three different temperatures (room temperature, 65 °C, and 85 °C) to determine the best temperature for the reaction. According to the UV–Vis absorption diagram (Fig. 2c), the adsorption rate rose with reaction temperature. Additionally, as the reaction temperature increased, the absorption peak shifted to shorter wavelengths, suggesting that the produced nanoparticles became smaller in size. As a result, 85 °C was the optimal temperature for this reaction. These results are consistent with those obtained in previous research (Aboelfetoh et al., 2017). High performance liquid chromatography (HPLC) analysis was used to investigate the mechanism of nanoparticle formation. The HPLC results revealed that the main compounds in the M. sativa extract were rutin (18.6 mg g⁻¹), p-coumaric acid (13.1 mg g⁻¹), quercetine (9.7 mg g⁻¹), and chlorogenic acid (6.4 mg g⁻¹). So, we can conclude that the biological activity of extract may attribute to rutin (Scheme. 1).

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Fig. 1

UV–Vis spectra of as-synthesized silver nanoparticles using M. sativa extract: (a) silver nitrate concentration, (b) time, and (c) temperatures.

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Fig. 2

XRD pattern of MSE-AgNPs (sample no. 10).

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Scheme 1

The possible mechanism of synthesis of silver nanoparticle using M. sativa extract.

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3.2 XRD analysis

The crystal structure and purity of the produced nanoparticles were determined through XRD analysis. The XRD analysis of sample no. 10 is represented in Fig. 2. As it can be seen, the XRD spectrum contains no impurities, and all peaks correspond to the synthesized silver nanoparticle, indicating the nanoparticles’ high purity as per the standard silver diffraction (JCPDS 01–087-0717). XRD analysis reveals peaks at 38.26°, 44.73°, 64.13°, and 77.68°, which correspond to (1 1 1), (2 0 0), (2 2 0), and (3 1 1) Bragg plates, respectively. The XRD analysis of the material confirms earlier findings. The obtained values of crystallite size estimated using Debye-Scherrer equation (Ebrahimzadeh et al., 2019) (D = nλ/βcosθ) where D is the average size, λ is the wavelength of the X-ray beam, β is the line broadening at half the maximum intensity and θ is the Bragg angle. According to this equation, the size of AgNPs is approximately 26 nm.

3.3 EDS, FESEM and TEM analysis

The energy scattering spectrum (EDS) of MSE-AgNPs is portrayed in Fig. 3 (sample no. 10). Silver is the predominant constituent of nanoparticles, as evidenced by a strong signal at 3 keV due to silver’s surface plasmon resonance. This EDS analysis revealed that the synthesized nanoparticles contain elements such as C and O, which are linked with the elements found in the M. sativa extract involved in the nanoparticle synthesis. The size, morphology, and uniformity of the synthesized nanoparticles may be determined using FESEM images. Fig. 4 illustrates the FESEM images of the synthesized nanoparticles (sample no. 10) at 1 µm, 500 nm, and 200 nm scales. As can be seen in the images, the majority of nanoparticles have a spherical and elliptical morphology with an average diameter of 20–50 nm. Moreover, in some areas, the nanoparticles are stuck together and form an irregular structure, which is attributable to the adhesion of the extracts on the surface of the nanoparticles. TEM analysis was used to evaluate the size and morphology of nanoparticles accurately. The TEM images (Fig. 5) of the synthesized nanoparticles reveal that the synthesized nanoparticles have a spherical morphology in a size range of 15–35 nm. The TEM image explicates the presence of secondary materials, exhibited by the dark shadows on the surface of AgNPs, which may be related to the M. sativa extract. Small size, spherical morphology, and uniform structures are characteristic of AgNPs synthesized using M. sativa extract, compared to nanoparticles produced in previous research (Rautela et al., 2019).

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Fig. 3

Energy dispersive X-ray spectroscopy representing the compositional analysis of MSE-AgNPs.

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Fig. 4

Field emission scanning electron microscopy (FESEM) images of the biosynthesized AgNPs using M. sativa extract at optimum condition (sample no. 10).

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Fig. 5

TEM images of synthesized Ag nanoparticles using M. sativa extract (sample no. 10).

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3.4 DLS and zeta potential analysis

DLS and zeta potential analyses indicate the particle size (hydrodynamic diameters) and the surface charge of the synthesized nanoparticles, respectively. Fig. 6a displays the DLS histogram of sample no. 10, according to which the hydrodynamic diameter of AgNPs is about 65–130 nm. The particle size analyzed by FESEM was smaller than the particle size obtained in the DLS analysis. This discrepancy in size may be due to the measurement of hydrodynamic diameter in DLS analysis. The surface charge on nanoparticles is critical for avoiding nanoparticle aggregation and boosting their stability (Kim et al., 2013). According to Fig. 6b, the zeta potential of the synthesized nanoparticles was –22.41 mV, which indicates the negative surface charge in the nanoparticles synthesized by the extract.

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Fig. 6

(a) Dynamic light scattering (DLS) and (b) zeta-potential measurement of MSE-AgNPs (sample no. 10).

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3.5 FT-IR spectrum

Fig. 7 illustrates the results of FT-IR analysis on M. sativa extract and synthesized nanoparticles (sample no. 10). FT-IR analysis revealed the functional groups in M. sativa extract and the synthesized nanoparticles. The FT-IR spectra of M. sativa extract (Fig. 7a) disclosed that the absorption bands at 3426, 2947, 1665, and 1334 correspond to the O–H stretching vibration, the C–H of hydrocarbon, the C = O of carbonyl bond, and the stretching C = C aromatic ring, respectively (Li et al., 2021). Additionally, Fig. 7b illustrated the FT-IR spectrum of AgNPs produced from M. sativa extract. The nanoparticles’ adsorption peaks were similar to those of the M. sativa extract. However, they had lower displacement and intensity than those of the M. sativa extract, which indicates the effect of M. sativa extract functional groups on the formation of nanoparticles (Shaik et al., 2018).

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Fig. 7

FT-IR spectrum of M. sativa extract (a) and biosynthesized MSE-AgNPs (b).

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3.6 Antibacterial and antifungal activities

The antibacterial and antifungal activities of M. sativa extract and MSE-AgNPs are summarized in Table. 2 and Table 3, respectively. According to antibacterial results, MSE-AgNPs had excellent antibacterial activity and their antibacterial property was stronger against Gram-negative than against Gram-positive bacteria. Indeed, P. aeruginosa exhibited the highest antibacterial activity with MIC and MBC values of 62.5 and 250 μg/mL, respectively. The lowest antibacterial property was noticed in S. aureus and S. epidermidis with MIC values of 1000 and 1000 μg/mL, respectively. Because gram-negative bacteria have a thinner layer enclosing their membrane and a lipopolysaccharide layer surrounding them, nanoparticles can enter and kill them more readily (Slavin et al., 2017). Additionally, the data indicated that nanoparticles are more efficient against C. albicans than bacteria, presumably because fungi belong to the eukaryotic group, while bacteria belong to the prokaryotic group (Marulasiddeshwara et al., 2017). This research found that the methanolic extract of M. sativa had no antibacterial effect on the tested bacteria and fungi (up to the 50 mg/mL concentration). This study demonstrates that silver can considerably enhance the antibacterial and antifungal effects of M. sativa extract when mixed with it. However, the mechanism through which AgNPs exert antibacterial action is unknown. Our argument is as follows: By and large, the antibacterial activity of nanoparticles is proportional to their shape and size. The smaller the nanoparticles and the larger the surface-to-volume ratio, the more likely the nanoparticles will attach to the microorganism and affect it (Soleimani et al., 2018). Due to their tiny size, nanoparticles may easily infiltrate microorganisms and impair their functioning by connecting to functional regions of key proteins and binding to nucleic acids. Further, they can induce oxidative stress in microorganisms and disrupt the operation of critical microorganism proteins. As microorganisms become more disordered, the function and permeability of the membrane change and microorganisms begin to ruin (Dangi et al., 2020).

3.7 Antioxidant activity

In the organism, oxidative stress arises when the number of free radicals produced exceeds the capacity of antioxidants to neutralize them (Sies, 2020). Oxidative stress is detrimental to many vital molecules in the body, such as nucleic acids, proteins, and lipids. AgNPs and plant extracts have been demonstrated to have a significant level of antioxidant activity in previous investigations (Fouda et al., 2020). There are several ways for assessing a substance’s antioxidant activity, one of which is the DPPH method. DPPH is reduced by absorbing hydrogen atoms from the hydroxyl group, resulting in a change in the color of the solution. This color change from purple to yellow indicates the substance’s antioxidant capacity (Brand-Williams et al., 1995). The present study investigated the percentage of DPPH inhibition as seen by nanoparticles and alfalfa extract in three concentrations (Fig. 8). The results demonstrated a direct link between nanoparticle concentration and DPPH inhibition percentage, with higher concentrations resulting in higher DPPH inhibition percentages. The percentage of DPPH inhibition rose from 25% to 78% when the nanoparticle concentration was raised from 8.6 to 43 μg/mL. By increasing the concentration of M. sativa extract from 1 to 5 mg/mL, the percentage of DPPH inhibition rose from 9 to 32%. Our results reveal that using M. sativa extract to synthesize AgNPs significantly improves their antioxidant properties. Consistent with our findings, Jalilian et al. discovered that AgNPs generated from Allium ampeloprasum extract had greater antioxidant activity than AgNPs synthesized from pure Allium ampeloprasum extract (Jalilian et al., 2020).

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Fig. 8

DPPH inhibition percentage at different concentrations of extract and MSE-AgNPs.

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3.8 Cytotoxicity of biogenic silver nanoparticles

This study analyzed the toxicity of AgNPs synthesized using M. sativa extract against human fibroblasts at 3, 5, 12.5, 25, 50, 100 µg/mL concentrations via the MTT colorimetric method. Moreover, cell viability percentage was assessed after 24 h. The presence of cells was assessed after 24 h. Treatment of human fibroblasts with different concentrations of AgNPs showed that the cytotoxicity of the synthesized AgNPs was dose-dependent. Indeed, the most cytotoxic effects were observed at concentrations of 50 μg/mL and 100 μg/ml, and the amount of inhibitory concentration (IC50) was 18.22 μg/mL (Fig. 9).

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Fig. 9

The results of the MTT assay in human fibroblasts cells treated with MSE-AgNPs after 24 h. Results were reported as survival percentage compared to control samples (p < 0.001***, p < 0.01**, p < 0.05*).

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3.9 Photocatalytic activity

The photocatalytic activity of biogenic AgNPs was investigated by observing the degradation of cationic and anionic organic pollutants, such as thymol blue (TB) and malachite green (MG), in an aqueous solution under UV and sunlight radiations. Fig. 10 illustrates the photocatalytic degradation of TB and MG contaminants. In the absence of MSE-AgNPs or light, virtually no pollutants were degraded after 100 min, indicating that the self-degradation component was irrelevant. Fig. 10 depicts the details of the degradation performance of TB and MG pollutants under UV and sunlight irradiations. As can be seen, the contaminant degradation percentages of the MG dye are nearly 94.37% and 82.65% under UV and sunlight irradiation after 100 min, respectively. Additionally, a review of the catalytic data revealed that TB was degraded by 90.12 percent and 74.22 percent in 100 min when exposed to UV and sunlight, respectively. Comparison of the results indicated that in the presence of AgNPs, a pollutant with a positive charge (cationic) degraded more rapidly than a dye with a negative charge (anionic). The mechanism of pollutions via optimized biogenic AgNPs by superoxide anion and hydroxyl radicals is presented below (Scheme. 2):

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Fig. 10

Photocatalytic degradation of thymol blue (TB) and malachite green (MG) organic pollutants by MSE-AgNPs.

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Scheme 2

Mechanism of photocatalytic degradation of organic pollutions by MSE-AgNPs.

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