Search
Close this search box.

Green synthesis of metal nanoparticles and study their anti-pathogenic properties against pathogens effect on plants and animals – Scientific Reports

The nanoparticles synthesized by green method were characterized by various characterization techniques.

Phytochemicals screening analysis

At first, the screening of extracted phytochemicals was done by using the standard methods obtained from various studies. The screening showed the presence of all active compounds necessary for the reduction, dispersion and stabilization of metal ions. The obtained extract from the citrus leaves were contained the phytochemicals such as flavonoids, phenols, steroids, glycosides and terpenoids. Several studies have already been reported about the presence of alkaloids, saponins, tannins, sterols and flavonoid in citrus extract. Flavonoids are most desiring and key component among all phytochemicals. In fact they played a dual role, as a reducing agent and as an antibacterial agent due to their inherent antipathogenic characteristics. Flavonoids also have antioxidative, cytotoxic, chemopreventive, and antiprnoliferative properties23. The list of extracted phytochemicals in accordance with the relevant studies is given in Table 2.

Table 2 Phytochemicals present in leaf of Citrus sinensis.

Distribution of particle sizes, polydispersity index and zeta potential

The molecule size was calculated by using DLS methods, based on the Brownian motions of the particles. Figure 3a,b respectively demonstrate the average particle size distribution for Cu-NPs and Ag-NPs. The sizes of particles were varying in size from nano meter to milli meter range in a multi-modal manner. The average size of copper and silver particles were notices about 510 nm and 470 nm respectively, at a zeta potential of − 41.6 mV and − 30.6 mV respectively. The Zeta potential values showed the colloid stability of both types of nanoparticles, as these values were > ± 30 mV22.This ensured that the particles were evenly distributed throughout the suspension and had a high negative potential from the Nano meter to the micro range. The stability of particles is well analyzed by zeta potential, while the particle size distribution in nano sciences is more articulate with polydispersity index (PDI) values. The polydispersity Index (PDI) of Ag-NPs and Cu-NPs was noted about 0.312 and 0.258 respectively. The values show that the synthesised particles are highly polydisperse26.

Figure 3
figure 3

The particle size distribution of (a) Cu-NPs and (b) Ag-NPs.

Surface characterizations

The surface morphology of synthesized copper and silver nanoparticles was examined using scanning electron microscopy (SEM). The external morphological investigation through SEM revealed the formation of Ag-NPs, and Cu-NPs at the nano to micrometric scale. The rough surface and random clusters with cylindrical form for both types of particles was observed. The tiny agglomerations with constant repetition and even deposition was also noticed. SEM images also revealed the irregular spherical morphological features of the biosynthesized nanoparticles as shown in Fig. 4b,e. However, the sizes of all particles were noticed within the nanometric sale by zeta size analysis as described in the previous Section “Distribution of particle sizes, polydispersity index and zeta potential”. More SEM images at higher magnification in their respective box are added to deeply analyze the connectivity of particles. Which showed the dense coating and homogeneous connectivity between the particles over the fabric structure. However, they were observed within the range of nanometric scale. Moreover, the TEM analysis were also performed to better clarify the sizes and morphologies of nanoparticles as shown in Fig. 4a,d. The TEM analysis estimates the sizes of copper and silver particles between 400 and 500 nm and report the morphologies nearly spherical. As aforementioned, SEM analysis shows the nanoclusters of particles, ranging in 500 nm. While during TEM analysis, it seems that the separate particles of copper and silver are broken parts from their respective clusters27.

Figure 4
figure 4

(ac) TEM analysis, SEM analysis and EDX spectra of silver particles (df) TEM analysis, SEM analysis and EDX spectra of copper particles.

Additionally, the elemental compositions of Cu-NPs and Ag-NPs were also estimated to found the amount of metal in percentage. The elemental composition of EDX was estimated using spectrum analysis also uncover additional information about the makeup and components of particles as shown in Fig. 4c,f. Except of oxygen and carbon, some other peaks of impurities in least amount were also noticed such as Ca, Mg, and Cl. The existence of trace elements with low quantities is normal behavior during elemental analysis28.

Justification for the formation of copper and silver particles

The UV–Visible spectroscopy was conducted to justify the synthesis of Cu-NPs and Ag-NPs.

The aqueous solution of nanoparticles was mixed with constant ratio (1:2) in distilled water. Subsequently, were mixed well and prepared for UV analysis. The UV spectrum obtained from the synthesized nanoparticles were noticed at 339 nm and 415 nm for copper and silver respectively. While the UV–Vis absorbance spectrum of orange peels extract was notes at λ max was noted 320 nm due to the respective signal of phenolics groups Fig. 5a29. In fact, the significant shifts in values and peaks of metal particles as compared to orange extract values is due to the changes in the morphology, size or surface microstructures of silver and copper nanostructures.

Figure 5
figure 5

(a) UV–Vis Spectrum of the orange peels extract, synthesized copper and silver nanoparticles, (b) XRD peaks of silver nanoparticles and (c) XRD pattern of Cu-NPs.

Moreover, the XRD analysis was done to justify the formation of crystalline nature of coper and silver particles. The phase purity of manufactured Ag particles was confirmed by exact indexing of all the peak intensity to the silver structure, as shown in Fig. 5b. The four peaks for Ag-NPs appeared at 2 values of 77.5, 64.5, 44.3, and 38.1, respectively. These peaks were attributed to cubic-shaped diffraction planes (3 1 1), (2 2 0), (2 0 0), and (1 1 1), according to data from the International Diffraction Centre (data number JCPDS 04-0783 card)30. No significant peaks were observed for other impurities, such as silver oxide. Figure 5c represents the XRD spectrum of Cu-NPs. A precise identification of every diffraction peak to the copper structure reveals the elemental composition of Cu particles. Cu diffraction planes (2 2 0), (2 0 0), and (1 1 1) are characterized by the occurrence of Cu diffraction pattern (2θ) at 74.2, 59.5, and 43.3°. From either the presence of peak position, the copper particles crystalline structure was investigated. Because no distinct impurity peaks were found, other than the development of the Cu2O peak (2θ) at 38°, the widening of the peaks instead indicated the synthesis of Cu particles at the nano range, respectively31.

The extract of orange peels was used as a bio-reductant to synthesized the nanoparticles of copper and silver. Therefore, FT-IR spectroscopy was employed to confirm this reduction process. An analysis of the FT-IR spectroscopy analyzed the presence of functional groups on green synthesized silver and copper particles. The Fig. 6 is illustrating the respective FT-IR spectra of orange peels extract coated cotton fabric, copper particles coated and silver particles coated fabrics. The absorption peaks around 2950, 3331, 2115, 1636, and 597 cm−1. On 2950 are C–H stretching vibration absorption peaks in cellulose. While, the broad absorption band on 3670 cm−1 corresponds to the O–H stretching frequency, while at 1636 cm−1 depicts the C=O stretching of the carbonyl group. The peak 1059 cm−1 was noted due to the to the link of alcohols or esters (C–O–H or C–O–R)32.

Figure 6
figure 6

The FT-IR spectra of orange peels extract, copper particles and silver particles coated fabrics.

The in-vitro analysis of synthesized nanoparticles against plants pathogens

The In-vitro analysis of the biosynthesized silver and copper nanoparticles was carried out by using disc diffusion method. The bacterial strains P. carotovorum, was used to check the antibacterial potential of biosynthesized Ag-NPs and Cu-NPs. Nutrient agar was used as a culture media. Different dilutions of the synthesized nanoparticles were used to analyze zones of inhibitions against the bacterial strains. The zone of inhibition against different concentrations of silver particles coated potato slices samples S1 (0.25), S2 (0.50) and S3 (1.00 g) and different concentrations of copper particles coated potato samples S7 (0.25), S8 (0.50) and S9 (1.00 g) are shown in Fig. 7a,b.

Figure 7
figure 7

Growth inhibitions in response to (a) Cu-NPs and (b) Ag-NPs against plant pathogens (P. carotovorum) and (c) graphical representation of zone of inhibition values for samples coated with silver and copper particles.

The silver particles coated potato sample S3 (1.00 g) showed the maximum zone about 16 mm. In a similar study, the antibacterial activity was noted against P. carotovorum by silver nanoparticles. Their results revealed that the Ag-NPs showed largest inhibition zone of about with the 14.33 mm33. While the copper particles coated sample S9 (1.00 g) showed the zone about 14 mm. It means the silver particles are little bit more effective as compared to copper particles. Azam et al., conducted a comparative analysis of copper and silver particles against different pathogens. Where, silver particles coated substrate showed better performance as compared to copper particles34. However, the overall efficiency of both particles is quite effective against bacterial strains P. carotovorum. Figure 7c is showing the bar graphs with standard errors against inhibition zone of all silver and copper particle coated potato samples. The group S3 and S9 contains the zone of inhibition values against Ag-NPs and Cu-NPs coated potato samples. During the observation the tail of error bar of silver particles coated sample S3 (1.00 g) group is not coinciding with the head of the error bar of copper coated S9 sample. It means there is a insignificant difference between two groups, which implies that silver and copper particles coated samples have different zone of inhibition range. The ANOVA analysis at 95% confidence interval was applied. The P value for group control sample and copper coated sample was calculated as 0.047 which is P < 0.05. Hence the P value was less than 0.05 which means null hypothesis is insignificant and there is significant difference between the values of sample group S3 and S9. So, there is significant difference between the inhibition Zone against Ag-NPs and Cu-NPs.

The in-vivo antagonistic potential of nanoparticles against plant pathogens

The potato slices coated with different concentrations of silver particles coated potato slices samples S1 (0.25), S2 (0.50) and S3 (1.00 g) and different concentrations of copper particles coated potato samples S7 (0.25), S8 (0.50) and S9 (1.00 g) were also subjected to In-vivo Antagonistic potential. The bacterial strains of (P. carotovorum) with constant concentration 50 µl were applied over each sample (silver particle coated potato slice S3 (1.00 g), copper particles coated potato slice S9 (1.00 g) and uncoated potato slice). Afterwards, the slices were put in petri plates and covered with para-film and incubated for 24–48 h at 35 °C. The diameter of the infectious zones with measured values are shown in Fig. 8a–c and their values of infection in graphical representation are shown in Fig. 8d respectively. No or almost zero zone of infection was observed on potato slice coated with silver particles S3, while the potato slice coated with copper particles S9 showed slight zone of infection. The reason we have already described in previous section (see section In-Vitro antagonistic), where silver particles proved more effective against pathogens as compared to copper particles. Furthermore, the clear and large zone of infection was seen on uncoated potato slice. It means nanoparticles are quite effective against the bacterial strains of (P. carotovorum). The tail of error bar on control sample group is not coinciding with the head of the error bar of copper coated error bar. It means there is significant difference between two groups, which implies that the coating of copper over the potato sample significantly reduce the infection against the bacterial strains of (P. carotovorum). Also, there is no group in the place of silver coated sample (as there was no zone of infection). So, A huge difference between each group is present and promotes to the significant difference. The ANOVA analysis at 95% confidence interval was applied. The P value for group control sample and copper coated sample was calculated as 0.029 which is P < 0.05. Hence the P value was less than 0.05 which means null hypothesis is insignificant and there is significant difference between the control and copper coated sample. So, there is significant difference between the infection zones.

Figure 8
figure 8

Potato slices with zone of infection caused by P. carotovorum (a) uncoated sample, (b) coated with copper particles and (c) coated with silver particles and (d) bar graphs showing the values of their respective zone of infections.

Moreover, the area of slice without infection in percentage (area free from bacterial attack) was also calculated by using the following Eq. (2).

$${text{Area}};{text{without}};{text{infection }}left( {% {text{age}}} right) = frac{Outer; Zone – Inner; Zone}{{Outer ;Zone}}; times ;{100}$$

(2)

The calculated values of percentage of clear area from bacterial strains are given in Table 3. The silver particles coated samples showed almost 100 percent clear area (i.e. not a single spot or colony of bacterial strain). While there was 87 percent bacterial free area was calculated for potato slice coated with copper particles. In case of potato slice having no coating of particles showed less bacterial free area, which is only 54.5 percent.

Table 3 Calculated values of zone of infection, and area without infection in percentage with sample codes.

In-vitro potential of synthesized nanoparticles against human pathogens

To evaluate the effectiveness of the coated textiles for antibacterial properties, both qualitative and quantitative test were conducted.

Reduction factor (quantitative test)

The quantitative technique according to AATCC-100 method was used to measure bacterial resistance against S. aureus and E. coli strains. Figure 9 presents the reduction percentage of the bacterial cultures on the treated and untreated textile samples. The effectiveness was checked against different concentrations of silver particles coated fabric samples S4 (0.25 g), S5 (0.50 g) and S6 (1.00 g) and different concentrations of copper particles coated fabric samples S10 (0.25 g), S11 (0.50 g) and S12 (1.00 g). The control sample was ineffective against the tested microorganisms. All of the treated samples showed higher reduction in percentage against both type of bacteria, as the amount of Cu-NPs (from 0.25 to 1 g) and Ag-NPs (from 0.25 to 1 g) on the fabric increased. The maximum reduction about 99.99% was found in case of both types S. aureus and E. coli bacterial colonies. It was noteworthy that all fabric samples coated with silver (S4 to S6) and copper particles (S10 to S12) showed about 99.99% reduction against E. coli as the compared to S. aureus. It means that E. coli is more susceptible to metal particles than to S. aureus. The reason is E. coli can survive less in open environment (cause less infections), easily vulnerable to antibiotics due to its interactive membrane. While the S. aureus can stay longer and resist a range of antibiotics and cause serious infections and leads to different physical rheological responses35,36.

Figure 9
figure 9

Antibacterial activity in terms of log CFU/ml (left) and percentage reduction (right) of fabrics treated with silver and copper nanoparticles and untreated cotton fabric.

Figure 10 provides additional evidence for the aforementioned trend by displaying the development of bacteria concentrations for silver coated fabric sample S6 (1 g of particles) and copper coated fabric sample S12 (1 g of particles). The untreated cloth was shown to be inefficient against bacterial growth when compared to textiles coated with copper and silver particles. The copper and silver particles coated samples showed maximum reduction in bacterial colonies against both type of pathogens (E. coli and S. aureus). At greater concentrations, colony reductions showed a substantial increase, with more than 99 percent efficiency for both species of bacteria37.

Figure 10
figure 10

Images of concentration of bacterial growth for the (a,b) copper particles, (c,d) for silver particles and (e,f) for untreated fabrics.

The silver particles coating on cotton fabric in present study showed better performances compared to previously reported study, where the incorporation of Ag NPs into cotton fabrics using UV photo-reduction was performed38. Their results also support the declaration about increase in concentration has direct relation on the reduction of antimicrobial activity of E. coli. Several researches have been conducted for the analysis of antimicrobial activities of Ag-NPs coated bandages, and their impact on bacterial strains. The exact mechanism of reduction or inhibition of bacteria growth is still partially understood. In fact, some vibrant concepts involve the release of Ag+ and interaction with cell walls. Moreover, these silver ions can also interact with released -SH groups from cellular excretions; and leads to further inactivation of proteins. Hence, the released Ag+ ions may again combine another protein when the current protein is decomposed. The silver ions also expediate the production of oxidized radicals; which can penetrate easily into cell wall structure39.

Zone of inhibition test (qualitative measurements)

Zone of inhibition test was also used to assess the samples antibacterial abilities. Both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria were incubated for 24 h at 37 °C in the dark, all fabric samples had distinct inhibitory zones, as seen in Fig. 11a,b. The effectiveness was checked against different concentrations of silver particles coated fabric samples S4 (0.25 g), S5 (0.50 g) and S6 (1.00 g) and different concentrations of copper particles coated fabric samples S10 (0.25 g), S11 (0.50 g) and S12 (1.00 g). The textiles treated with silver nanoparticles (S4 to S6) had the greatest antibacterial zones against the strains of S. aureus and E. coli, whereas the fabrics treated with Cu-NPs (S10-S12) exhibited a smaller zone of inhibition. The average values were computed by conducting three readings of each sample. The outcomes showed that the free-standing nature of the copper and silver particles led to considerable disinfection of both bacterial strains, where S. aureus demonstrating more sensitivity than E. coli. As an illustration, using copper and silver particles raised the area of inhibition for E. coli from 4.5 to 10.7 mm, while increasing the area of inhibition for S. aureus from 6.5 to 14 mm as shown in Fig. 11c. It should be noted that the increase in inhibition zone with the increase in the concentration of nanoparticles had already been discussed in some previously published research works30. The combination of physical and chemical action of bacteria with particles is assumed to be the cause of coated textiles antibacterial properties. Through endocytosis procedures, the nanoparticles are absorbed by the cells. Ionic species are produced inside the cells during the nanoparticles degradation, increasing the cells ability to absorb ions40. Silver is showing good antimicrobial ability. In fact, the less antipathogenic effect of copper coating over the substrate as compared to silver was due to the less stability of copper. The similar effect of antimicrobial effectiveness was observed in some relevant studies. Where the in-situ deposition of copper and silver particles was performed to achieve the electrical conductivity and antimicrobial effectiveness. The reason for low electrical performance and bioactive performance was due to the susceptibility of copper particles to oxidation and carbonization41.

Figure 11
figure 11

Inhibition zones (a) against S. aureus, (b) E. coli., (c) graphical representation of Zone of inhibition around all samples.

The group S6 contains the Ag-NPs coated fabric samples showing the zone of inhibition values against S. aureus, E. coli, whereas group S12 contains the Cu-NPs coated fabric samples showing the zone of inhibition values against S. aureus, E. coli. While comparing the zone of inhibition, values against S. aureus were between S6 and S12. The tail of error bar of silver coated S6 sample group (zone of inhibition around S. aureus black bar) is not coinciding with the head of the error bar copper coated S9 sample of (zone of inhibition around S. aureus black bar). It means there is significant difference between two groups, which implies that silver and copper particles coated samples have different zone of inhibition range. The P value between these two groups was observed as 0.037 which is P < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group (zone of inhibition around S. aureus black bar) and copper coated S9 sample of (zone of inhibition around S. aureus black bar).

In the same way, while comparing the zone of inhibition values against E. coli between S6 and S12. The tail of error bar of silver coated S6 sample group (zone of inhibition around E. coli green bar) is not coinciding with the head of the error bar copper coated S9 sample of (zone of inhibition around E. coli green bar). It means there is significant difference between two groups, which implies that silver and copper particles coated samples have different zone of inhibition range. The P value between these two groups was observed as 0.029 which is P < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group (zone of inhibition around E. coli green bar) and copper coated S9 sample (zone of inhibition around E. coli green bar).

Antifungal activity of treated samples

In order to assess the effectiveness of various fabric samples against the A. niger fungus, the AATCC-100 method was utilized in this study. Figure 12a–d showed the results related to fungus growth against each particle coated sample and percentage reduction in fungal spore germination for each fabric specimen. However, it was observed that fabrics with particle coatings were better in combating fungi when compared to untreated samples. Silver coated fabrics had the greatest inhibition of fungal growth among the particles-coated samples with antifungal effectiveness of approximately 77%. In fact, the present study showed better antipathogenic properties of silver particles overall. The statement can be further justified from a related study; where green synthesized silver particles showed almost the same reduction in percentage of fungus36. The group S6 contains the Ag-NPs coated fabric samples showing the reduction percentage of fungal activity values against A. niger, whereas group S12 contains the Cu-NPs coated fabric samples showing the reduction percentage of fungal activity values. While comparing the reduction percentage of fungal activity values against A. niger between S6 and S12. The tail of error bar of silver coated S6 sample group is not coinciding with the head of the error bar copper coated S9 sample. It means there is significant difference between two groups, which implies that silver and copper particles coated samples have reduction percentage of fungal activity values. The P value between these two groups was observed as 0.013 which is P < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group (percentage of fungal activity values against A. niger) and copper coated sample S12.

Figure 12
figure 12

Images shows fungus growth against (a) silver particles coated samples, (b) against copper particles coated samples, (c) raw cotton and (d) percentage reduction in fungal spore germination for each fabric specimen.

Antiviral effectiveness

The Behrens and Karber method was used to measure the antiviral effectiveness. Starting with initial viral titer of infectivity to determine the decrease in viral titer for coronavirus. The viral infectivity titer log is shown in Fig. 13 for both 0 h and 6 min. Overall, it showed that all treated samples (S4-S6 and S10-S12) with nanoparticles had sharply reduced viral infectious titer more than double as compared to untreated samples. However, there was no considerable difference of the titer amount in samples treated with either Ag-NPs or Cu-NPs. It indicates both silver and copper nanoparticles are almost equally effective in reducing viral infection in tested cell lines.

Figure 13
figure 13

Reduction in viral infectivity titer (a) and percentage adsorption (b) calculated from viral infectivity at a contact time of 0 and 60 min.

One possible mechanism for the suppression of viruses and the antiviral effects seen involves the interaction between particles and glycoproteins on the viral surface. In a recent research silver particle coated fabric were fabricated by photo deposition method. The couple effect of Ag0/Ag+ redox active agent exhibits 97% viral reduction specific to SARS-CoV-242. The group S6 contains the Ag-NPs coated fabric samples showing the virus adsorption in percentage by nanoparticles, whereas group S12 contains the Cu-NPs coated fabric samples showing the virus adsorption in percentage by nanoparticles. While comparing the virus adsorption in percentage between S6 and S12. The tail of error bar of silver coated S6 sample group is not coinciding with the head of the error bar copper coated S9 sample. It means there is significant difference between two groups, which implies that silver and copper particles coated samples have significant difference in virus adsorption in percentage. The P value between these two groups was observed as 0.03 which is P < 0.05. Hence the P value was less than 0.05 which means there is significant difference between silver coated S6 sample group and copper coated sample S12.