Silver nanoparticles as antimicrobials: A comparative analysis of green and traditional chemistry synthesis methods
Karla Vizuete, Dayanna Gabriela Cabascango, Tamia Xaymara Iza García, António Machado, Fernanda Pilaquinga, Lenys Fernandez, Patricio J. Espinoza-Montero, Alexis Debut
TL;DR
This study compares green and traditional methods for making silver nanoparticles, finding differences in stability and antimicrobial effectiveness against bacteria.
Contribution
The study provides new insights into how synthesis methods affect nanoparticle stability and antimicrobial performance.
Findings
Green-synthesized AgNPs showed increased antimicrobial activity over time against S. aureus and E. coli.
Traditional AgNPs demonstrated greater colloidal stability compared to green AgNPs.
Silver ion release correlated with antimicrobial efficacy, highlighting the role of Ag⁺ in antibacterial effects.
Abstract
Silver nanoparticles (AgNPs) have garnered attention due to their antimicrobial properties and applications in nanomaterials. The objective of this study is to compare the antimicrobial activities of AgNPs synthesized using green and traditional methods with silver ions (Ag+). The characterization of AgNPs was conducted through the utilization of UV-Vis spectroscopy, energy-dispersive X-ray spectrometry, transmission electron microscopy, X-ray diffraction, and differential pulse voltammetry (DPV). AgNPs presented quasi-spherical nature and well-dispersed characteristics, with a mean diameter around 10nm and 25nm for the traditional and green methods respectively. DPV measurements showed a decline of the area under the curve from 1.79 μA mm-2 to 0.7679 μA mm-2, indicating limited colloidal stability of green AgNPs. In contrast, traditional AgNPs demonstrated stability over time,…
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Fig 9- —http://dx.doi.org/10.13039/501100006368Universidad de Las Palmas de Gran Canaria
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Taxonomy
TopicsNanoparticles: synthesis and applications · Gold and Silver Nanoparticles Synthesis and Applications · Nanomaterials for catalytic reactions
Introduction
As humanity endeavors to mitigate pollution, green chemistry —with its twelve principles—has emerged as a potential solution for the development of sustainable technologies since the 1990s [1]. According to the U.S. Environmental Protection Agency (EPA), green chemistry is defined as the design of processes and chemical products that reduce or eliminate the use or generation of toxic substances. This definition can be extended to all stages of a product’s life cycle, including its design, manufacture, use, and disposal [2].In the field of nanotechnology, traditional chemical and physical synthesis techniques have been partially replaced by green chemistry methods because they consume high amounts of energy, use toxic and hazardous reagents, generate toxic by-products, and require complex laboratory equipment and synthesis conditions [3].
Green synthesis of nanomaterials has demonstrated considerable potential at the laboratory scale due to its simplicity. This approach utilizes clean, safe, and environmentally friendly processes to produce nanomaterials [4,5]. In the specific case of nanoparticle formation, it has been demonstrated over the past decades that biological systems, parts, or their extracts, including plants [6,7], algae [8], fungi [6,9], and bacteria [10], can act as reducing or stabilizing agents for the synthesis of nanoparticles [11,12]. The use of solvents, reducing agents, and non-toxic stabilizers has facilitated the prospective applications and use of nanoparticles obtained by green chemistry in fields such as environmental remediation [13], photocatalysis [14], sensors [15], solar cells [16], and energy storage [17]. Moreover, numerous authors have reported the biocompatibility, low toxicity, and antimicrobial activity of these materials, which have been employed in a wide range of biomedical applications, including diagnostics [18], wound healing and dressing [19], immunotherapy [20], regenerative medicine [21], and targeted drug delivery [22] at the laboratory level. It is also noteworthy that the appeal of green nanoparticle synthesis lies in the fact that the majority of scientific articles cite it as a cost-effective method [23–29]. However, to the authors’ knowledge, few economic analyses have been conducted to substantiate this claim. While it is acknowledged that nanoparticles synthesized with green chemistry have the potential to offer numerous advantages, the technique is nevertheless subject to certain limitations. These limitations encompass the selection of eco-friendly raw materials, synthesis conditions, storage modalities, optimization and quality of the product, concerns regarding long-term toxicity, and considerations for large-scale implementation [30–32].
Despite the existence of these documented limitations, silver nanoparticles (AgNPs) have been and continue to be among the most extensively studied in terms of green synthesis methods using biological systems, parts, or their extracts. A remarkable property of these particles is their antimicrobial activity, a property that has been recognized and documented for millennia [33]. For instance, numerous civilizations, including the Persians, Phoenicians, Greeks, Romans, Macedonians, and Egyptians, utilized silver in various ways to preserve food and water, treat post-operative infections, manage ulcers, facilitate wound and burn healing, and so forth. This practice persisted throughout the medieval period, from AD 500–1500, and continues to the present day [34,35]. Concerning the antibacterial activity of silver nanoparticles synthesized through green chemistry, laboratory studies have demonstrated the effectiveness of these particles against a diverse range of Gram-positive and Gram-negative bacteria [36,37]. In parallel with their antimicrobial potential, silver nanoparticles have raised concerns regarding toxicity, particularly in relation to dose, particle size, surface chemistry, and exposure route. Studies have reported cytotoxic effects in mammalian cells, as well as ecotoxicity in aquatic organisms, with outcomes strongly influenced by nanoparticle dissolution and coating [38,39]. These findings underscore the importance of context-specific toxicological evaluation, which remains beyond the scope of the present study. In light of the aforementioned considerations, a substantial effort has been dedicated to elucidating the mechanism of action of silver nanoparticles concerning their antibacterial effect. However, the precise mechanisms underlying this phenomenon remain to be fully elucidated and are the subject of ongoing research and debate. In addition, numerous studies indicate that shape, size, coating, and surface charge are the primary parameters affecting antimicrobial activity [40,41]. The release of silver ions (Ag^+^) and the generation of reactive oxygen species (ROS) have also been identified as key antimicrobial effectors of AgNPs [42,43], among others. The impact of Ag^+^ release on the efficacy of the antimicrobial activity of AgNPs has been a subject of debate, raising questions about the true effectiveness of nanoparticles in this regard [44,45]. In order to gain insight into this unknown phenomenon, a variety of approaches and methodologies have been employed. However, the results of these investigations have been inconclusive or contradictory [45–50]. Therefore, it is essential to understand the amount of Ag^+^ that coexists with AgNPs to accurately assess their impact on the antimicrobial activity of silver ions and silver nanoparticles. The most common technique employed for this purpose is inductively coupled plasma mass spectrometry (ICP-MS). A notable drawback of ICP-MS is the high capital and operating costs, so not all laboratories can afford this type of equipment [51].
In this context, the main objective of the present study is to compare the antimicrobial activity exhibited by nanoparticles synthesized using green chemistry, traditional chemistry, and silver ions at two defined time points: the day of synthesis and thirty days later. The second objective is to estimate the amount of Ag^+^ present in a solution of AgNPs synthesized by green and traditional chemistry using a differential pulse cyclic voltammetry technique. This approach is economically advantageous in comparison to ICP-mass spectrometry while maintaining the capacity to assess the impact of ion release on the antimicrobial activity of these nanoparticles. Additionally, this study briefly discusses the cost-effectiveness of using silver nanoparticles synthesized by green and traditional chemistry, to provide readers with a more comprehensive understanding of the concept of cost-effective green synthesis.
In this work, we used Bursera graveolens leaves to obtain green synthesized AgNPs. B. graveolens, also known as “palo santo”, is one of the most common native trees in the tropical and dry forests of Ecuador and its trunk and leaves are frequently traded in local markets [52]. This tree is widely used for both spiritual and therapeutic purposes. It is used to treat conditions such as acne, skin pimples, styes, rheumatism, swelling, bone pain, coughs, ringworm and insect bites [53]. It has also been used before to synthesize AgNPs using green chemistry. Thanks to its antioxidant properties, B. graveolens leaves extract can act as reducing and capping agent in AgNPs manufacture [54].
Materials and methods
Silver ion solution
Silver nitrate (Sigma Aldrich, > 99%) stock solution was prepared in type 1 distilled water (resistivity of 18.2 MΩ·cm, conductivity less than 0.056 µS/cm, total organic carbon content usually below 30 ppb).
Nanoparticle synthesis
Synthesis of silver nanoparticles (AgNPs) using green chemistry.
The B. graveolens leaves were purchased in Sangolquí, Pichincha-Ecuador local market (−0.3345085,-78.4496945). Leaves were carefully washed with distilled water and subsequently dried in an oven set at 25°C for 5 days. The dried organic material was ground and stored at 4°C, avoiding exposure to sunlight. For the preparation of the extract, 0.5 g of the leaves were mixed with 20 mL of distilled water at 60°C under constant stirring for one hour. The mixture was then centrifuged at 2224 xg for 20 minutes. The supernatant was filtered through Whatman 41 filter paper under vacuum. The extract was stored at 4°C in the dark.
For nanoparticle synthesis, 150 mL of a 5 mM AgNO_3_ solution were mixed with 3 mL of the leaf extract and heated at 70°C for 1 hour, corresponding to a loading dose of 0.75 mmol of AgNO_3_. The pH was adjusted to 9.5 ± 0.5 using 1% NaOH (Sigma Aldrich) solution dropwise, and the solution was further agitated and heated at 70°C for another hour. An additional silver nanoparticles sample was synthesized using a 15 mM AgNO_3_ solution, corresponding to a loading dose of 2.25 mmol of AgNO_3_, following the same procedure afore described.
Synthesis of silver nanoparticles (AgNPs) using traditional chemistry
The synthesis of silver nanoparticles (AgNPs) using traditional chemistry via sodium borohydride was based on the previous study realized by Khatoon et al. (2023), with slight modifications [55]. Briefly, AgNPs were synthesized using 150 mL of a 5 mM AgNO3 (Sigma Aldrich) solution as a precursor agent, corresponding to a loading dose of 0.75 mmol of AgNO_3.. An aqueous solution of sodium borohydride (NaBH_4, Sigma Aldrich) was prepared at a concentration of 0.5 mg mL^-1^. The AgNO_3_ solution was immersed in an ice bath, and the reducing agent was added dropwise under constant stirring until the color of the solution stabilized. Subsequently, the solution underwent a centrifugation process, after which the precipitate was thoroughly rinsed with distilled water. The precipitate was redissolved in 100 mL of distilled water and stored at 4°C in the dark. The same procedure was followed to prepare AgNPs at a concentration of 15 mM, corresponding to a loading dose of 2.25 mmol of AgNO_3_,
Nanoparticle characterization
The UV-vis characterization of AgNPs was conducted using a Cary 60 double-beam UV-Vis spectrophotometer (Agilent Technologies, USA) equipped with polystyrene cells, which had a 1 cm optical path. The measurements were conducted within the range of 300–800 nanometers.
Energy-dispersive X-ray spectrometry (EDS) and elemental mapping were performed in a field emission scanning electron microscope (Mira 3, Tescan, Czech Republic) using an EDS detector (X-Flash 6|30, Bruker, Germany) with a 123 eV resolution at Mn Kα. The AgNPs micrographs were obtained using a Transmission electron microscope (Tecnai G2 Spirit Twin, FEI, Netherlands) coupled with an Eagle 4k HR camera at 80 kV. The X-ray diffraction (XRD) analysis of the AgNPs was performed in a diffractometer (EMPYREAN, PANalytical, United Kingdom) operating in a θ − 2θ configuration (Bragg-Brentano geometry) equipped with an X-ray tube of copper (Kα radiation λ = 1.54056 Å) at 45 kV and 40 mA. Highscore© software with Crystallography Open Database (COD) was used for data interpretation [56].
Differential pulse voltammetry (DPV)
Differential pulse voltammetry (DPV) was employed to monitor the stability and evolution of traditional and green silver nanoparticles (AgNPs) [57]. The oxidation signal of Ag^0^/Ag^+^ at 0.5 V (vs. Ag/AgCl) was used to track the presence and stability of AgNPs. For this purpose, a potentiostat (1400, CH Instruments, USA) and a single-compartment three-electrode electrochemical cell equipped with a working electrode of vitreous carbon (3 mm diameter), a counter electrode of graphite, and a reference electrode of Ag/AgCl (3 M KCl) were used. Before each measurement, the working electrode was polished with alumina powder of grain size 1.0, 0.3, and 0.05 μm in succession, followed by rinsing with abundant 18 MΩ deionized water. A pH 5 acetate buffer was utilized as the supporting electrolyte, i.e., background. Voltammograms were recorded in the potential range of 0.0 to 1.2 V (vs. Ag/AgCl) with a scan rate of 100 mV s^-1^ at the synthesis day and 30 days after (n = 3). Prior to each measurement, the reaction medium was purged with nitrogen gas for five minutes to eliminate dissolved oxygen.
Antimicrobial activity analysis
Bacteria isolates and growth conditions.
All potential treatments were then studied for their antibacterial activities against two types of bacteria, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, from the bacterial collection of Microbiology Institute of the Universidad San Francisco de Quito (IM-USFQ). These bacterial species stored at −80°C were cultivated on Mueller-Hinton agar (MHA) medium at 37°C for 24 hours before each assay. These strains were selected because S. aureus ATCC 25923 (Gram-positive) and E. coli ATCC 25922 (Gram-negative) are internationally recognized reference quality control strains recommended by CLSI and EUCAST guidelines for antimicrobial susceptibility testing, ensuring that our results are standardized, reproducible, and comparable with other studies.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).
The microdilution method for minimum inhibitory concentration (MIC) assays was performed, as described by Wiegand and colleagues [58], under the considerations established by the Clinical Laboratory Standards Institute (CLSI) [59]. Briefly, 10 μL of each treatment (green AgNPs, traditional AgNPs, and Ag^+^) at different molar concentrations were placed in a 96-well plate to determine MIC and MBC values, followed by 190 μL of Mueller-Hinton broth (MHB) containing bacteria at a final concentration of 1x10^5^ colony-forming units (CFU) mL^-1^. This resulted in final treatment concentrations in the wells of 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 10, and 15 mM. The plates were then incubated at 37°C for 24 hours. Additionally, 200 μL of MHB medium with bacteria was placed as a positive control and 200 μL of just MHB medium as a negative control (sterility control). The results were measured using the Biotek Instruments ELx808IU spectrophotometer at 570 nm (OD570) and the lowest concentration of each treatment without bacterial growth was classified as MIC [60]. Finally, the minimum bactericidal concentration (MBC) was identified by removing 50 μL of each treatment into the 96-well plate, replacing it with 50 L of resazurin (0.015%; m/v), and finally incubating the 96-well plate at 37°C for 1–2 hours until a complete color change (i.e., resazurin blue to pink color) was observed in the positive control (MHB medium plus bacteria without treatment). The MBC value was determined when the blue resazurin color remained unchanged indicating no metabolism and microbial death, as previously described by Elshikh and colleagues [61]. All tests were performed at least 6 times and with two replicates. These assays were also performed in two phases. The initial phase of the experiment was conducted at a concentration of 15 mM, while the subsequent phase involved a concentration of 5 mM, in two independently prepared treatments. All these assays were realized on days 1 and 30 later following the synthesis procedure.
Statistical analysis
For pairwise comparison between control and treated samples in MIC assays, the Wilcoxon non-parametric test was used through R studio version 4.0 (https://www.rstudio.com/products/rstudio/download/) using several R packages (“ggpubr”, “rstatixs”, “openxlsx”, and “tidyverse”) [62,63]. All p-values <0.05 were considered significant.
Results
Silver nanoparticles were synthesized by green and traditional methods, and their formation was confirmed by UV-Vis spectroscopy. The UV-Vis absorption spectra of the prepared samples by green and traditional chemistry techniques are shown in Fig 1. It displays a single peak centered at 395 nm for AgNPs synthesized by traditional chemistry using NaBH_4_ for both concentrations 5 and 15 mM of AgNO_3_. In contrast, the peak for those synthesized using B. graveolens extract is centered at 400 nm, as well, for both concentrations studied.
(a) and (c) UV-Vis spectra of AgNPs synthesized by traditional chemistry with 5 and 15 mM, respectively.(b) and (d) UV-Vis spectra of AgNPs synthesized by green chemistry with 5 and 15 mM, respectively.
The EDS spectrum obtained from the selected area of each sample exhibits the presence of Ag. The energy of the silver emission was recorded at 2.98 keV (Fig 2). Elemental mapping also confirms the presence of Ag in each sample.
EDS spectra of AgNPs synthesized with NaBH4 as reducer agent (traditional chemistry), AgNPs synthesized with B. graveolens as reducer agent (green chemistry), and AgNO3 solution, all at concentration of 5 and 15 mM.
TEM micrographs were obtained on days 1 and 30 following the synthesis procedure. The TEM micrographs obtained on days 1 and 30 post-synthesis serve to substantiate the presence of quasi-spherical silver nanoparticles in both traditional and green chemistry experiments, employing 5 mM and 15 mM concentrations of AgNO_3_, respectively.
The mean diameter of the nanoparticles was calculated using Image J. Table 1 summarizes the arithmetic average diameter of the synthesized nanoparticles.
Table 1: Mean diameter of the synthesized AgNPs using traditional and green chemistry.
The XRD diffractograms of AgNPs obtained by green and traditional methods are shown in Fig 3a and 3b, and the XRD pattern of silver ions solution is shown in Fig 3c. The utilization of Highscore software has enabled the identification of zerovalent silver in both synthesis methods, as indicated by the COD database code 90110607. The (111) and (200) planes were identified at approximately 38° and 44.3°, respectively, for green and traditional synthesis techniques. Furthermore, silver nitrate, COD database code: 1509468, was found in the silver ion solution. Using the Scherrer equation, the crystallite grain size was estimated to be 27 and 29 nm for green and traditional synthesized AgNPs, respectively.
XRD patterns of a) traditionally synthesized AgNPs, b) green synthesized AgNPs, and c) silver ions.
Differential pulse voltammograms are illustrated in Fig 4. Fig 4a shows the voltammograms of green synthesized AgNPs colloidal suspension at 5mM and B. graveolens extract. After 30 days, a considerable decrease in the voltammetric signal at 0.5 V (vs. Ag/AgCl) was observed in the voltammogram of the green synthesized AgNPs colloidal suspension. In contrast, the voltammograms obtained for AgNPs suspensions obtained by the traditional method (Fig 4b) show a signal at 0.5 V (versus Ag/AgCl), which is practically maintained from days 1–30.
Differential pulse voltammogram of a) Green AgNPs 5Mm day 1 and 30 and b) Differential pulse voltammogram of traditional AgNPs 5 mM day 1 and 30 (A: Peak area at 0.5 V for each voltammogram).
The antibacterial activity of green AgNPs, traditional AgNPs, and silver ions was evaluated by determining the MIC and MBC for both E. coli ATCC 25922 and S. aureus ATCC 25923 according to the Clinical & Laboratory Standards Institute (CLSI) guidelines.
For phase 1 (green-AgNPs, traditional-AgNPs, silver ions at 15mM, and plant extract), planktonic growth of E. coli ATCC 25922 and S. aureus at days 1 and 30 days after the synthesis are shown in Figs 5 and 6. Figs 7 and 8 show the planktonic growth of E. coli ATCC 25922 and S. aureus in phase 2 (green AgNPs, traditional AgNPs, silver ions at 15 mM and NaBH_4_) on days 1 and 30 after synthesis.
Inhibition of E. coli ATCC 25922 planktonic growth using silver nanoparticles synthesized by traditional chemistry and green chemistry, silver ions, and plant extract at days 1 and day 30 from phase 1 (15mM).The bars indicate the percentage of planktonic growth from the lowest to the highest concentration, the internal number indicates the percentage of inhibition, and the asterisks indicate the level of significance. The blue frame indicates the minimum inhibitory concentration (MIC), the purple frame indicates the minimum bactericidal concentration (MBC), and the orange frame indicates when MIC and MBC are the same concentration * p < 0.05; ** p < 0.01; * * * p < 0.001; **** p < 0.0001, (ns) non-significant.
Inhibition of S. aureus ATCC 25923 planktonic growth using silver nanoparticles synthesized by traditional chemistry and green chemistry, silver ions, and plant extract at day 1 and day 30 from phase 1 (15mM).Minimum inhibitory concentration (MIC) in using silver nanoparticles synthesized by traditional chemistry and green chemistry, silver ions, and plant extract. The bars indicate the percentage of planktonic growth from the lowest to the highest concentration, the internal number indicates the percentage of inhibition, and the asterisks indicate the level of significance. The blue frame indicates the minimum inhibitory concentration (MIC), the purple frame indicates the minimum bactericidal concentration (MBC), and the orange frame indicates when MIC and MBC are the same concentration * p < 0.05; ** p < 0.01; * * * p < 0.001; **** p < 0.0001, (ns) non-significant.
Inhibition of E. coli ATCC 25922 planktonic growth using silver nanoparticles synthesized by traditional chemistry and green chemistry, silver ions, and NaBH4 (reducing agent of traditional synthesis method) at day 1 and day 30 from phase 2 (5mM).The bars indicate the percentage of planktonic growth from the lowest to the highest concentration, the internal number indicates the percentage of inhibition, and the asterisks indicate the level of significance. The blue frame indicates the minimum inhibitory concentration (MIC), the purple frame indicates the minimum bactericidal concentration (MBC), and the orange frame indicates when MIC and MBC are the same concentration * p < 0.05; ** p < 0.01; * * * p < 0.001; **** p < 0.0001, (ns) non-significant.
Inhibition of S. aureus ATTC 25923 planktonic growth using silver nanoparticles synthesized by traditional chemistry and green chemistry, silver ions, and NaBH4 (reducing agent of traditional synthesis method) at day 1 and day 30 from phase 2 (5mM).The bars indicate the percentage of planktonic growth from the lowest to the highest concentration, the internal number indicates the percentage of inhibition, and the asterisks indicate the level of significance. The blue frame indicates the minimum inhibitory concentration (MIC), the purple frame indicates the minimum bactericidal concentration (MBC), and the orange frame indicates when MIC and MBC are the same concentration * p < 0.05; ** p < 0.01; * * * p < 0.001; **** p < 0.0001, (ns) non-significant.
In phase 1, the B. graveolens plant extract exhibited no antimicrobial activity on the first day or the thirtieth day for either S. aureus or E. coli as had been anticipated. The minimal inhibitory concentration (MIC) values of green and traditional AgNPs were 1 mM on days 1 and 30, respectively. However, higher antimicrobial activity of green AgNPs was found at day 30 compared to day 1. An increase in the inhibition of the planktonic growth of E. coli and S. aureus was observed, with an initial inhibition percentage of 86% and 76% compared to 93% and 87%, at days 1 and 30, respectively, for each bacterium. In contrast, traditional AgNPs demonstrated comparable inhibition of planktonic growth on days 1 and 30. Similarly, silver ions antimicrobial effect exhibited the same tendency, first a similar antimicrobial effect compared to green and traditional AgNPs, and second a notable augmentation in the inhibition of the planktonic growth of day 1 compared to day 30 for E. coli and S. aureus (see Figs 5 and 6).
In the second phase of the study, a decline in the initial antimicrobial activity was observed for both E. coli and S. aureus (at days 1 and 30) when utilizing green chemistry-synthesized AgNPs. In contrast, the Traditional method demonstrated comparable antimicrobial activity to that observed in phase 1. Furthermore, the investigation revealed a dose-dependent inhibition effect for silver ions, as evidenced by the findings presented in Figs 7 and 8. Moreover, as shown in Supplementary S1 and S2 Tables, the MIC and minimum bactericidal concentration (MBC) values are also similar in both traditional and green chemistry approaches.
To summarize, Table 2 exposes the obtained minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the assays against S. aureus ATCC 25923 and E. coli ATTC 25922, for all treatments that were evaluated in this present study.
Table 2: Summary of the results obtained in the Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays against S. aureus ATCC 25923 and E. coli ATTC 25922. Legend – MIC: minimum inhibitory concentration, MBC: Minimum bactericidal concentration for each bacterium.
Discussion
Silver nanoparticles have garnered significant attention from the scientific community due to their intrinsic antimicrobial properties. This heightened focus has resulted in a substantial presence of these particles within the domain of nanostructured materials research [64,65]. This assertion is supported by a significant number of scientific publications and patents [66]. In addition, a considerable volume of research has demonstrated that the dimensions and morphology of AgNPs, which are inherently determined by the synthesis conditions, have a substantial impact on their effectiveness as an antimicrobial agent, in terms of activity and cellular uptake [40]. In this sense, the primary objective of this study was to comparatively analyze the antimicrobial activity of silver ions and nanoparticles synthesized via green and traditional chemistry. In addition, the study sought to assess the impact of released silver ions on antimicrobial effectiveness.
The synthesis of silver nanoparticles was confirmed by UV-Vis. The UV-Vis absorption spectra of the nanoparticles synthesized by traditional and green methods are displayed in Fig 1. In all cases, a single peak around 400 nm was observed, confirming the spherical shape of the synthesized silver nanoparticles [67,68]. As demonstrated in Fig 1a and 1c, the spectra of 5 mM and 15 mM AgNPs synthesized via traditional chemistry present a single peak centered at 395 nm. In contrast, the spectra of AgNPs synthesized through green chemistry (see Figs 1b and 1d) exhibit a peak centered at 400 nm. These results are consistent with the conclusions drawn in other studies [69,70]. The difference in the peak positions is attributed to the size and shape of the nanoparticles, and the use of stabilizing agents inherent to the plant extract used for the synthesis [71,72]. The findings of this study are consistent with a previous investigation that employed B. graveolens plant extract for AgNPs green synthesis. This earlier study indicated that the secondary metabolites of B. graveolens can function as a reducing agent in the formation of silver nanoparticles, thus preventing aggregation, collision and coalescence through interparticle interactions, and reported a surface plasmon resonance peak centered at 400 nm [54]. Thereby, a blue shift in the peak position indicated smaller nanoparticle sizes, while a red shift was associated with larger nanoparticles, in agreement with TEM observations [73]. In addition, for the traditional synthesis method (see Fig 1c and 1d), the absorption band of the 15 mM AgNPs was observed to be wider than 5 mM AgNPs, suggesting size variability. Furthermore, a significant decrease in absorbance was also observed in AgNPs using traditional chemistry compared to the green method at 5 and 15 mM. This may be due to the absence of a stabilizing agent and also to a reduced nucleation, resulting in less nanoparticle formation [73,74].
The EDS spectra collected from specific areas of each sample, as shown in Fig 2, clearly indicated the presence of silver, as the silver emission energy peak is identified at 2.98 keV, corresponding to the L 1 shell of Ag [75]. Elemental mapping analysis also provided additional evidence supporting the presence of silver within the sample.
As illustrated in Fig 9, transmission electron microscopy (TEM) images of green and traditional AgNPs were presented on days 1 and 30. In all cases, the TEM images revealed the presence of quasi-spherical nanoparticles that exhibited well-dispersed characteristics, a finding that aligns with the results previously reported in the UV-vis analysis. As demonstrated in Fig 9, the green synthesis of nanoparticles at concentrations of 5 and 15 mM resulted in the formation of AgNPs with particle sizes of 25.61 7.83 nm and 23.47 5.95 nm, respectively. It is important to emphasize that the morphology and size of the obtained green silver nanoparticles remained constant 30 days after the synthesis day. Besides, TEM images revealed the presence of an organic layer surrounding green-synthesized AgNPs. The occurrence of this layer can be attributed to the organic compounds present in the B. graveolens extract. These compounds act as a stabilizing agent, preventing agglomeration and thus increasing size [76,77]. Conversely, TEM micrographs of conventional chemistry AgNPs at concentrations of 5 and 15 mM demonstrated nanoparticle formation with a size of 7.70 2.90 nm and 8.13 4.02 nm, respectively. In contrast with green synthesized AgNPs, traditional chemistry AgNPs exhibited a slight increase in size before 30 days of synthesis, reaching 10.70 7.86 nm for the 5 mM concentration and 11.32 6.53 nm for the 15 mM concentration. As previously stated, the absence of a stabilization agent in this synthesis could be a contributing factor to the observed increase in size over time.
TEM images of AgNPs synthesized with NaBH4 as reducer agent (traditional chemistry) and AgNPs synthesized with B. graveolens as reducer agent (green chemistry) at days 1 and 30.The AgNPs were observed at concentrations of 5 and 15 mM of AgNO3.
XRD confirmed that green and traditional synthesis methods produced mainly Ag^0^ (Fig 3), since no silver oxide was found, or if present, its concentration was below the detection limit. The application of Highscore software in conjunction with the COD database facilitated the identification of Ag^0^ as a cubic system within the F m −3 m group space. These findings are consistent with those of earlier studies on green and traditional AgNPs [78–80]. In addition, the crystalline size estimated from the Scherrer equation showed a good correlation with the TEM particle size determination.
Electrochemistry is a relatively straightforward, cost-effective, rapid, and portable analytical method that has garnered significant interest in the analysis of metal nanoparticles [81]. Therefore, this could be considered as a strong tool to evaluate the transformation of Ag^0^ to Ag^+^ in an AgNPs suspension. Differential pulse voltammograms of the plant extract, green, and traditional AgNPS are exhibited in Fig 4. Fig 4a present the voltammograms of B. graveolens plant extract and green synthesized AgNPs (5 mM). It is important to highlight that the voltammogram of the plant extract demonstrated an almost complete dissipation of the voltammetric signal. This finding suggests that the antioxidant species initially present in the B. graveolens extract have been significantly depleted, indicating its use as a reducing and capping agent for AgNPs synthesis [57]. Furthermore, Fig 4a shows the evolution of the green synthesized AgNPs from Ag^0^ to Ag^+^, as evidenced by the measurement of the current density signal at 0.5 V (vs. Ag/AgCl) in the electrolytic medium over a period of 30 days. The current density value for the Ag^0^/Ag^+^ signal decreased from day 1 to day 30, indicating that AgNPs convert back into silver ions (Ag^+^), suggesting instability of the nanoparticulate material in the colloidal medium [82]. This result contrasts with the voltammograms obtained for AgNPs suspensions obtained with the traditional method (5 mM) (Fig 4b). In this case, the current density at 0.5 V, Ag^0^/Ag^+^ oxidation was practically maintained from day 1 to day 30. Fig 4 also reports the peak areas calculated at 0.5 V for each of the voltammograms, these values were similar to the concentration of AgNPs in suspension [83]. The area under the curve decreased for the sequence of voltammograms of the green synthesized AgNPs suspensions, from 1.79 A mm^-2^ (day 1) to 0.7679 A mm^-2^ (day 30), again indicating the instability of the nanomaterial. While the areas of the AgNPs obtained by traditional chemical synthesis, around 31 A mm^-2^, exhibited no significant variation over a 30-day period. Thereby suggesting a higher stability of traditional AgNPs (mostly Ag^0^) compared to green AgNPs (mixture of Ag^0^ + Ag^+^). Green and traditional AgNPs at 15 mM concentration were not tested by differential pulse voltammetry (DPV) because electrode surface saturation was observed at this concentration [84].
On the other hand, despite the extensive documentation of the inhibitory effect of AgNPs on microbial growth, a notable variability in outcomes was observed when employing diverse microbiological techniques to assess the antimicrobial activity of these nanoparticles [85]. The most frequently applied antimicrobial assay techniques in most studies are those based on agar diffusion procedures, such as disk diffusion and well diffusion [86,87]. The primary objective of these microbiological techniques is to produce qualitative results by indicating the presence or absence of antimicrobial activity through the inhibition of the microorganism in culture growth, which provides a relative indication of antimicrobial effectiveness [88]. The prevalence of these methodologies can be attributed to three factors: their cost-effectiveness, the minimal requirement for specialized equipment, and the lower need for microbiological expertise [89,90]. Nonetheless, it must be noted that agar diffusion assays show inherent limitations, which have the potential to impact the result of the antimicrobial test. The variability in results can be attributed to several factors, particularly the diffusion of the antimicrobial agent due to its molecular weight, solubility, thickness and uniformity of the agar, as well as the incubation conditions [86,87]. Furthermore, the interpretation of the inhibition zones obtained by agar diffusion assays is subjective and has the potential to introduce bias to the results [90,91]. For instance, Chung et. al. (2023) tested ten different nanomaterials against ten distinct microbial strains using both the agar well diffusion method and the resazurin-based broth microdilution assay, with a total of 100 tests conducted (excluding triplicates). As a result, no antimicrobial effect was observed in 76 nanoparticle suspensions, despite the documented antimicrobial activity of these nanomaterials. Whereas the resazurin-based broth microdilution assay yielded 75 positive results for antimicrobial activity. It was also observed that all the positive antimicrobial results obtained from the agar well diffusion method were consistent with the results obtained using the resazurin-based broth microdilution assay, with one exception [92]. In this study, the authors indicated that direct physical contact between the nanoparticles and microbes is a prerequisite for the observation of antimicrobial function. Likewise, the degree of the diffusion ability of different nanoparticles through the solid agar is dependent on the physico-chemical properties of the material, particularly the size. Therefore, it is hypothesized that smaller particles will demonstrate higher diffusion efficiency in comparison to larger particles [93]. This increased diffusion efficiency is expected to result in a stronger antimicrobial effect [94]. Additionally, agar diffusion methods are not adequate for determining the minimum inhibitory concentration (MIC), as well-established by CLSI guidelines [59,90]. This is due to the inability to quantify the amount of the antimicrobial agent that diffuses into the agar medium [95]. Also, the diffusion coefficient of the material is not always known, and there may be differential diffusion rates from the various components of the material [91,92,94,96].
In light of the aforementioned assertions, the broth microdilution method emerges as a more suitable technique. This is primarily due to its quantitative nature, which facilitates the determination of the minimum inhibitory concentration (MIC) as the concentration of the antimicrobial agent is known [97]. Moreover, there are numerous authorized guidelines for the execution of dilution antimicrobial susceptibility testing of a variety of microorganisms. The most prominent standards are stipulated by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [59,98].
The present study evaluated the antibacterial effects of Ag^+^ and AgNPs on planktonic growth of S. aureus and E. coli. The antibacterial effects were measured by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the broth microdilution method (see Table 2), where a similar antibacterial effect was observed against S. aureus ATCC 25923 by green/traditional AgNPs and Ag^+^ (MIC and MBC values ≈ 1 mM). However, a slight difference was detected against E. coli ATCC 25922, where traditional AgNPs evidenced lower MIC and MBC values on day 30 (≈ 0.5 mM) and Ag^+^ ions evidenced MIC and MBC values of 0.5 and 0.75 mM, respectively, on both days 1 and 30. This result was surprising due to this species as well as other Gram-negative bacteria ability to extrude antibacterial agents through the porins and efflux transport systems [99].
Despite the extensive utilization of AgNPs as an antimicrobial agent, its precise antimicrobial mechanisms of AgNPs remain to be fully elucidated [100,101]. It is imperative to acknowledge that the effect of AgNPs on a particular organism is contingent on several factors. The physicochemical properties of the particles encompass a range of factors, including but not limited to: morphology, dimensions, surface modifications, and dissolution. Furthermore, the bacterial strain in question, the toxicity criterion under assessment (i.e., growth inhibition or complete eradication), the nature of the evaluation method, the concentration of the AgNPs, the composition of the medium, temperature, pH, ionic strength, binding partners, and the presence of light or oxygen must be taken into account [42,102,103]. In consideration of the aforementioned elements, the most probable mechanism is hypothesized to include the following three factors: (i) significant interference with cellular processes caused by direct damage to the cell membrane, (ii) disruption of molecules within the cell, and (iii) oxidative stress triggered by metal-induced reactive oxygen species (ROS), ultimately leading to free radical formation and widespread damage to the cell [64]. The initial accumulation of AgNPs in the cell wall/membrane has been demonstrated to induce alterations in cell structure, manifesting finally as cell wall and membrane disruption. This process is preceded by a series of morphological changes, including cytoplasmic shrinkage, membrane detachment, the appearance of numerous electron-dense pits, an increase in cell membrane permeability, membrane depolarization, and a decrease in respiratory potential [46,104]. Over time and depending on the medium, AgNPs will undergo a series of processes including but not exclusively, nucleation, suspension, and dissolution. These processes result in the generation of multiple coexisting species derived from silver, including AgNPs/Ag-ions, Ag-ions/complexes, and suspended/aggregated AgNPs [49]. It has been demonstrated that free diffused silver ions get inside the cell and interact with biomolecules such as enzymes, nucleic acids, proteins, DNA, and RNA, among others, causing the inactivation of the biological system [46,105,106]. Concurrently, the augmentation of Ag^+^ in the medium with dissolved oxygen is anticipated to result in an escalation of cellular oxidative stress which is related to the generation of abnormal levels of reactive oxygen species (ROS) and free radicals. ROS can chemically modify by oxidation a variety of macromolecules, altering their structure and therefore their function can be affected (i.e., hyperoxidation of proteins, lipids, DNA, and cell membrane), usually resulting in cell toxicity and death [40,107]. For example, an excess of ROS and free radicals have been demonstrated to cause the following: glutathione (GSH) oxidation, which is an antioxidant that prevents cellular damage; direct damage to the mitochondrial membrane, which consequently relays signaling and blocks the mitochondrial respiratory function of bacteria; dysfunction of the electron transport chain and proton motive force due to the inactivation of membrane-bound enzymes, impacting ATP synthesis and, thereby, affecting many vital cell functions; and DNA damage [42,46].
The documented impact of Ag^+^ on the antimicrobial effect of AgNPs has prompted several authors to consider AgNPs as reservoirs for silver ions, which are considered the main responsible for the antimicrobial activity. For instance, Xiu et al. (2012) probed the lack of toxicity of AgNPs when synthesized and tested under strict anaerobic conditions. These specific conditions preclude Ag^0^ oxidation and Ag^+^ release. When the anaerobic synthesized AgNPs were exposed to an aerobic environment, which promotes the transformation of AgNPs to silver ions, an enhanced toxicity of the AgNPs was observed. These authors of the study concluded that the observed antimicrobial activity was attributable to the release of Ag^+^ alone and that the toxicity of AgNPs is very sensitive to the presence of air. This finding suggests that no direct, particle-specific effects contributed to the observed toxicity [94]. Despite the similar MIC values for green and traditional AgNPs, the present study yielded analogous results: an increased antimicrobial activity of green AgNPs was observed at day 30 compared to day 1.The result of the antimicrobial test along with those of DPV, were an increase of Ag^+^ was evidenced demonstrating the transformation of some AgNPs to Ag^+^ over time, prove that silver ions haver a critical role in the antimicrobial activity of silver nanoparticles as its release occurs gradually [94,104,108]. In accordance with the aforementioned assertions, Cavassin et al. (2015) ascribed the remarkable in vitro inhibitory effect of citrate AgNPs and chitosan AgNPs against susceptible and multidrug-resistant bacteria to the rapid oxidation process of these particles and its consequent release of Ag^+^, and to the high surface charge of the particles [109]. Also, Salayová et al. (2021), reported similar findings when they tested green synthesized nanoparticles using plant extracts (Brassica nigra seeds, Capsella bursa-pastoris leaves, Lavandula angustifolia leaves, Origanum vulgare leaves, and Berberis vulgaris root) against S. aureus CCM 4223, Listeria monocytogenes CCM 4699, E. coli CCM 3988, Salmonella enterica ser. Typhimurium CCM 7205, and Pseudomonas aeruginosa CCM 3989 [45].
This gradual and medium-dependent release has the potential to be exploited for the manufacture of nanodispositives capable of long-acting, slow-release of Ag^+^. For instance, Fan et al. (2021) demonstrated the efficacy of the chitosan-AgNPs nanogel in the treatment of bacterial infections. Therefore, it is anticipated that the nanogel will also provide an effective treatment modality for implant infections in clinical settings [110].
On the other hand, Li et al. (2017) demonstrated that the antibacterial activity of Ag^+^ was consistently stronger than that of commercial 5 nm and 20 nm AgNPs against E. coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, S. aureus ATCC 6538, and Staphylococcus epidermidis ATCC 12228 under aerobic conditions [111]. Choi and colleagues demonstrated the higher antimicrobial activity of silver ions through their superior toxic effects in planktonic microbial growth of E. coli K-12 compared to commercia AgNPs [112]. Likewise, Kędziora et al. reported that Ag^+^ remain highly effective even at low concentrations, being consistent with the results from our study where the minimum inhibitory concentration was equal or below 1 mM [50]. This is in agreement with our preliminary planktonic evaluation of Ag^+^, which generally showed lower MIC values than green and traditional AgNPs, in particular, against E. coli ATCC 25922. These findings contribute to the ongoing discourse surrounding the true applicability of AgNPs as an antimicrobial agent, as silver ions exhibit comparable, if not superior, antimicrobial activity.
It is important to acknowledge that a slight difference in the initial antimicrobial activity was observed in phase 2 between green and traditional chemistry AgNPs, both against E. coli and S. aureus (see Fig 7 and 8), where traditional AgNPs revealed lower MIC and MBC values, specifically against E. coli on day 30 (see Table 2). As traditional AgNPs were synthesized using NaBH_4_, this reducing agent can also act as a stabilizing agent. As a result, a more efficient and controlled reduction of Ag^+^ to Ag^0^ is obtained, which highly influences the stability, morphology, size of AgNPs and of course have important repercussions on the antimicrobial activity. For instance, in a separate study, the agar plate method was employed to assess the antibacterial properties of green and chemically synthesized AgNPs against Salmonella typhimurium, P. aeruginosa, E. coli, Burkholderia cepacia, and S. aureus. The authors reported that chemically obtained AgNPs exhibited substantial antimicrobial activity against all the bacterium strains that were tested. The diameter of the inhibition halo ranged from 0.70 to 7.00 millimeters, with a standard deviation of 0.1 millimeters. In contrast, AgNPs synthesized using Pelargonium domesticum (green synthesis) did not demonstrate any antibacterial activity, at any concentration, against S. typhimurium, P. aeruginosa, or B. cepacian. In the presence of E. coli and S. aureus, green synthesized AgNPs demonstrated a diameter of the inhibition halo that was measured to be 1.63 mm ± 0.1 mm and 1.3 mm ± 0.1 mm, respectively, at a concentration of 0.8 mM AgNPs. The findings of this study exhibit the superiority AgNPs obtained by traditional chemistry [78] and are in agreement with the results of the present study, were traditional AgNPs demonstrated superior and more consistent antibacterial activity compared to green-synthesized nanoparticles. This phenomenon may be attributed to the inherent stability of traditional AgNPs, a hypothesis that was also supported by the results of the DPV analysis (Figs 4-8 and Table 2).
These findings are in contrast with the results of previous studies conducted by Singh et. al (2018), which reported superior antibacterial activity using Brassicaceae plant extracts obtained through green synthesis methods compared to those AgNPs obtained by traditional chemistry against E. coli, P. aeruginosa, Kocuria, Myroides and Promicromonospora using agar-well diffusion method. Moreover, according to their evaluation, it was posited that plant extracts, in and of themselves, do not interfere with the innate antimicrobial activity of AgNPs [102]. In a notable finding, Shaik et al. (2016) reported that the chemically synthesized AgNPs exhibited comparable or slightly lower antibacterial activity compared to the green synthesized AgNPs determined by modified Kirby-Bauer disk diffusion method. It is noteworthy that the antimicrobial activities of the green-synthesized AgNPs were found to increase in proportion to the volume of plant extract utilized [113]. Likewise, Urnukhsaikhan and colleagues (2021) reported similar antimicrobial activity of biosynthesized AgNPs from Carduus crispus against both Gram-positive and Gram-negative bacteria, postulating the consistency of the antimicrobial activity of AgNPs across different bacterial groups tested by well diffusion method [114]. In 2021, Csakvari and colleagues showed similar antimicrobial activities of AgNPs using cannabis leaf extracts against both E. coli and S. aureus using a disc agar diffusion method [115]. Nevertheless, it is crucial to acknowledge the observations made by Wasilewska and colleagues. The study’s findings indicated the absence of antimicrobial activity in AgNPs synthesized with radish extracts applying minimal inhibitory concentration (MIC) protocols in accordance with the CLSI. In contrast, AgNPs synthesized in the presence of onion, garlic, potato, and apple extracts exhibited the greatest effectiveness against the tested microorganisms (S. aureus strain ATCC 6538, B. cereus strain ATCC 10987, E.coli strain ATCC 11229, and Candida krusei strain ATCC 30135) [116].
The conflicting results previously reported on the antimicrobial efficacy of silver nanoparticles (AgNPs) synthesized through green or traditional methods, as well as the influence of silver ions and their role in the antimicrobial activity, underscore the necessity for a more profound comprehension of nanoparticle behavior in biological environments. These findings further highlight the significance of employing rigorous, quantitative testing methods to advance the clinical and practical application of silver-based nanomaterials.
In addition to these findings, it is essential to address the often-overlooked economic dimension of nanoparticle synthesis methods. Although green synthesis is frequently portrayed in the literature as a cost-effective alternative, it is imperative to underscore that a multitude of publications refer to a latter’s comparatively reduced cost relative to traditional chemistry methods. It is important to note that there is a recurrence in bibliographic references about cost effectiveness of AgNPs, which generates a citation chaining effect. A review of the extant literature reveals a paucity of articles that specifically and thoroughly address the costs associated with the topic in question. Consequently, it is deemed relevant to allocate a section to elucidate these costs, with the objective of enhancing clarity and comprehension in this field. In the domain of scientific research, it has been observed that certain suppliers offer a 25 mL of 50 nm average particle size silver nanoparticle solution with a concentration of 0.02 milligrams per milliliter, prepared by traditional chemistry methods, at a cost of approximately 2,000. This expenditure signifies a substantial increase in investment when juxtaposed with the acquisition of the products directly from a supplier. While a reduction in cost is anticipated in the context of industrial production, it is imperative to consider that the green chemistry method exhibits limited stability in its ability to reproduce the synthesis method, in contrast to the “traditional” chemical method. Therefore, for green chemistry to be considered cost-effective, it is imperative to implement scalable syntheses on an industrial scale and ensure reproducibility of results. In the contemporary context, the designation of such methodologies as “low-cost/cost-effective” could be no longer valid.
Conclusions
This study underscores the complex nature of silver nanoparticles as antimicrobial agents. While both green and traditionally synthesized AgNPs demonstrated inhibitory effects against E. coli and S. aureus, traditionally synthesized nanoparticles exhibited more consistent and stable antimicrobial activity over time. The results of the study also confirm the critical role of silver ions (Ag^+^) in the antimicrobial efficacy of AgNPs. An increase in the inhibition of planktonic growth was observed in conjunction with the release of greater quantities of Ag⁺ into the medium. Furthermore, the findings highlight the importance of selecting appropriate and standardized antimicrobial testing protocols, such as broth dilution, to accurately determine the activity of AgNPs. It is also important to recognize a prevailing perception of green synthesis of silver nanoparticles as a cost-effective alternative to traditional chemical methods. Despite its frequent citation as a cost-effective measure, this assertion frequently originates from the practice of citation chaining rather than from comprehensive cost analyses. A closer examination reveals that green synthesis can incur significantly higher expenses, particularly in research settings. The elevated costs are attributable to the labor-intensive nature of the process, the need for biological materials, and the requirement for advanced characterization equipment. Moreover, the standardization, reproducibility and scalability of green methods are still significant limitations. Therefore, unless these challenges are addressed the label of “cost-effective” for green synthesis methods is may be currently misleading and should be reconsidered in academic and practical discussions.
Supporting information
S1 TableBacterial planktonic growth inhibition Day 1.(DOCX)
S2 TableBacterial planktonic growth inhibition Day 30.(DOCX)
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