Green synthesis of novel chitosan-graphene oxide-silver nanoparticle nanocomposite for broad-spectrum antibacterial applications

Abstract

In this study, a novel nanocomposite composed of chitosan, graphene oxide, and silver nanoparticles (CS-GO-Ag NPs) is presented and synthesized using pulsed laser ablation. Characterization techniques, including X-ray diffraction (XRD), Field Emission Scanning Electron Microscopy (FE-SEM), Energy Dispersive Spectroscopy EDX, and UV-visible spectroscopy, confirm successful synthesis. The XRD results reveal that the CS-GO-Ag NPs exhibit crystalline structures with an average crystallite size of approximately 30 nm. FE-SEM analysis shows a heterogeneous mixture of nanoparticles, ranging in size from 32.38 to 174.9 nm. The EDX spectra elucidate the elemental composition, while UV-Vis spectroscopy indicates strong interactions among the components, yielding a direct bandgap of 2.33 eV and an indirect bandgap of 1.2 eV, suggesting efficient light absorption and emission for applications in Light emitted Diode (LEDs) and solar cells. Furthermore, the CS-GO-Ag NPs nanocomposite displays enhanced antibacterial activity against both gram-negative and gram-positive bacteria when it compared to its individual components. According to characterization tests, well-dispersed silver nanoparticles were formed into a ternary nanocomposite, successfully. The enhanced antibacterial capabilities including increased bacterial cell membrane disruption and oxidative stress can be attributed to the synergistic interaction between the components. The outcomes highlight the CS- GO-Ag NPs nanocomposite's potential as a promising antimicrobial agent for numerous applications including the biomedical fields and water treatment industries.

Introduction

In the last few decades, nanomaterials have attracted a lot of interest, mainly for their potential in developing novel antibacterial agents. These multifunctional materials improve their physical and chemical properties, thus improving their antibacterial efficiency. Graphene oxide (GO), silver nanoparticles (Ag NPs), and chitosan are prominent among these materials due to their antibacterial properties.

Chitosan is a polymer that occurs naturally from chitin and is well-known for its antibacterial and biodegradable properties [1]. Chitosan is extensively used in medical applications such as wound healing and drug delivery [2]. Research denotes that chitosan nanoparticles have a highly effective interact with tissues due to their inherent antibacterial properties [3]. A carbon-based nanomaterial called graphene oxide (GO) obtained through the chemical exfoliation of graphite.

GO has a variety of oxygen-containing groups, including epoxy, carboxyl, and hydroxyl groups, which provide reactive sites for modifications and improve its antibacterial potential [4].

Studies have confirmed that GO exhibits antibacterial properties due to these groups, which harm bacterial cell membranes and cause oxidative stress that results in cell death [5–7].

Ag NPs, silver nanoparticles, are well known for their strong antibacterial effects. Recent findings highlighted that silver (Ag)-based nanocomposites are widely recognized in medical and environmental applications. This is due to their synergistic properties derived from the unique behaviour of silver nanoparticles (AgNPs) and their combination with other materials making them effective in decreasing the activity of microorganisms. AgNPs represents a promising materials in addressing bacterial infections and antimicrobial resistance [8, 9]. Recent advancements feature Silver nanoparticles mechanisms that justify their antibacterial activity are multifaceted and operate at molecular and cellular levels [10]. The Key mechanisms are, first the membrane disruption, silver nanocomposites release silver ions, which are the primary agents responsible for antimicrobial activity. AgNPs interact with bacterial cell membranes causing increased membrane permeability [8]. This leads to structural damage and ultimately causing cell death. This is due to leakage of cellular contents and disrupting vital processes as the small size of AgNPs (often under 50 nm) enables them to penetrate cell membranes effectively and disrupting lipid bilayers [11] Second, Generation of Reactive Oxygen Species (ROS), AgNPs catalyze the production of reactive oxygen species (ROS) (e.g., hydroxyl radicals, superoxide anions, and hydrogen peroxide) [9, 10] that provoke oxidative stress, damaging bacterial membranes, proteins, DNA and lipids within microbial cells leading to structural breakdown. This oxidative damage is mainly fatal as it targets multiple cellular components instantaneously [11]. Third, protein and DNA binding, from the AgNPs, silver ions released and bind to sulfur- and phosphorus-containing compounds in proteins and DNA disrupting enzymatic functions. This prevents replication and transcription processes within microbial cells [12]. Forth, biofilm inhibition: microorganisms form protective matrices called biofilm. It has been found that Ag nanocomposites are remarkably effective against biofilms [9, 11] which are typically resistant to antibiotics. As they prevent biofilm formation by penetrating the biofilm matrix, disrupting their structural integrity and reducing the expression of genes such as quorum-sensing signals required for biofilm development and killing microorganisms within the biofilm [10, 12]. Fifth, synergistic effects in composites, in nanocomposites, Ag is often combined with materials such as silica, polymers or metal oxides [10]. These combinations improve antimicrobial antibacterial efficiency. In the current work, a combining of AgNPs with graphene oxide creates graphene oxide-silver (GO-Ag) composites demonstrate enhanced dispersibility and stability as well as reducing the agglomeration issues of individual Ag NPs. Such composites show higher effectiveness at lower concentrations owning to improved ROS production and physical disruption of bacterial membranes [9, 10]. Sixth, shape and surface effects, recent publishing indicated the functionalization effects of shape and surface of AgNPs on their antimicrobial efficiency. For instance, triangular nanoparticles and those with active surface features have shown superior activity due to greater surface area and interaction potential [12].

These mechanisms collectively qualify silver nanocomposites that can prevail over microorganisms resistance mechanisms which typically used by them against conventional antibiotics and also making AgNPs as one of the most successful against water/wastewater treatment that pollutants with organic materials and heavy metals [13]. Tuning nanoparticle size, shape, and composition could be further enhanced their antimicrobial potential. This makes AgNPs as promising agents in the fight against resistant infections [12].

As a result, Ag NPs demonstrate a broad-spectrum activity by interacting with bacterial cell membranes and metabolic processes, producing reactive oxygen species, and influencing bacterial enzymes [14, 15]. The antibacterial capabilities of silver nanoparticles are enhanced by integrating them with chitosan and graphene oxide. Silver nanoparticles are deposited onto graphene oxide and chitosan using pulsed laser ablation techniques, permitting precise control over particle size and distribution, which enhanced their efficiency in biological applications [16]. This method enables fine-tuning of nanoparticle size and distribution, improving their performance in electronic and medical fields [17]. According to recent researches, the combining chitosan, graphene oxide, and silver nanoparticles significantly improves antibacterial activity in the comparison to individual components, making such composites promising for advanced antibacterial applications [18, 19]. Moreover, researches confirm the potential of chitosan-based composites in the treatment of wastewater and the administration of targeted medications [20, 21].

This study presents a novel nanocomposite incorporating chitosan, graphene oxide, and silver nanoparticles, aiming to obtain better antibacterial activity.

Pulsed laser ablation method was used to synthesize the nanocomposite and its structural, optical, and antibacterial properties were characterized. The results reveal the nanocomposite's superior antibacterial activity compared to its individual components, highlighting its potential for different biomedical applications.

Experimental

Chitosan powder (molecular weight 1500 g/mol), Acetic acid, Graphene oxide from Life Sciences (GP5053), Silver metal (99.99% purity), Distilled water. In details, for synthesis of Chitosan-Graphene Oxide (CS-GO) Solution, dissolve 2 g of chitosan powder in 280 ml of distilled water and 35 ml of concentrated acetic acid to obtain a chitosan solution, and add 1 mg/ml graphene oxide solution to the chitosan solution under vigorous stirring. In the final, sonicate the solution to ensure homogeneous dispersion of graphene oxide. Also, for synthesis of Chitosan-Graphene Oxide/Silver Nanoparticles (CS-GO/Ag NPs) Nanocomposite, a piece of high-purity silver metal is placed into the CS-GO solution, and pulsed laser ablation (PLA) is performed using an Nd: YAG laser with a power of 80 mJ, 500 pulses, and a wavelength of 1064nm. This process facilitates the generation of silver nanoparticles directly within the CS-GO solution, resulting in the formation of the CS-GO/Ag NPs nanocomposite. And for the control samples, prepare a CS-GO solution without silver nanoparticles for comparison, and prepare a chitosan/silver nanoparticles solution using the same PLA process but without adding graphene oxide.

We using these instruments to amylases the nanocomposites, the FE-SEM as a type of electron microscopy using a field emission as the electron source and providing a high-resolution imaging technique. By scanning images with a focused electron beam FE-SEM produces detailed surface images of samples with superior resolution, enhanced contrast, and greater sensitivity compared to conventional SEM. Therefore, it ideal for studying fine surface structures at nanoscale. Also, to analyze the crystalline structure of (CS-GO/Ag NPs) Nanocomposite the X-ray diffraction (XRD) technique is used. XRD ’s work based on a directed X-rays at sample, and the crystal lattice diffracted produces a pattern that can be used to determine the material’s phase, composition, and structure. UV-Vis (Ultraviolet-Visible) Spectroscopy is an analytical technique that measures the absorption of ultraviolet and visible light by a (CS-GO/Ag NPs) Nanocomposite sample. The absorption spectrum typically shows peaks in the UV range due to transitions in their electronic transitions.

Finally, the resulted compounds are separately deposited on three aluminum plates by a dip deposition method for characterizing via Field emission scanning electron microscopy (FE-SEM), (German origin and model ZEISS LEO 912.). X-ray diffraction (X-ray diffraction) (XRD) ((type Analytical X 'Pert Pr) of British origin). FE-SEM images were used to measure the grain size, while XRD measurements determined the crystal size of Ag NPs and the CS-GO/Ag NPs nanocomposite. The UV-Vis Spectroscopy (UV-Vis) (Located in the Faculty of Science for Women in the Department of Laser Physics) measurements are performed using an Aquarius (Cecil Company) spectrophotometer and room temperature range from 200 to 1100 nm to obtain the optical properties.

Result and discussion

Structural properties

The X-ray diffraction (XRD) results indicate that the nanocomposite of chitosan, graphene oxide, and silver nanoparticles exhibit crystalline structures. Figure 1 show a broad XRD peak at 2θ = 25° is often indicative of a poorly crystalline, which aligns with the characteristics of rGO confirming its presence in all three samples. This peak corresponds to the (002) plane, which reflects the interlayer spacing between graphene sheets. From Fig. 1, it can be noticed a weak peak at 2θ = 10.5° corresponding to (001) plane that belongs to GO. This peak does not show high intensity which might be due to the GO nanoparticles preparation conditions that attributed to the intercalation with water and the presence of oxygen functionalities such as epoxide and hydroxyl groups. This populates on the basal plane of the carbon sheet and the thermal process that occurred in the ablation technique making the peak undetectable [22].

Silver nanoparticles (Ag NPs) have peaks at 2θ = 38° and 40° are indicative of silver nanoparticles, suggesting their successful incorporation into the nanocomposite. While the typical peak for Ag NPs is found around 2θ = 44°, variations may occur due to factors such as particle size, strain, or impurities.

Due to several physicochemical and biological factors, the size of AgNPs in nanocomposites plays a key role in controlling their antimicrobial features. This factors for instance is surface area-to-volume ratio [23]. Where smaller AgNPs have a higher surface area-to-volume ratio, permitting for better interaction with microbial cell membranes as this increasing enhances the silver ions (Ag⁺) release, which are primarily responsible for antimicrobial activity [8]. This means more active sites are available for binding to microbial surfaces when particle size decreases and consequently, enhances their ability to disrupt cellular processes, leading to more efficient antimicrobial action [13, 23].

The identified peaks at 2θ = 38° and 40° correspond to the Miller indices (111) and (200), respectively. The peaks observed for the chitosan-graphene oxide-silver nanoparticles (CS-GO-Ag NPs) at 2θ = 4.15° and 16.99° correspond to chitosan. The average crystallite size calculated for the CS-GO-Ag NPs nanocomposite is approximately about 30 nm. These findings align with previous studies [24–26]

Morphological studies

The FE-SEM analysis as shows in Fig. 2 revealed a heterogeneous mixture of nanoparticles with varying sizes ranging from 32.38 to 174.9 nm. While some particle aggregation was observed, the overall distribution appeared relatively uniform. The presence of both spherical and elongated particles suggests a diverse morphology. These findings highlight the potential influence of particle size and distribution on the nanocomposite's properties and performance. The results are consistent with the results obtained from XRD and with previous studies [27, 28].

The energy dispersive spectroscopy (EDX) analysis showed in Fig. 3 and the corresponding Table 1, provides insights into the elemental composition of the CS-GO-Ag NPs nanocomposite. The EDX spectra denotes significant peaks corresponding to carbon (C), silver (Ag), and oxygen (O), with carbon being the most abundant element. The high carbon peak reveals the substantial presence of cellulose (CS) and graphene oxide (GO), which are essential composite components. The significant Ag Kα peak highlights the significance of silver nanoparticles (Ag NPs) in the composite’s structure validating their successful integration. Moreover, a minor signal (O Kα) linked to oxygen suggests the presence of some oxygen bound to cellulose and oxygen in graphene oxide. The weight percentages of 74.71% carbon, 21.59% silver, and 3.70% oxygen are quantitatively presented in the EDX data. These results supported the proposed composition of the CS-GO-Ag NPs composite. It is worth to mention that these findings align with earlier researches, emphasizing the substance of carbon, silver, and oxygen in the material's overall properties [29, 30].

Optical properties

The UV–visible spectroscopy as in Fig. 4 illustrates the absorbance spectra of various nanocomposites, including chitosan (CS), graphene oxide (GO), and silver nanoparticles (Ag NPs), highlighting their optical properties. The spectrum for GO exhibits a prominent peak at 245 nm, indicative of significant light absorption associated with π-π* transitions of the C=C bonds in its structure. In contrast, the absorbance spectrum for CS shows minimal absorbance, reflecting the low light-absorbing capacity typical of biopolymers. The GO-Ag NPs composite demonstrates enhanced absorbance due to the synergistic effects of reduce graphene oxide as shoulder at 283 nm and silver nanoparticles peek at 400 nm, likely attributed to surface plasmon resonance (SPR) associated with the Ag NPs. The CS-Ag NPs spectrum reveals moderate absorbance, suggesting that the incorporation of silver enhances the optical characteristics of the chitosan matrix. Notably, the CS-GO-Ag NPs composite exhibits the most complex absorbance profile, indicating a significant enhancement in optical activity due to the synergistic interplay of all three components.

Also, it can observe in Fig. 4 the decrease in the absorption peak of Ag NPs in the presence of GO can be the interaction between GO and Ag NPs induce aggregation or clustering of the Ag NPs. This can lead to changes in the size distribution and shape of the Ag NPs, which can affect their SPR properties as shown in FESEM images. Additionally, the peak at 283 nm is typically associated with rGO due to its π-π* transitions, this indicated as in XRD the sample have rGO interaction with chitosan and sliver nanoparticles.

When it comes to the absorption coefficient, the reason is that the amount of optical absorption begins to increase when the wavelength exceeds the amount of the cut wavelength because the incoming photons with energy greater than the energy band gap value cause an increase in the practical value of the absorption coefficient, which increases with the increase in the laser ablation rate, as shown in Fig. 5.

The prepared materials CS-GO-Ag NPs nanocomposite have an absorption coefficient value α > 10⁴ to meet the conditions of direct transmission. The absorption coefficient amount begins to increase slightly in the energy region lower than the energy band gap, and then gradually decreases [31, 32].

Where α is the Absorbance coefficient, h is the Planck constant, ν is the photon’s frequency, B is a constant, n is 1/2 for allowed direct transitions and 2 for indirect transitions, and Eg is energy bandgap.

Then, by using Tauc plot analysis as in Fig. 6. The optical bandgap values were determined by extrapolating the linear portion of the (α⋅hν)ⁿ versus hν curve to the photon energy axis [33].

• The direct bandgap was found to be 2.33 eV, suggesting efficient photon absorption for optoelectronic applications like LEDs and solar cells.

• The indirect bandgap was calculated to be 1.2 eV, indicating that the material can also facilitate carrier transport across a lower energy threshold.

Antibacterial affect

The antibacterial properties of CS, CS-GO nanocomposite, and CS-GO-Ag NPs nanocomposite using some of gram-negative and gram-positive (E. coli) (S. aurous) strains, Klebsiella, and Pseudomonas is spread on nutrient agar plates. The sterilization of media is done by autoclaving at 121°C, 15 pound/inch² for 15 min. Then poured into a tube and/or Petri dishes then incubated for 24 h at 37°C after that kept at 4°C. The sample (CS-GO-Ag NPs) is then placed on plates in triplicates for 24 h at room temperature. After the incubation period, the inhibition length (i.e. the diameter of inhibition zone surrounding the samples) is measured to determine anti-bacterial activity. It is observed from Fig. 7 that when the polymer was applied alone to the bacteria, we obtained a low percentage of killing of Klebsiella (6 mm) and Pseudomonas aeruginosa (15 mm), and no effect on other species.

But when exposing the bacteria to polymer and graphene a killing rate is (35 mm) is obtained for Pseudomonas and Staphylococcus (16 mm), where it is found that the polymer combined with graphene affects Klebsiella. When the bacteria are exposed to a solution of polymer, graphene and silver all types used for the test are killed with higher killing rates than the previous cases (28 mm) for Klebsiella, Pseudomonas (38 mm), Staphylococcus aureus (20 mm) and E. coli (22 mm), while E. coli are affected only by the polymer with silver and graphene.

The CS-GO-Ag NPs nanocomposite exhibits robust antimicrobial activity against a wide range of microorganisms. Such nanocomposite combines the distinctive properties of each component to improve its antimicrobial effectiveness. The mechanisms behind the antimicrobial action of CS-GO-Ag NPs include: the synergistic action of (Ag NPs) as they are identified for their potent antimicrobial properties where Ag⁺ ions disrupt the electron transport chain in bacteria and prevent DNA replication, leading to cell death; (GO) as a physical disruptor that can interact with microbial cell membranes, causing membrane disruption or the formation of pores leading to leakage of cellular contents, structural damage and ultimately cell death. Finally, (CS) as a biopolymer with antimicrobial properties that inherent antimicrobial activity due to its positive charge and ability to interact with the negatively charged microbial cell surfaces, leading to membrane destabilization and cell death. Furthermore, ROS Generation can be enhanced by Ag NPs and GO (as illustrated previously in the introduction section), for instance, E. coli exposure to CS-GO-Ag NPs leads to ROS generation that damages bacterial membranes and DNA, impairing cell function and viability.

From the above it can be noticed that the CS-GO-Ag NPs composite structure permits for controlled release of the antimicrobial agents where the graphene oxide serves as a carrier for silver nanoparticles, and chitosan can help in the sustained release of silver ions and other antimicrobial agents to prolong the antimicrobial effect and ensures sustained action against microorganisms.

As a result, the antibacterial properties of newly synthesized agents have been carefully assessed and compared with that of individual components. Thus, the combined substances have a better and stronger effect against bacteria than alone, the product is environmentally friendly and can be used for water treatment, biomedical as well as in the food industry and many other applications. Figure 8 shows the killing rate of each type of bacteria by the nanocomposite CS- GO-Ag.

Conclusions

This study successfully synthesized a ternary nanocomposite comprising chitosan, graphene oxide, and silver nanoparticles. The CS- GO-Ag NPs nanocomposite demonstrated remarkable antibacterial activity against a broad spectrum of bacteria, surpassing the individual components.

The CS-GO-Ag NPs nanocomposites antimicrobial activity develops from the synergistic interactions of chitosan, graphene oxide, and silver nanoparticles. Multiple mechanisms of these nanocomposites exploit to combat microorganisms involve physical disruption of microbial cell membranes (via GO and CS), silver ions (Ag⁺) release, reactive oxygen species (ROS) generation, the electrostatic interactions leading to membrane destabilization, biofilm formation Inhibition and the controlled delivery of antimicrobial agents.

This combination of mechanisms makes CS-GO-Ag NPs a highly effective antimicrobial material that offers a promising solution for combating bacterial, fungal, and other microbial infections, particular those showed a high resistance to conventional antibiotics

The findings highlight the potential of this nanocomposite as a promising antimicrobial agent for various applications, including water treatment and biomedical fields.

Author contributions

Hamsa N. Naser (Data curation [equal], Investigation [equal], Methodology [equal], Writing—original draft [equal]), Mulamahawsh Anfal Fadhil (Data curation [equal], Investigation [equal], Validation [equal], Writing—original draft [equal]), Tebark Abd Zaid Hassoun (Data curation [equal], Writing—review & editing [equal]), Rafea Tuama Ahmed (Methodology [equal], Validation [equal]), and Amer Al-Nafiey (Project administration-Lead, Writing—review & editing [equal])

Conflict of interest: The authors declare that they have no conflicts of interest.

Funding

This research received no external funding.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References