One-pot fabrication of Ag @Ag2O core–shell nanostructures for biosafe antimicrobial and antibiofilm applications

Structural analysis and chemical bonds formation

X-ray diffraction (XRD)

The X-ray diffraction (XRD) spectrum of nano-composite (Ag@Ag₂O)NCsis given in Fig. 2a. The peaks observed at the diffraction angles (2θ): 26.2°, 32.9°, 38.1°, 55.2°, 65.7° and 69.1° corresponding to (110), (111), (200), (220), (311) and (222) set of Ag₂O lattice planes (cubic structure), respectively [JCPDS card No. 76-1393]. The high intense peak (111) may refer to the arrangement of lattice atoms in an ordered structural fashion¹⁵. Whiles, the peaks observed at (2θ): 44.34° and 77.34° correspond to the metallic Ag (200) and (311), respectively in the face-centered cubic structure [JCPDS card No. 04-0783]¹⁶.On the other hand, the close overlap between Ag and Ag₂O diffraction peaks and the difficulty to distinguish between the Ag⁺ and Ag⁰ peaks at the diffraction angle of 38.1° inferred a formation of a hybrid structure^17,18. Sajjad Ullah et al.¹⁸ found that, the diffraction peak at around 38° could be assigned to metallic Ag and/or Ag₂O and proving the existence of Ag (38.1°, JCPDS card No. 65-2871 and 04-0783) and Ag₂O at the same time (38.0°, JCPDS card No. 41-1104) in the samples. The structure could possibly have an Ag₂O shell with Ag as the core with a decreasing gradient of oxygen from the surface to the core⁶. Despite the simplicity of preparation method, the silver element needs a special medium during its preparation. The individual crystallite size (t) was calculated using Scherrer’s formula¹⁹ given by Eq. (2).

where k is the Scherrer’s constant (0.89–0.9), λ is the X-ray wavelength, β is the full width at half maximum (FWHM) and θ is the Bragg angle¹⁹. According to Eq. (2), the sample crystallite size for the plan (111) is calculated and is found to be approximately 18.6 nm.

Raman analysis

The molecular structure and phase identification of the Ag@Ag₂Ocore shell are explored using Raman spectra. Figure 2b shows the Raman spectrum for the prepared nanopowder in a range from 0 to 4500 cm⁻¹. Two major peaks are clearly detected; the first one at 74.1 cm⁻¹ with an intensity of 7900 cps for Ag⁰ (Ag lattice vibrational mode) and the other one at 1046.7 cm⁻¹ with an intensity of 583 cps for Ag₂O (Ag–O stretching/bending modes)²⁰.

Fourier-transform infrared spectroscopy (FTIR):

FTIR analysis reveals the functional groups of the Ag@/Ag₂O nanocomposite synthesized using alkali chemical treatment (Fig. 2c).The broad band at3400 cm^-1indicates the O–H stretching vibrations of the hydroxyl groups²¹ corresponding to H-bonded alcohols and also to intramolecular H bonds which are most probably from water molecules²². The peaks at 2357 cm⁻¹and 1655 cm⁻¹prove the existence of O–H carboxylic acids²³ and OH bending, respectively²⁴. The band at 1387 cm⁻¹ assigned to O–H bend of carboxylate²⁵. The absorption band on 675 cm ⁻¹ is due to Ag–O stretching mode, which corresponds to Ag–O vibration in Ag₂O.Furthermore, the appearance of follower peak at 868.68 cm⁻¹corresponds to the metal–oxygen vibrations for the formation of (Ag@Ag₂O) NCs¹⁵; thus, synchronizing with the aforementioned XRD results; confirming the formation of (Ag@Ag₂O) NCs.

X-ray photoelectron spectroscopy (XPS)

The XPS data for the chemically prepared Ag@Ag₂O NCs is illustrated in Fig. 3. Figure 3a shows the general survey analysis of the nanopowder, which exhibits a major detected peak of the Ag3d at a binding energy of 368.34 eV with an atomic ratio of 48.5%. Also, O1s peak is detected at a binding energy of 530.81 eV with an atomic ratio of 29.75% and K2p peak is observed at a binding energy of 293.5 eV with an atomic ratio of 2.18%. Finally, C1s peak is measured at a binding energy of 285.21 eV with an atomic ratio of 19.56%. In Fig. 3b, the high resolution of the Ag3d spectrum displays two main strong bands. Such two bands can be further de-convoluted into two pairs of sub-peaks. The peaks at 367.98 eV with an atomic ratio of 44.01% and 373.96 eV with an atomic ratio of 28.75% are respectively assigned to Ag⁰ (3d5/2 and 3d3/2). The other set of bands is detected at 367.38 eV with an atomic ratio of 10.48% and 373.6 eV with an atomic ratio of 10.36% are attributed to Ag⁺ (3d5/2 and 3d3/2, respectively) in the nanocomposite. Figure 3c confirms the oxidation of the silver nanoparticles through the existence of the O1s spectrum at 529.39 eV with an atomic ratio of 47.64%, at 530.77 eV with an atomic ratio of 25.32% and at 531.4 eV with an atomic ratio of 23.19%. Finally, the results confirm that there are two different configurations of silver species, namely Ag₂O and Ag, which is consistent with many published reports^26,27,28. The detected elemental carbon in the main survey analysis may have originated from the ambient atmosphere itself. The adsorption of hydrocarbons from the surrounding atmosphere, which results in the creation of a thin carbon layer on surfaces, is most likely the source of the carbon contamination²⁹.

Morphological analysis

Scanning electron microscope (SEM)

Figure 4a and b illustrates the scanning electron microscope (SEM) surface morphological image of the nano-composite (Ag@Ag₂O) at different magnifications. The Ag₂O exhibited bundles of nearly spherical nanoparticles ranged from 19 to 59 nm and this result is nearly consistent with the result from XRD patterns in Fig. 2a. The silver oxide may have formed in as a solution mixture containing potassium hydroxide and n-propanol which have high oxidation potential³⁰. Also, the time spent since the beginning of the reaction, i.e. when adding silver nitrate to the oxidized mixture and until the end of the reaction is not enough to produce the silver oxide in its final form. Thus, it is an incomplete reaction that results in the precipitation of the silver nanopowders. This step entails the formation of a layer of silver oxide on the surface of the silver powder nanoparticles as a result of remaining in the oxidizing solution for a longer time³¹. Thus, it is logical to form an Ag/Ag₂O core shell compound of a spherical nature as a result of the lattice mismatch between silver metal and silver oxide⁶. However, the aggregation is more likely to occur due to too small size as shown in Fig. 4a and b. Generally, the smaller particle size is usually more beneficial for the antibacterial activity. Because the particle size is smaller, many more particles will be easily adsorbed on the surface of the bacterial cell membrane, and then successfully attack the cell, ultimately destroying the physiological functional groups of the cell³².

Transmission electron microscopy (TEM)

TEM has been employed to characterize the size, shape, morphology and crystallinity of the synthesized Ag@Ag₂O NCs. Figure 4c–i shows both TEM and HRTEM images of the Ag@Ag₂O NCs where regular spherical shaped nanoparticles with size range of 40–60 nm are detected which is in good agreement with the particle size calculated from the SEM images. In Fig. 4e, core shell nanoparticle is clearly indicating the existence of Ag as core and Ag₂O as shell shape. High-resolution TEM (HRTEM) in Fig. 4e shows the lattice fringes with a d-spacing of 2.33 Å, which matches to the (111) reflection of face-centered cubic (fcc) of Ag. The crystallinity of Ag@Ag₂O was observed by selected area emission diffraction (SAED), which was recorded by directing the electron beam perpendicular to NCs and shown in Fig. 4i. That reveals the polycrystalline nature of the Ag@Ag₂O. The elemental mapping of Ag and O elements shown in Fig. 4f–h shows a major distribution of Ag element with regular and minor distribution of O element. The detected multi diffraction spots in Fig. 4i may be referred to the dual lattices from both Ag and Ag₂O nanostructures.

Zeta potential

The surface charge of Ag@Ag₂O core shell was determined from Zeta potential by applying voltage across a pair of electrodes at either end of a cell containing the particle dispersed. The charged particles are attracted to the oppositely charged electrode and assessing the Zeta-potential value by − 5.34 mV (Fig. 5a). The Ag@Ag₂O NCs show slightly low surface charges which tend to form agglomerates³³. Moreover, the low surface charges of Ag@Ag₂O NCs reflect the urgent requirement of a capping agent to prevent such agglomeration and keep nanocomposites stable for a long time³⁴. However, upon antimicrobial application and cytotoxicity evaluation, the examined NCs were freshly prepared and examined after a short time of preparation (within 48 h of preparation). Subsequently, the prepared NCs, within such time, didn’t exhibit aggregation and were still stable. Additionally, several reports^35,36 synthesized AgNPs and other metal-NPs in the same range of zeta and also exhibited antimicrobial activity.

Fluorescence spectra

The fluorescence emission peak of Ag@Ag₂O NCs was detected using an excitation wavelength of 250 nm and appeared at about 498 nm in the visible range as shown in Fig. 5b. This fluorescence emission peak may be attributed to the relaxation of the electronic motion of surface plasmons³⁷. The sharpening behavior in the peak may be due to the core shell structure and coverage of Ag by Ag₂O, which prevents the nanopowder from combining with any water molecules as well as continuing the oxidation process³⁸.

The chemical mechanism

Based on the preceding experimental data, it is worth mentioning to explain the chemical mechanism of the nanocomposite (Ag@Ag₂O) formation as demonstrated in Eq. (3). The reaction of silver nitrate with potassium hydroxide produces silver hydroxide via the following mechanism²⁴:

The intermediate AgOH is thermodynamically unstable, and the Ag₂O is formed phase through many steps, as shown in the following Eqs. (4)–(7). Briefly, a part of AgOH may be reacting with the n-propanol, which acts as a wetting agent that decreases the recombination rate and the generation of silver propanoate (Ag-O₂CCH₂CH₃), as shown in Eq. (4), which is inferred from FTIR spectra as a sharp peak at 1655 cm⁻¹ and 3400 cm⁻¹ as shown in Fig. 2c ³⁹. Meanwhile, Ag-O₂CCH₂CH₃is reacted with the hydroxyl group of KOH producing silver ions (Ag⁺) in a continuous oxidation process [Eq. (5)]. The silver ion reacts with water and n-propanol in an alkaline medium via the presence of OH^– group to produce silver element (core); as shown in [Eq. (6a)]. Additionally, some of the silver ions re-interact with water and n-propanol for producing silver hydroxide as in [Eq. (6b)]. Therefore, the unstable silver hydroxide product (AgOH) is reduced to silver oxide (Ag₂O shell) as shown in [Eq. (7)].

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Antimicrobial efficiency of Ag@/Ag₂O NCs against planktonic pathogens

Considering the health problems associated with microbial contamination, it is vital to find out effective antimicrobial agents that are able to control their outbreak. Thus, the current study is concerned with the antimicrobial activity of Ag@Ag₂O NCs against some prokaryotic and eukaryotic pathogens. The sensitivity of the examined pathogens to different concentrations of Ag@Ag₂O NCs is shown through agar diffusion assay. Figure 6a and b illustrates the comparative results of antimicrobial activities of the Ag@Ag₂O NCs and their precursor. Also, it demonstrated a dose-dependent manner in which the antimicrobial activity of different concentrations of Ag@Ag₂O NCs against E. coli, B. cereus and C. albicans as representative models of pathogenic Gram-negative bacteria, Gram-positive bacteria and Fungi, as well as C. vulgaris control before treatment with Ag@Ag₂O NCs and C.vulgaris after treatment with 50 μg/mL of Ag@Ag₂O NCs are shown in Fig. 7A–E respectively. Generally, Ag NPs displayed considerable effectiveness indicated by halo zones which exceeded 1 mm, where any antimicrobial agent was evaluated as “good” atan inhibition zone greater than 1 mm⁴⁰. For all the examined pathogens, inhibition halos were directly proportional to the concentration of AgNPs. In addition, Gram-positive strains seemed to be more resistant than Gram-negative strains. That could be attributed to the lipophilicity of Ag NPs according to different cell wall polarity and compositional variations⁴¹.

As revealed by Pazos-Ortiz et al.⁴² the thickness of the cell wall increases the resistance of bacteria to the exposed NPs. The thick peptidoglycan layer of the Gram-positive bacteria’s wall, which is composed of teicoic acids and lipoteicoic acids, restricts the diffusion of NPs. Moreover, the tolerance response of each microbe depends on its metabolic properties. However, the cell wall of the Gram-negative bacteria is composed of thinner peptidoglycan layer together with lipoprotein and lipopolysaccharide, which together represent 25% of its mass. It is noteworthy to mention that the nosocomial infections and enteric fever are associated with P. aeruginosa, E. coli and S. typhi, respectively. Therefore, their inhibition is a pivotal issue. In agreement with our results^42,43 reported low reduction in S. aureus count (CFU/mL) and also halo zone in comparison to Gram-negative bacteria upon treatment by Ag@Ag₂O NPCs. Besides, Ag@Ag₂O NPCs biosynthesized by aqueous leaf extract of Eupatorium odoratum (EO) exhibited antagonistic performance coincident with the obtained results of current study⁴¹. In the same sense, D’Lima et al.⁶ reported that Ag/Ag₂O hybrid nanoparticles showed a considerable zone of inhibition against P. aeruginosa; declaring the enhancement of antibacterial activity upon combination with carbenicillin. In contrast, other studies reported higher susceptibility of Gram-positive bacteria for NPs treatment than Gram-negative one^11,44. Remarkably, a considerable halo of mycostasis was noticed against C. albicans. Despite the oligodynamic nature of silver ions, which is due to their higher activity at minute concentrations, a potent antifungal efficiency of 50 μg of Ag@Ag₂ONCs exhibited upon comparing with its precursor (Fig. 6); implying effectiveness in the treatment of COVID-19 post infections. Such fungal infections appeared recently in the second wave in India, in particular in patients who were put on mechanical ventilation in intensive—care units. The fungicidal property of Ag@Ag₂ONCs could be assigned to the damage of the glycoprotein-glucan-chitin cross-linkage of fungi cell wall followed by sever alterations in cellular biochemistry^11,45. In addition, it has been suggested that Ag nanoparticles interact with the proteins of the plasma membrane, which is responsible for keeping trans-membrane electrochemical potential gradient such as H⁺ ATPase protein. Such interaction leads to alterations of normal protein conformations and malfunctioning by blocking the regulation of H⁺ transport across the membrane, which ultimately hindering growth, restraining respiration and ending with death^46,47,48. In coincidence with our results, Mallmann et al.⁴⁹ highlighted similar results with inhibitory influence of Ag@Ag₂O NCs against several species of Candida. Otherwise, Elemike et al.⁴¹ demonstrated the dominant biocidal effectiveness ofAg@Ag₂O NCs in bacteria than fungi.

Evaluation of the as-prepared Ag@Ag₂ONCs against biofilm formation, biofilm disintegration and algal growth

Biofilms are multicellular sessile microbial communities embedded in a self-produced extracellular polymeric matrix (EPS) (e.g. DNA, proteins and polysaccharides) and attached toa living or inert substratum or interface. Actually, the viscoelastic nature of the EPS represents a serious concern, especially in water pipes, water purification systems and also in medical devices. Where, the biofilms have the capability to withstand different stress factors by the virtue of such feature. Hence, nanotechnology invasion has provided a significant tool to eradicate such problem at both environmental and medical levels⁵⁰. The inhibitory effect of different concentrations of as-synthesized Ag@Ag₂ONCs and their precursor salt on biofilm formation/ disintegration of both Gram-positive and Gram-negative bacteria was illustrated in Table 1. As noticed, P. aeruginosa biofilm was less susceptible for both treatments and under formation/ disintegration conditions, in comparison to S. aureus biofilm. As revealed by Hoseini -Alfatemi et al.⁵¹, P. aeruginosa and S. aureus biofilms were inhibited by 10 and 1 mg/mL of AgNPs, respectively; which makes our study characteristic. Where, 100 µg/mL suppressed (98.7% and 87.5%) and (93.1 and 74.8%) of S. aureus and P. aeruginosa biofilm synthesis and disintegration, respectively. Interestingly, Gram‐negative biofilms were comparatively more resistant to antibiofilm treatments than Gram‐positive as reported in several studies^42,51,52. Generally, Ag@Ag₂O NCs exhibited antibiofilm activity via several routes including, destruction of initial planktonic phase, damage of aggregated/sessile phase, disruption of EPS matrix, increasing of hydrophobicity of EPS and inhibition of quorum sensing system⁵³.

What is more, the inhibitory effect of Ag@Ag₂ONCs against algal growth of C. vulgaris was studied. C. vulgaris is involved among other algal genera which are responsible for various environmental issues such as eutrophication and biofouling, especially in the availability of high concentrations of contaminants and in association with direct sunlight⁵³. As illustrated in Table 1, Ag@Ag₂O NCs exhibited a drastic algicidal effect on the proliferation and viability of algae with 98.4% growth inhibition. Severe damage of chloroplasts could be proposed due to yellowish to pale green color of algal growth in the presence of Ag@Ag₂ONCs. Meanwhile, the control culture (without Ag@Ag₂ONCs) appeared green and flourished during 7 daysof incubation as shown in Fig. 7D and E. Disintegration of algal cell organelles, thylakoid disorder and plasmolysis are common features associated with the destructive effect of Ag@Ag₂ONCs on algal cell as stated by Duong et al.⁵⁴. Therefore, the employment of Ag@Ag₂ONCs in restriction the algal blooms could result in constraining of their environmentally adverse influence.

As general observations, Ag@Ag₂ONCs exhibited greater inhibitory activity than its precursor against all examined microbial forms. That could be assigned to the small size of nanoparticles and in relation to surface area. As pointed out by⁵⁵, the antagonistic activity of NPs derived from their penetration ability which depends on sizes that are less than 100 nm. In addition, the biocide activity of Ag@Ag₂ONCs uplifted linearly with increasing in Ag@Ag₂O NCs concentration, which implies dose-dependent manner. However, NPs type, concentration, size, aggregation state, surface charge, synthesis conditions and tested microbe consider being governing parameters influencing of the effective doses⁵¹.

Broadly, several strategies could be ascribed for NPs to display their toxicity against different microbial forms. The first strategy begins from puncturing and perforating the first protective barrier of the cell, which is cell wall, by interacting with its anionic components such as neuraminic acid, N-acetylmuramic acid, and sialic acid. However, as long as the NPs are smaller than 80 nm, their passage to cell membrane and later inside the cell is facile; causing phospholipid peroxidation, polysaccharides depolymerization and subsequently membrane detachment and integrity destruction^10,56. At this stage, cell permeability increases followed by intracellular components leakage and proton motive force dissipation. Once NPs occupies intracellularly, more destructive features were exerted concerning metabolism and biochemical activities¹⁰. AgNPs showed higher affinity for binding with thiol group of amino acids; forming extra –S–S– bonds. By such way, deformation of protein configuration occurs, leading to proteins denaturation and ribosomes inactivation^56,57. Further, NPs bind with nucleic acids such genomic and plasmid DNA; causing blockage of DNA replication and repair processes. With continuous release of Ag⁺ ions and their oxide from Ag@Ag₂O NCs, set of reactions (e.g., Fenton and Haber–Weiss reactions) are continuously and intensively generating Reactive Oxygen Species (ROS) such as hydroxyl radicals (OH⁻), superoxide radicals (O₂⁻) and singlet oxygen (¹O₂). Under such oxidative stress, massive damage to the cell takes place and eventually lead to cell death. Tee et al.⁵⁸ and Pazos-Ortiz et al.⁴² referred to the complexity of the mechanisms by which NPs exhibit their antagonistic influence. Figure 1b represents schematic illustration on the destructive effect of Ag@Ag₂O NCs against different microbial forms.

Cytotoxicity assessment

After 72 h of incubation of the Ag/Ag₂O NPs and silver nitrate precursor with normal renal epithelial Vero cells, it was found that their estimated safe doses on cell viability were 13.43 ± 1.63 µg/mL and 0.075 ± 0.001 µg/mL, respectively. This indicates that Ag/Ag₂O NPs ismore safe than silver nitrate source. However, at 100 µg/mL of Ag/Ag₂O NPs or silver nitrate caused death in Vero cells by 79.69% and 91.09%, respectively, as it is shown in Fig. 8a. Moreover, severe collapse in the normal spindle shape of silver nitrate-treated cells, at 25 µg/mL, confirmed its cytotoxicity in comparison to the normal morphology of Ag/Ag₂O NPs-treated cells and untreated control healthy cells (Fig. 8b)¹⁴. The lower cytotoxicity of the prepared NPs, at < 13 µg/mL, may be related to their particle size (≥ 40 nm), negatively particle charge and high agglomeration potential (Fig. 4a,b) which results in increasing their size thus decreasing their cellular uptake and diminishes ROS generation⁵⁹.

In support of this issue, Liu et al.⁶⁰ found that Ag NPs with size of 55 nm generated less ROS than 15 nm Ag NPs. Moreover, silver NPs’ tendency to agglomeration increases in culture medium⁶¹. Besides, based on the previous finding, corona formation which is mediated by adsorption of fetal bovine serum (FBS), from culture medium, on silver NPs, mainly limits their cytotoxicity via reducing their cellular uptake^59,62. All these factors contribute to minimize the cytotoxicity effect of Ag/Ag₂O NCs on normal cells. This higher safety of Ag/Ag₂O NPs on human normal cells (Fig. 1c) lends credibility to their biomedical applications compared to bulk silver nitrate.