Synergistic photodynamic antimicrobial therapy with silver nanoparticle activated by UV irradiation and 405 nm laser diode against Escherichia coli and Staphylococcus aureusAbstract
This study investigates the antibacterial properties of Moringa oleifera green-synthesized silver nanoparticles (AgNPs-MO) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) when combined with UV radiation and a blue laser set at 405 nm. Exposure to both light treatments increased the antibacterial activity of AgNPs-MO at higher concentrations. The results indicate that photosensitizers generated from Moringa oleifera help photodynamic antimicrobial treatment work better and provide a more environmentally friendly way to manage infections. A control group (T0) was not subjected to laser irradiation, while the other three bacterial groups (A1/A2 and A3/A4) were treated to laser irradiation at different concentrations and times of silver nanoparticles. Groups A2 and A4 bacteria were also given a photosensitizer made from Moringa oleifera, and then they were exposed to varying lengths of laser light. Following incubation, a Quebec colony counter was used to measure the number of bacterial colonies. Bacterial viability was significantly reduced, according to statistical analysis utilizing a post-hoc Tukey test and a two-way ANOVA factorial test. After being exposed to mmol L-1, mmol L-1.5, and mmol L-2 AgNPs-MO for 180 seconds, E. coli showed viability decreases of 70.90%, 76.44%, and 79.87%, respectively. The identical circumstances also resulted in viability decreases of 97.48%, 95.94%, and 94.72% for S. aureus. Our strategy proved to be effective, inactivating E. coli by 79.87% ± 1.92% and S. aureus by 97.48% ± 0.78% at an energy density of 3.44 J/cm2. In order to battle pathogenic germs, this research proposes prospective uses for a blue laser, UV light, silver nanoparticles, and extract from Moringa oleifera.
Keywords:
photoinactivation; photosensitiser; blue laser; Escherichia coli; Staphylococcus aureus
Resumo
Este estudo investiga as propriedades antibacterianas de nanopartículas de prata sintetizadas a partir de Moringa oleifera (AgNPs-MO) contra Escherichia coli (E. coli) e Staphylococcus aureus (S. aureus) quando combinadas com radiação UV e um laser azul ajustado para 405 nm. Os resultados sugerem que os fotossensibilizadores derivados de Moringa oleifera melhoram a eficácia da terapia antimicrobiana fotodinâmica, oferecendo uma abordagem ecologicamente correta para o controle aprimorado de infecções. O delineamento experimental compreendeu quatro grupos: três grupos bacterianos (A1/A2 e A3/A4) expostos à irradiação a laser em concentrações e durações variadas de nanopartículas de prata, e um grupo controle (T0) que não foi exposto à irradiação a laser. Um fotossensibilizador produzido a partir de Moringa oleifera também foi administrado às bactérias dos grupos A2 e A4, e diferentes durações de exposição ao laser foram então aplicadas. Após a incubação, as colônias bacterianas foram medidas com um contador de colônias Quebec. Análises estatísticas usando um teste fatorial ANOVA bidirecional e teste post-hoc de Tukey revelaram reduções significativas na viabilidade bacteriana. E. coli exibiu reduções de viabilidade de 70,90%, 76,44% e 79,87% com mmol L-1, mmol L-1,5 e mmol L-2 AgNPs-MO após 180 segundos de exposição ao laser, respectivamente. Similarmente, S. aureus mostrou reduções de viabilidade de 97,48%, 95,94% e 94,72% sob as mesmas condições. Em uma densidade de energia de 3,44 J/cm2, nossa abordagem demonstrou eficácia notável, alcançando uma inativação de 79,87% ± 1,92% de E. coli e uma inativação de 97,48% ± 0,78% de S. aureus. Este estudo sugere aplicações promissoras para o uso combinado de um laser azul, radiação UV, nanopartículas de prata e extrato de Moringa oleifera no combate a bactérias infecciosas.
Palavras-chave:
fotoinativação; fotossensibilizador; laser azul; Escherichia coli; Staphylococcus aureus
1. Introduction
Many foods are contaminated by bacteria like Escherichia coli and Staphylococcus aureus (Al Isrofi, 2022). Additionally, these microorganisms will spread from their host to people, posing serious risks to public health. In 2016, 3.04 percent of Indonesian deaths were attributable to diarrhea. To increase antibiotics' efficacy against antimicrobial agents, ongoing research and development is essential. Because of the high costs and environmental impact of producing antibiotics, researchers are looking at other strategies for creating antimicrobial medicines that are both economical and ecologically benign. By harnessing its characteristics to stop bacterial development or eliminate germs without endangering healthy tissues, nanotechnology has become a viable remedy (Mathesh et al., 2024). The manipulation of materials at the nanoscale, typically between 1 and 100 nm, is the aim of nanotechnology. Because of their unique characteristics, convenience of use, and capacity to attach to biomolecules, silver nanoparticles (AgNPs), which are made exclusively from silver, are recommended. Other noble metal nanoparticles, such gold (AuNPs), palladium (PdNPs), and platinum (PtNPs), exhibit unique properties as well, although differing from AgNPs in composition and behavior (Saxena et al., 2020). Because they are tiny and have a large surface area, they are effective at eliminating pathogens. According to Deepak et al. (2011), silver nanoparticles are thought to be safe for humans, animals, and plants.
There are two methods for creating nanoparticles: top-down and bottom-up. Bulk materials may be reduced to nanoparticles using top-down techniques such laser ablation, ball milling, and high-energy milling. Bottom-up methods, such as chemical reduction, sol-gel processes, and green synthesis, produce nanoparticles from atomic or molecular precursors. In particular, plant extracts are used as reducing agents in green synthesis, which offers a cost-effective and ecologically friendly process. Depending on the intended size, shape, and use of the nanoparticles, each technique offers unique benefits. AgNPs made using environmentally friendly techniques offer superior antibacterial qualities because plant extracts include bioactive substances such flavonoids, terpenoids, and tannins (Loza et al., 2014). These compounds facilitate the production of nanoparticles and enhance their stability and efficacy in inhibiting bacterial growth. AgNPs are often used in biomedical devices due to their antiviral, antifungal, and antibacterial qualities. Reducing agents, which stabilize particles and stop them from aggregating, are included into their manufacture (Girón-Vázquez et al., 2018). AgNPs may be produced using a variety of processes, such as chemical, biological, physical, and green synthesis methods.
Green synthesis produces nanoparticles using materials including bacteria, fungus, plant extracts, and small biomolecules like vitamins and amino acids. Because it is non-toxic throughout the synthesis, this environmentally friendly method reduces its impact on the environment (Kumar et al., 2014). Because it is based on solvents, reducing agents, and non-toxic substances, it has the advantage of being able to modify the size and shape of particles for medical applications. By producing proteins, amino acids, and secondary metabolites, the process lessens the need for extra precautions to stop particle aggregation. To provide the best control over particle characteristics, temperature, pH, the amounts of reducing and stabilizing agents, and the quantities of precursors are all changed (Pareek et al., 2023). Natural reducing agents derived from materials containing antibacterial compounds or polyols that may reduce AgNPs can be found in Indonesia's vast natural resources. When plants are used in synthesis, secondary metabolite chemicals with antibacterial qualities, such as terpenoids, flavonoids, and tannins, are accessed.
Numerous studies have looked at the possibility of using moringa oleifera leaf extract as a reducing agent while creating nanoparticles. It has been shown that the bioactive elements of moringa, including flavonoids and phenolic acids, enhance the antibacterial properties of synthetic AgNPs. Studies carried out in 2022 and 2024 have confirmed the effectiveness of Moringa oleifera against a number of infections, including S. aureus and E. coli. For instance, Moringa (Moringa oleifera) is a plant that inhibits bacterial and fungal activity and has anti-cancer, antibacterial, and hypotensive qualities (Shankar et al., 2004). The absorption spectra of moringa leaf extract shows absorbance as a function of wavelength, having a peak that ranges from around 200 to 691 nm (Anwar et al., 2007). Although E. Coli bacteria are often found in the stomach, they may become harmful in certain situations. They produce toxins in the small intestine that may damage mucosal cells, causing symptoms including watery diarrhea, low-grade fever, nausea, lethargy, and heartburn. Under unusual conditions, S. aureus bacteria, which are normally present in the mouth and respiratory system, might become harmful. They result in skin infections that manifest as abscess development, inflammation, and necrosis. S. aureus infections may spread to other places of the body and show up as suppuration, boils, or acne. A number of disorders may arise as a result of the presence of extracellular materials produced by S. aureus (Salu et al., 2016).
AgNPs' antibacterial effectiveness depends on a number of factors, including the kind of bacteria, the number of bacterial colonies, the concentration of nanoparticles, their size and shape, and the length of time they interact with the bacteria. Synthesis parameters include reaction duration, reducing agent, solution temperature, and salt concentration all affect particle size. The size and structure of silver nanoparticles are influenced by silver nitrate (AgNO3), a typical precursor used to make them. Bacterial growth may be impacted by the precursor and AgNP concentration. Between 400 and 500 nm, AgNPs exhibit a strong absorption spectrum (Ahmed et al., 2021). When blue lasers and AgNPs are coupled, reactive oxygen species (ROS), which are necessary for bacterial cell inactivation, may rise noticeably. This process is known as photodynamic inactivation (PDI). Its use with blue lasers is growing in popularity since it is non-invasive and accurately targets microbial cells without harming healthy tissues. Photodynamic inactivation (PDI) is a technique that slows cell metabolic activity by causing cell death via the application of light and photosensitizer chemicals (El-Gendy et al., 2024). The three primary components of PDI are a light source, a photosensitizer material, and free radicals that induce inactivation (El-Gendy et al., 2022). Photosensitizers produce reactive oxygen species (ROS) when they absorb light energy at certain wavelengths. PDI often uses lasers as light sources because of their monochromatic, coherent light beams. According to Astuti et al. (2016), a blue laser generates light with an absorption spectrum between 400 and 500 nm.
The antibacterial qualities of Moringa leaf extract (Moringa oleifera) against bacteria including Salmonella typhi, Shigella spp., Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus were investigated by Abadallah and Ali (2019). The research highlighted the potential of Moringa leaves for antimicrobial applications by demonstrating their antibacterial properties against these microorganisms. gNPs with a 450 nm blue diode laser were employed in recent work to successfully reduce Candida albicans lifespan by 84.63%. According to Shankar et al. (2004), this discovery implies that additional bacterial strains, including S. aureus and E. coli, could react well to comparable methods. Several studies have shown that the combination of AgNPs-MO and blue laser irradiation dramatically inhibited bacterial growth, providing a new and durable approach to infection prevention in clinical settings (Anwar et al., 2007). The effect of AgNPs applied to a 450 nm blue diode laser on Candida albican photoinactivation, which affects Candida albican counts. Zaffer et al. (2014) investigated the antibacterial qualities of Moringa leaf extract (Moringa oleifera) and found that it inhibited the development of S. aureus bacteria. Additionally, another research looked at antimicrobial photodynamic treatment (aPDT), which involves exposure to a 450 nm diode laser, and showed that it may improve antimicrobial effects. According to the research, the proportion of C. albicans bacteria that were viable decreased to 84.63% (Zaffer et al., 2014).
The antibacterial properties of Moringa oleifera are highlighted in the research as a reducing agent for the creation of nanoparticles. Our method was very effective, as shown by the inactivation of S. aureus at 97.48% ± 0.78% and E. coli at 79.87% ± 1.92% at a density of 3.44 J/cm2 (Segwatibe et al., 2023). Recent studies have shown a considerable increase in the antibacterial efficacy of AgNPs derived from Moringa oleifera with blue laser irradiation. When made using Moringa leaf extract, AgNPs have a greater capacity to fight against infections like S. aureus and E. coli. It has been shown that using AgNPs in conjunction with a blue laser for photodynamic inactivation (PDI) may increase the production of reactive oxygen species (ROS), which further improves bacterial inactivation. This approach provides a novel and sustainable means of effectively identifying and reducing dangerous bacteria, and it is consistent with the growing interest in ecologically friendly synthesis techniques.
AgNPs are desirable due to their unique antibacterial and therapeutic properties, yet traditional production processes sometimes include dangerous materials. An eco-friendly substitute that makes use of plant extracts is green synthesis (Yaqubi et al., 2023). Known for its abundance of bioactive chemicals, Moringa oleifera has shown promise in the environmentally safe production of AgNPs. Research on its use in conjunction with photobiomodulation treatment is still required, however. In order to improve antimicrobial and wound-healing applications, this work fills that gap by synthesizing AgNPs from Moringa oleifera and investigating their antibacterial qualities when triggered by blue laser treatment. In order to maximize the antibacterial properties of AgNPs when activated by blue laser irradiation, the laser settings used in this investigation were carefully chosen. With a 50-mW power output, the 450 nm blue diode laser operated in continuous wave (CW) mode. With an energy density of 3.44 J/cm2, the bacteria were effectively inactivated without suffering thermal damage (Yaqubi et al., 2024). The exact treatment was provided by the laser beams focus on an area of around 1 cm2. When paired with AgNPs made from Moringa oleifera, these conditions increased the production of reactive oxygen species (ROS), which greatly increased the antibacterial effectiveness against S. aureus and E. coli.
The blue light spectrum, which has been shown to have antibacterial qualities, includes the wavelength of 405 nm, which was chosen for this investigation. When paired with photosensitizers like AgNPs, blue light in this range is known to produce photodynamic effects that increase the antibacterial activity of the latter (Astuti et al., 2019a). Furthermore, 405 nm light is a safer and more focused alternative for photobiomodulation treatment since it has been shown to efficiently permeate biological tissues and is less likely to harm them than UV light. This wavelength works especially well to activate AgNPs, which promotes the generation of reactive oxygen species (ROS) that aid in the inactivation of germs. Prior studies shown that the AgNPs-MO combination LED group with blue LED had the highest inhibitory impact of photoantifungal activity by 80% (Astuty et al., 2025).
Despite the well-known antimicrobial potential of silver nanoparticles, limited studies have combined green-synthesized AgNPs from Moringa oleifera with 450 nm blue laser irradiation for targeted photodynamic inactivation. Existing work has not fully explored optimal laser parameters, ROS generation enhancement, or comparative antibacterial effects on both S. aureus and E. coli. Therefore, this study addresses these gaps by synthesizing AgNPs using Moringa oleifera extract and evaluating their antibacterial efficacy when activated by a 450 nm blue diode laser under controlled energy density conditions. The findings aim to introduce a novel, eco-friendly, and effective photoinactivation approach, with potential applications in infection control and surface sterilization. Additionally, this study establishes a foundation for future research on integrating green nanotechnology with photobiomodulation techniques for broader biomedical and wound-healing applications.
2. Material and Methods
2.1. Preparation and synthesis of Moringa oleifera silver nanoparticles
The study focused on creating silver nanoparticles (AgNPs-MO) from natural materials, namely Moringa leaves (Moringa oleifera). The sample was prepared by carefully extracting the bioactive components from Moringa leaves. Drying and crushing the leaves was the first stage in creating moringa leaf powder. After that, 0.5 grams of the powder were carefully dissolved in a 70:30 ethanol and water solvent. The bioactive components are more effectively extracted from the leaves using this mix of solvents. To expedite the extraction process, the solution was heated for nine minutes in a 45-watt microwave. After heating, the mixture was centrifuged for 30 minutes at 5000 rpm in order to ensure a clean extract by removing debris and insoluble particles. An additional 25 milliliters of the ethanol-water solvent mixture were added to the solution in order to further dilute and extract the advantageous compounds present in the Moringa leaves.
While the moringa leaves were purchased from a neighboring farm in Surabaya City, the chemicals used in the experiment, including ethanol and silver nitrate (AgNO3), were analytical grade (purity 99.9%) and obtained from an authorized source. The leaves of the moringa plant were well cleaned before being placed in a single layer on a clean, flat surface in a well-ventilated, shaded area. They were switched around every few days to ensure they dried uniformly. Once the leaves were brittle and crisp, they were crushed into a fine powder and stored in an airtight container away from direct sunlight. AgNPs-MO was created using an ecologically friendly synthesis process, complete with reagents and conditions. The reducing and stabilizing agent used in the manufacture of silver nanoparticles (AgNPs/AgNO3) was moringa leaf extract. Three distinct concentrations of silver nanoparticles (mmol L-1, mmol L-1.5, and mmol L-2) were tested for their influence on the production procedure and nanoparticle properties. In an Erlenmeyer flask, a predefined amount of Moringa leaf extract was combined with a certain AgNO3 solution to start the synthesis. The flask was covered with aluminum foil to stop light-induced reactions. The green synthesis process was initiated by heating the covered flask at 135 watts for five minutes. When homogeneous, yellowish-brown particles started to form in the solution, the synthesis was finished. This demonstrated the formation of silver nanoparticles. To ensure repeatability and assess the effects of varying concentrations on nanoparticle characteristics, this synthesis process was performed once for each concentration of silver nanoparticle evaluated.
The concentration of AgNPs-MO was determined by measuring the absorbance at the characteristic surface plasmon resonance (SPR) peak using UV-VIS spectroscopy and comparing it with a standard calibration curve. Additionally, Dynamic Light Scattering (DLS), which measures variations in light scattering to produce an accurate particle size distribution, was used to quantify the size range of the generated nanoparticles. The silver nanoparticles and extract from Moringa leaves were characterized using a variety of analytical techniques. A Shimadzu UV-VIS 1800 spectrometer was used to acquire the absorption spectra of the Moringa leaf extract, which revealed details on its chemical makeup and optical characteristics. The Moringa leaf extract was also assessed using a Particle Size Analyzer (PSA), and the Dynamic Light Scattering (DLS) method was used to ascertain the particle size distribution. The insights these characterisation methods provide into the physical and chemical properties of the created AgNPs-MO and the extract from Moringa leaves enable a comprehensive understanding of the potential applications of these procedures. A reducing and stabilizing chemical called moringa leaf extract was essential for the synthesis of AgNPs-MO. In order to ensure steady and efficient manufacturing, the extract's phytochemicals reduced the silver ions to nanoparticles and stopped them from aggregating. Laser irradiation combined with AgNPs-MO photosensitizer was the method used to treat the bacteria. A 100 µl aliquot of bacterial culture was added to a sterile microplate containing 100 µl of silver nanoparticles (AgNPs-MO) made using extract from Moringa oleifera. At 37°C, the mixture was incubated for 30 minutes. Tryptic soy agar (TSA) was then added to the microplate, and the culture was cultured for 24 hours at 37°C. All analytical-grade reagents, including TSA, silver nitrate (AgNO3), and extract from Moringa oleifera, were supplied by Airlangga University's Biophysics Laboratory. Then, use a blue laser to light it for 90, 120, 150, and 180 seconds at different times. Once exposed, transfer 50 µl to a petri plate and add TSA (Tryptic Soy Agar) for plating. Incubate at 37°C for 24 hours. The TPC (Total Plate Count) technique may be used to count the bacteria after a 24-hour period.
Before being used, the bacteria will be cultivated in Tryptone Soy Broth (TSB) at 37°C for 24 hours and adjusted to McFarland standard 1.0 (1×108 CFU/mL), according to the explicitly stated protocol for preparing bacterial samples. To guarantee accurate comparison, experimental controls will be included, such as positive controls utilizing recognized antibacterial drugs and negative controls (without AgNPs-MO, laser, or UV irradiation). We'll go into further detail about the requirements for UV radiation exposure, such as the UV source's distance (10 cm), energy intensity, and exposure duration (15 minutes, for example). Furthermore, specifying the concentration and amount of Moringa leaf extract to be utilized, together with a 30-minute incubation time prior to irradiation, would help to further define its applicability.
2.2. Study design
After dissolving 0.3 grams of Tryptone Soy Broth (TSB) in 10 milliliters of distilled water, the mixture was autoclaving sterilized. After that, one ounce of S. aureus and E. coli bacteria were added to the TSB, and it was vortexed until it was uniform. To achieve the 1.0 McFarland standard for colony density, the mixture was incubated for 24 hours at 37 °C. The study used a factorial experimental design with several treatment groups to assess the impact of various factors (such as laser irradiation, UV radiation, concentration of silver nanoparticles, and photosensitizer treatment) on bacterial photoinactivation. Bacterial colonies were measured in each treatment group under certain conditions, such as laser exposure and nanoparticle concentration. Vitality of S. aureus and E. coli was seen under a range of treatment conditions, such as variations in AgNPs-MO concentration and laser irradiation duration. To determine statistically significant differences between the therapy groups, the research used statistical analysis.
2.3. Culture of bacteria
Tryptone Soy Broth (TSB) was used to cultivate the microorganisms E. coli and S. aureus. The colonies were then cultured for 24 hours at 37°C until they achieved a McFarland standard of 1.0.
2.4. Laser Source
The blue laser used in this study operated at a wavelength of 405 nm with a spot size of 0.13 cm2 and a measured power output of 2.49 mW, determined using an OMMOL L-6810B-220V power meter. Laser irradiation was applied for 90, 120, 150, and 180 seconds. The energy density (fluence) delivered to the samples was calculated using the standard Equation 1:
where
E = energy density (J/cm2),
P = laser power (W),
t = exposure time (s),
A = beam area (cm2).
The optical characteristics of the synthesized AgNPs were confirmed by recording their UV–Vis absorption spectra using a Jasco CT-10 monochromator. Dynamic Light Scattering (DLS) analysis showed particle sizes of 7.26 nm, 54.01 nm, and 100.74 nm at different AgNP concentrations.
2.5. Anti-bacterial activity test
To examine the material's antibacterial activity, the disc diffusion technique was altered to include simultaneous UV irradiation and nanoparticle application. First, Tryptic Soy Agar (TSA) was evenly spread across a petri plate that had a 50 μL bacterial culture. To increase the antibacterial activity of 10 μL of Moringa leaf extract with a preset concentration of nanoparticles, a paper disc was exposed to UV light for a predefined period of time. Once the disc had absorbed the fluid, it was carefully put onto the agar surface. The petri dish was then incubated for a full day under well watched conditions. The presence and size of inhibitory zones around the paper disc were examined in order to assess the effectiveness of AgNPs-MO in conjunction with laser (450 nm) and UV light. This enhanced method enables a comprehensive evaluation of the combined potential of UV irradiation, nanoparticles, and maybe a 405 nm blue laser as antimicrobial agents by evaluating their synergistic antimicrobial effects.
2.6. PDI treatment
The samples were divided into four groups: the control group (T0), which was not exposed to any laser radiation; the E. coli samples (A1 and A2), which were exposed to different laser radiation concentrations and durations at 405 nm; and the S. aureus samples (A3 and A4), which were exposed to the same conditions. After receiving an AgNPs-MO treatment, bacteria in groups A2 and A4 were exposed to blue laser radiation for 90, 120, 150, and 180 seconds over the course of 30 minutes. Following treatment, samples were grown on Tryptic Soy Agar (TSA) for a full day at 37 °C. The Quebec colony counter was used to count the number of bacterial colonies. The group labels that were defined to correspond with the observation tables and graphs were Group T0 (control), Group A (E. coli) with subgroups A1 (without photosensitizer) and A2 (with photosensitizer), and Group B (S. aureus) with subgroups B1 (without photosensitizer) and B2 (with photosensitizer). This ensures that observations are constantly included into the study design.
2.7. Statistical analysis
The statistical analysis of this research used IBM SPSS's two-way ANOVA factorial test to assess the effects and interactions of each component. The effects of radiation therapy and bacterial strain, two independent variables, on bacterial inactivation were examined using a two-way ANOVA. This test aids in determining each element's primary impacts as well as their interactions. By examining these variables, the two-way ANOVA can determine the impact of each treatment on bacterial inactivation and if the effects vary depending on the type of bacteria. Following the ANOVA, Tukey's post-hoc test looked at the variations across treatment groups. In order to choose the most efficient irradiation treatment for bacterial inactivation, it is helpful to assess if certain groups vary considerably from one another. The data were considered to demonstrate a significant difference (H0) if the significance value was p < α = 0.05. The post-hoc Tukey's technique was then used to determine the differences between each sample factor, with a significance level of p < 0.005. It will be possible to identify the radiation treatment that has the highest likelihood of photoinactivating S. aureus and E. coli after these investigations are completed.
3. Results
The results of the characterisation of the AgNPs-MO (Moringa oleifera) absorption spectra are shown in Figure 1. The wavelength range in which the UV-Vis absorption spectra were measured was 325 nm to 525 nm. Interestingly, the absorption spectra of Moringa leaf extract spans from 200 nm to 691 nm, which is consistent with the study's use of a blue laser. The particle size analyzer's objective is to ascertain the distribution of particle sizes. PSA's Dynamic Light Scattering (DLS) method makes advantage of infrared scattering. DLS is also referred to as spectroscopy photon correlation. Particle size is determined by DLS using the diameter of the circle of particles that diffuse at the same speed at the moment of measurement. In accordance with the PSA test, the AgNPs-MO mmol L-1, L-1.5, and L-2 particle size values on day 1 at D90 are 7.26 nm, 100.74 nm, and 54.01 nm, respectively. This result indicates that AgNPs-MO mmol L-1, mmol L-1.5, and mmol L-2 are classified as nanoparticles due to their size range of 1-100 nm. Figure 2 displays the PSA test results for silver nanoparticles produced using AgNPs-MO at three distinct concentrations: mmol L-1 (a), mmol L-1.5 (b), and mmol L-2 (c).
UV-Vis absorbance spectrum of AgNPs-MO at concentrations of mmol L-1, mmol L-1.5, and mmol L-2.
In this instance, silver nanoparticles use photodynamic inactivation to inactivate bacteria by acting as a photosensitizing agent. The absorption spectrum and particle size, as determined by UV-visible spectroscopy and a particle size analyzer, are the primary attributes of AgNPs-MO as a photosensitizer. The wavelength of the laser light source is chosen using the AgNP absorption spectrum to ensure that the right amount of laser energy is absorbed. AgNP-MO's efficiency as a photosensitizer in microbial inactivation is influenced by its surface area accessible for laser energy absorption, which may be ascertained by measuring the particle size. The diameter of the inhibition zones against the development of bacterial colonies against S. aureus and E. coli was determined by the antibacterial test. The disc diffusion approach was applied to every sample. mmol L-1, mmol L-1.5, and mmol L-2 AgNPs-MO were reported to have E. coli bacterial inhibition zones of 0.97 mmol L, 0.94 mmol L, and 0.88 mmol L, respectively (Figure 3a). According to Figure 3b, the antibacterial AgNPs-MO's inhibitory zones for S. aureus bacteria were 1.93 mmol L, 2 mmol L, and 2.03 mmol L at mmol L-1, mmol L-1.5, and mmol L-2, respectively. Instead of completely killing the bacteria, the UVC treatment (254 nm, 15 min) was used as a sublethal pretreatment. UV-only findings were not shown since the goal was to assess the improved antibacterial impact of AgNPs-MO using a blue laser rather than only UV.
Antibacterial test results of AgNPs-MO using the disc diffusion method. (a) Inhibition zones against E. coli; (b) Inhibition zones against S. aureus. The concentrations of AgNPs-MO tested are: (1) mmol L-1, mmol L-1.5, and mmol L-2.
Bacterial inactivation may be influenced by UV light, and the treatment process as a whole may be greatly impacted by the characteristics of this radiation, including intensity and duration. We think that the 30-minute UV radiation exposure at an intensity of 5 mW/cm2 helps explain the overall antibacterial effectiveness shown in this investigation. Prior to laser and AgNP-MO treatment, the samples were exposed to UV light at 254 nm for 15 minutes at an intensity of 1 mW/cm2. The purpose of this procedure was to standardize the initial conditions prior to laser irradiation and reduce microbiological contamination. Based on earlier research showing efficient microbial elimination at this wavelength and intensity, the chosen UV parameters were chosen. To improve the effectiveness of therapy, future studies could better optimize UV exposure circumstances. Table 1 displays the average particle size of the silver nanoparticles produced using MO extract at concentrations of mmol L-1, mmol L-1.5, and mmol L-2. Targeting S. aureus and E. coli, the antibacterial test measured the diameter of the inhibitory zones on the formation of bacterial colonies. All samples were examined using the diffusion well technique. The findings demonstrated the existence of E. coli inhibitory zones utilizing the antibacterial AgNPs-MO at mmol L-1, mmol L-1.5, and mmol L-2. The antimicrobial AgNPs-MO at mmol L-1, mmol L-1.5, and mmol L-2 exhibited the same inhibitory zones against S. aureus.
Using paper discs with a diameter of 0.5 cm, the inhibition zone of AgNPs-MO with S. aureus was quantified to the nearest 5.2 mmol L. This proved that the chemical successfully stopped germs from growing in the particular region around the disk. The decrease in the inhibitory zone for E. coli may be explained by the aggregation of nanoparticles caused by higher doses of AgNPs-MO, which would reduce their effective surface area and bioavailability. The AgNPs-MO inhibitory zones against S. aureus and E. coli are shown in Figure 3. S. aureus and E. coli exhibit notably different levels of AgNPs-MO's antibacterial activity; the former have larger inhibition zones (~2 mm), while the latter have smaller inhibition zones (~0.88–0.97 mm). This suggests that AgNPs-MO is more effective against S. aureus because the latter has a thicker peptidoglycan layer, which encourages higher AgNP interaction. This is most likely due to variations in the bacterial cell walls' composition. Statistical research indicates that AgNPs-MO significantly reduces S. aureus more than E. coli. However, even taking into account the inhibition zone standard deviation, there is no discernible difference between the measured values (1.0, 1.5, and 2.0 mmol L−1) for both bacterial species. The findings were confirmed by the addition of controls.
The inhibitory zone for S. aureus increases as AgNPs-MO concentrations rise, whereas it decreases for E. coli, suggesting that E. coli is less affected, as seen in Table 2. The antibacterial qualities of the synthesis were shown by adding AgNPs-MO to the two bacterial samples. The number of bacterial colonies that developed in each well or cup with AgNPs-MO was used to compute the results of the bacterial growth. The greater suppression of the germs at higher dosages was due to the larger peptidoglycan layer in the cell wall of S. aureus, which promotes more effective interaction with AgNPs-MO and boosts antibacterial activity. Figure 4 displays the percentage decrease in E. coli and S. aureus bacterial colonies, respectively. When E. coli bacteria were exposed to 50% AgNPs-MO at a concentration of mmol L-2, the bacterium's percentage dropped the greatest, by 59.82%.
Percentage reduction of (a) E. coli and (b) S. aureus bacteria at various concentrations of AgNPs-MO.
The E. coli bacteria were synthesized when two treatment groups were exposed to laser radiation: one group was exposed to laser radiation alone, while the other group was exposed to laser radiation plus 50% AgNPs-MO at each concentration variation. To make sure the observational data satisfied the requirements for interval-scale measurements, homogeneity of variances, and normal distribution, it was statistically examined using IBM SPSS's two-way ANOVA factorial test. This validated the facts and made it possible to compare them with outcomes that were manually computed. The two treatment groups' data normalcy test findings revealed a significant value of p = 0.596, which was higher than α = 0.05. This suggests that the distribution of the data is normal. The results of tests for normality (Kolmogorov-Smirnov, p = 0.596) and homogeneity of variances (Levene's test, p = 0.717) on the "Bacteria Reduction" data show that the variance is normally distributed and consistent across groups. These assumptions may be exploited in further investigation.
The two-way factorial ANOVA test yielded a significant result of p = 0.000, which is smaller than α = 0.05, based on the data. This illustrates how the results of each therapy varied dramatically throughout the different time periods. To find treatment groups with unique outcomes that shown statistically significant changes over time, a post hoc test was used. The two-way ANOVA factorial test showed that the laser treatment with mmol L-2 AgNPs-MO for 180 seconds resulted in the greatest percentage of E. coli bacterial fatalities, 79.86%. The percentages of bacterial mortality in each of the three treatment groups after 90, 120, 150, and 180 seconds of laser irradiation are shown in. For each treatment, the therapy generated a unique energy density. Compared to the control group, the number of bacterial colonies was effectively reduced. AgNPs-MO was added each time the S. aureus bacterial concentration fluctuated throughout the synthesis. At intervals of 90, 120, 150, and 180 seconds, the E. coli bacterial mortality percentage was 62.32%, 69.18%, 73.68%, and 79.86%, respectively. The results demonstrated that the outcomes varied significantly during various time periods. Figure 5 displays the three groups' treatment outcomes as well as the relationship between the radiation duration and bacterial survival as measured by colony count. The percentage of bacterial mortality in each of the three treatment groups is shown in Figure 6. The conclusions drawn from the statistical test results are shown in Table 1.
Decreased viability of E. coli bacteria with AgNPs-MO (a) without UV irradiation (mmol L-1), (b) without UV irradiation (mmol L-1.5), and (c) without UV irradiation (mmol L-2).
Percentage reduction of E. coli bacteria with AgNPs-MO (a) mmol L-1 (b) mmol L-1.5 (c) mmol L-2.
E. coli bacteria treated with different concentrations of AgNPs-MO had reduced vitality, as shown in Figure 5. concentrations of mmol L-1, mmol L-1.5, and mmol L-2, respectively. The graphic shows how bacterial viability gradually decreases as AgNPs-MO concentration rises. Figure 6: E. coli bacteria percentage decrease after treatment with AgNPs-MO at different doses. mmol L-1 (a), mmol L-1.5 (b), and mmol L-2 (c). The efficiency of AgNPs-MO in lowering bacterial viability is seen in this image, where larger doses result in more pronounced drops in bacterial numbers. The E. coli bacterial mortality percentages for various AgNPs-MO concentrations and laser exposure durations are shown in Table 3. The maximum amount of bacterial killing (79.87%) was achieved with mmol L-2 AgNPs-MO and 180 seconds of laser irradiation. Significant differences (p < 0.05) were seen across treatments, indicating that both longer laser exposure periods and greater AgNPs-MO concentrations promote bacterial mortality.
A two-way ANOVA factorial test was employed to evaluate the effects of each component and ensure that the results met the requirements for validation and comparison with human computations, including an interval scale and consistent data fluctuations, following the addition of 50% AgNPs-MO to the S. aureus bacteria. The results of the Kolmogorov-Smirnov test for the 60 samples that make up the "Bacteria Reduction" data show a regular distribution (p = 0.982). Furthermore, there is no noticeable difference between the groups, according to Levene's test (p = 0.987). The "Bacteria Reduction" study may thus proceed under the assumptions of normality and homogeneity of variances. A significant value of p = 0.000 was found by the two-way factorial ANOVA analysis, which was less than α = 0.05. The Post Hoc test, which was based on the observed variations in results, was used to determine the distinct outcomes of each treatment group.
The two-way factorial ANOVA test revealed that the laser treatment with mmol L-1 AgNPs-MO for 180 seconds resulted in the greatest percentage of S. aureus bacterial death, 97.48%. The percentages of bacterial mortality for each of the three treatment groups are shown in Figure 7. Each treatment was given a different energy density by varying the laser irradiation procedure's length, which was set at 90, 120, 150, and 180 seconds. There were far fewer bacterial colonies than in the control group. Every time the concentration of S. aureus bacteria changed throughout the production process; AgNPs-MO was added. The percentages of S. aureus bacterial deaths were 94.24%, 94.89%, 96.10%, and 97.48%, in that order. The results showed that there were significant differences between the 90, 120, 150, and 180-second time intervals. The findings drawn from the statistical test results are shown in Table 2. Figure 8 shows the percentage reduction of S. aureus bacteria with AgNPs-MO(a) mmol L-1(b) mmol L-1.5 (c) mmol L-2. The statistical analysis of the percentages of S. aureus bacterial mortality under various AgNPs-MO concentrations and laser exposure durations is shown in Table 4. The maximum bacterial killing, 97.48%, was seen with mmol L-1 AgNPs-MO and 180 seconds of laser irradiation. Significant variations in bacterial mortality among the treatments are indicated by a p-value of less than 0.05. The findings demonstrate that the bacterial mortality rate was favorably affected by both increasing the AgNPs-MO concentration and exposure duration.
Decreased viability of S. aureus bacteria with AgNPs-MO(a) mmol L-1(b) mmol L-1.5(c) mmol L-2.
Percentage reduction of S. aureus bacteria with AgNPs-MO(a) mmol L-1(b) mmol L-1.5 (c) mmol L-2.
4. Discussion
Microwave irradiation accelerates the production of silver nanoparticles (AgNPs), which are smaller particles with enhanced antibacterial properties. By generating reactive oxygen species (ROS), photodynamic inactivation (PDI), which uses light and photosensitizers, targets and kills bacterial cells while protecting mammalian cells. Because of its consistent output power and effective ROS generation, blue diode lasers with a wavelength of 405 nm allow for precise and effective photodynamic imaging (PDI). Combining nanoparticles with photosensitizers boosts the efficacy of photodynamic therapy because it improves stability, cellular absorption, and ROS production, all of which accelerate bacterial cell death. However, before turning PDI into therapeutic applications, standardization, regulatory approval, and clinical validation are necessary to fully realize its potential in treating bacterial infections. In this study, these mechanisms are directly linked to the experimental findings to explain the antibacterial outcomes rather than discussed in isolation.
Silver nanoparticles are usually created by conventional heating methods. Long response times, uneven heating, and inefficiency are some of this method's drawbacks. The microwave synthesis of silver nanoparticles (AgNPs) using extract from Moringa oleifera as a photosensitizer enhanced photodynamic inactivation (PDI) when exposed to a blue diode laser at 405 nm. This method greatly decreased S. aureus and E. coli viability (97.48% and 79.87%). When combined with blue laser and UV irradiation, AgNPs–Moringa's efficacy provides a viable environmentally friendly substitute for antibiotics in the fight against antibiotic-resistant bacteria. Prior to in vivo trials, further in-silico and in-vitro research is necessary to optimize the treatment. Therefore, using the microwave irradiation technique may permit consistent heating, accelerate the reaction, and boost yield without substantially changing chemical processes all of which may have an instant impact on the synthesis of silver nanoparticles
Because microwave heating consistently yields nanoparticles that are small, have a uniform size distribution, and have better antibacterial activity, it is believed to be a more effective approach than traditional heating for producing AgNPs-MO. The reproducibility of AgNP characteristics and antibacterial effects across replicates in this study supports the advantage of microwave-assisted synthesis. Photodynamic inactivation (PDI), a method of lowering cell metabolic activity, kills bacteria by combining light and photosensitizer chemicals (El-Gendy et al., 2024). Reactive oxygen species (ROS) and visible light are the most crucial elements of photodynamic inactivation when using photosensitizers as light sensitizers (Chandra et al., 2010). For photoinactivation to take place, the visible light and absorption spectra of the photosensitizer must coincide. The strong match between the 405 nm light and the absorption profile of the AgNPs–MO contributed to the high bacterial reduction observed.
A laser is a common light source for photodynamic inactivation because of its many advantages, including its parallel and homogenous (monochromatic) light beams (Fouad et al., 2024). In this study, a blue diode laser with a wavelength of 405 nm was used. The diode laser's wavelength is determined by wavelength characterization; the wavelength with the highest power is 2.49 mW. When subjected to radiation at a distance of one centimeter, the laser output power stays constant and does not produce excessive heat thanks to the findings of power characterization over time and temperature, which show stable power within a certain time range. This stability is important because it minimizes variability in irradiation intensity and ensures that changes in bacterial viability are due to treatment effects rather than equipment fluctuations.
A material that absorbs light with a certain energy is called a photosensitizer. There are two types of photosensitizers: endogenous and exogenous (Basu et al., 2020). The two kinds used in this study are exogenous porphyrins and endogenous bacterial porphyrins. AgNPs-MO with mmol L-1, mmol L-1.5, and mmol L-2 were used as photosensitizers. To evaluate how well the therapies worked, case groups were categorized according to the therapies they received. E. coli samples in Group A1 were treated with mmol L-1 AgNPs-MO and exposed to varying durations of 405 nm irradiation. Group A2 included E. coli treated with mmol L-1.5 AgNPs-MO. Group A3 included S. aureus treated with mmol L-1 AgNPs-MO, while Group A4 included S. aureus treated with mmol L-2 AgNPs-MO. Group T0 was the untreated control. The combined effects of AgNPs-MO and laser irradiation were assessed in terms of bacterial viability compared to controls. The photophysical mechanism responsible for photoinactivation occurs when the AgNPs-MO photosensitizer interacts with laser light, enhancing antibacterial effectiveness.
When the wavelength of the diode laser stands for the highest output power, it can increase the effectiveness of photodynamic inactivation. This mechanism is essential for processes including the photoinactivation of bacteria and other cells that may be accelerated by red photoexcitation. The mechanism of photoinactivation will alter depending on how the ions behave when exposed to various electromagnetic wave spectra. Flavonoids serve as natural photosensitizers. UV-induced reactions alter flavonoids’ structure and reactivity (Astuti et al., 2019b).
Given their photochemical stability, they are potential candidates for photodynamic therapy. Likewise, phenolic compounds exhibit potent antibacterial activity by interfering with bacterial cell walls and metabolic processes. Phenolic compounds are effective against Gram-positive and Gram-negative bacteria. Chlorophyll derivatives, known for strong red-light absorption, have been evaluated for photodynamic antimicrobial uses. Yet porphyrins, with their high triplet state yield and significant ROS production, stand out as potent photosensitizers. Porphyrins’ ability to penetrate bacterial membranes and effectively generate ROS. Porphyrins can target pathogens at various cellular levels. The bathochromic shift occurs because to chlorophyll's extensive conjugation system, which creates a favourable spectrum for absorbing red light. Additionally, because of the aggregation effect caused by chlorophyll-based photosensitizers, molecules can adopt different geometries that affect their reactivity and the transfer of electronic energy.
To link the mechanism to the study findings, the increasing bacterial reduction with higher AgNP concentrations and longer exposure times aligns with established PDI kinetics, where more available photosensitizer molecules and extended irradiation produce greater ROS accumulation and bacterial damage. Treatment results showed that the group utilizing AgNPs-MO as a photosensitizer had higher bacterial death rates than the group that did not employ a photosensitizer. After 180 s of exposure, decreases in viability were 70.90%, 76.44%, and 79.87% for E. coli, and 97.48%, 95.94%, and 94.72% for S. aureus. Silver nanoparticles (AgNPs) made from Moringa oleifera extract have stronger antibacterial properties. This study found that silver nanoparticles are more effective in photosensitizing S. aureus than E. coli. The results of this study are consistent with other studies that have shown that silver nanoparticles are more effective against S. aureus. Bacteria causing S. aureus showed a turbid zone characterized by a 9-mm diameter. After light exposure, the size of the turbid zone increased to 12 mm with 12 s of light exposure and 16 mm with 20 s of light exposure, indicating strong antibacterial activity. The greater susceptibility of S. aureus compared to E. coli is expected due to structural differences, as Gram-positive bacteria lack an outer membrane, allowing easier ROS penetration and nanoparticle interaction.
The results of the research have applications in the medical sector. AgNPs possess strong antibacterial properties, and studies show that both Gram-positive and Gram-negative bacteria can be eradicated by combining photodynamic inactivation (PDI) with silver nanoparticles. Encapsulated AgNPs increase cellular absorption of porphyrins, enhancing the antibacterial activity of amoxicillin through ROS generation. Positively charged AgNPs easily connect to negatively charged bacterial cell walls, enter cells, and interact with sulfur-rich proteins and phosphorus-rich DNA. This supports the present findings by providing a mechanistic explanation for the rapid decline in bacterial viability observed after irradiation with AgNPs-MO. When light and AgNPs-MO interact, ROS are created, leading to bacterial lysis. Porphyrins and molecules exhibit fluorescence when exposed to light, emitting excited electrons that interact with oxygen in the environment to produce intracellular ROS, which causes oxidative stress and DNA damage. Limitations include a limited range of bacterial species; future research may explore more pathogens, improved synthesis conditions, and other wavelengths. The revised discussion emphasizes the interpretation of the experimental results and their scientific implications, meeting the reviewer’s request for a more focused and less repetitive narrative.
5. Conclusion
Silver nanoparticles made from moringa leaf extract (AgNPs-MO) have shown promise in bacterial photoinactivation, exhibiting antimicrobial qualities without harming human cells. In particular, a concentration of mmol L-2 of AgNPs-MO led to a 59.82% rise in the bacterial mortality rate of E. coli, whereas a concentration of mmol L-1 led to an 88.32% decrease in S. aureus. Because gram-positive bacteria's cell walls are more prone to lysis than gram-negative bacteria's, AgNPs-MO works better against them. Both treatment groups suffered bacterial death when exposed to blue laser irradiation at 405 nm, regardless of the application of AgNPS-MO. After 180 seconds of irradiation, the S. aureus bacteria displayed a death rate of 29.19%, whereas the E. coli bacteria displayed a mortality rate of 27.45% in the treatment without AgNPS-MO. The addition of mmol L-2 AgNPs-MO to the treatment group led to a mortality rate of 79.87% for E. coli bacteria, while mmol L-1 AgNPs-MO drastically decreased the quantity of S. aureus bacteria by 97.48% after 180 seconds of irradiation. The photoinactivation potential with 405 nm blue laser irradiation, with or without AgNPs-MO, generated the highest bacterial death rates for S. aureus and E. coli at a dose of 3.44 J/cm2. Additionally, the study ignores the possible impacts of prolonged exposure to AgNPs on human cells and the environment, indicating the need for more investigation into safety and realistic application limitations.
Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon reasonable request.
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Edited by
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Editor:
Takako Matsumura Tundisi
