Antimicrobial activity of synthesized graphene oxide-selenium nanocomposites: A mechanistic insight

Nanoparticles have recently gained interest as an anti-bacterial agent due to their large surface area/volume ratio and potential to compromise the integrity of bacterial cell membranes. Due to its versatility and anti-bacterial activity, graphene-based materials have drawn significant interest in biomedical applications. One of the greatest threats to life in the modern technological era is the pervasiveness of infectious diseases since bacteria cells are constantly updating themselves to resist antibiotics. In this presented study, GO-Se nanocomposite has been synthesized using polymer solution via a simple dispersion method. The structural and physicochemical properties of nanocomposite were investigated in detail. Staphylococcus aureus, Proteus vulgaris, and Bacillus subtilis bacterial strains were employed to study the anti-bacterial activity of GO-Se nanocomposite. The results show that the synthesized nanocomposites have good efficacy as an anti-bacterial agent. UV-vis spectroscopy, FTIR spectroscopy, HRTEM, XPS, and Raman spectroscopy were used to analyze the as-prepared GO and GO-Se nanocomposite.


Introduction
Graphene oxide (GO), chemically exfoliated from oxidized graphite, is a promising material in biomedicine and other fields. GO has outstanding amphiphilicity, surface-enhanced Raman scattering (SERS) property, aqueous processability, fluorescence quenching ability, and surface functional ability (Potts et al. 2011). The peculiar chemical structure of GO, which comprises sp 2 carbon character surrounded by sp 3 carbon character and the hydrophilic groups containing oxygen, is responsible for interesting features of the GO (Zhou et al. 2010). Many investigations have been reported on anti-bacterial coatings based on GO nanocomposites. The incorporation of GO into biocompatible polymers, which can provide anti-bacterial coatings, is one of the most promising applications (Fauzi et al. 2021). It is still a task to identify nanocomposites with outstanding anti-bacterial capabilities and low toxicity. Graphene oxide may be used in a variety of materials, including ceramics, polymers, and other elements. The anti-bacterial activity of a graphene oxide nanocomposite is enhanced by incorporating chalcogens (Niranjan et al. 2022). Se is a chalcogenide element with exceptional physical, chemical, and biological characteristics. It can act as an enzyme's catalytic center for biocatalysts. Se ions are steadily liberated from nanoparticles in aqueous system and absorbed via cell membranes, resulting in direct interactions with nucleic acid functional groups such as carboxyl (-COOH), mercapt (-SH), and amine (-NH) groups, as well as proteins (Shoeibi and Mashreghi 2017). These interactions have a wide range of consequences, including alterations in cell structure and abnormal enzyme activity, which disrupt normal physiological functions (Sibhghatulla et al. 2019). Se nanoparticles (Se NPs) have the potential to develop into innovative dietary supplements with enhanced biodegradability, reduced toxicity, and systemic elimination from the human body. Se NPs are selenium species with remarkable preventive and therapeutic capabilities with a wide range of biological activities, such as anti-bacterial and antifungal agents (Sakr et al. 2018). Se NPs have shown antibacterial viability toward both Gram-positive and Gram-negative bacteria (Boroumand et al. 2019). Shar et al. (2020) created rGO-Se nanocomposite for acetone gas detection, while Ahmed et al. (2020) used a stable rGO-Se nanocomposite as an electrode material for devices storing energy. Here, we present an easy approach for producing GO-Se nanocomposite. In addition, the Staphylococcus aureus, Proteus vulgaris, and Bacillus subtilis bacterial strains were used to test the GO-Se nanocomposite's anti-bacterial efficacy, which demonstrates these hybrid nanocomposites as excellent and biocompatible anti-bacterial agents. UV-Vis spectroscopy, FTIR, Raman spectroscopy (defects and polycrystalline form), HRTEM (morphology), and XPS (elemental composition) were used to study the GO-Se nanocomposite. Agar-well diffusion method was employed to examine the GO-Se nanocomposite's inhibitory zones for anti-bacterial characteristics.

Synthesis of graphene oxide
The GO was synthesized by modified Hummer's method which comprises graphite's oxidation, introducing oxygen molecules into pure carbon graphene (Hummers and Offeman 1958). Thus, the obtained graphene oxide was chemically exfoliated and oxidized graphite layer was further exfoliated through ultrasonication in the presence of aqueous solution of PDADMAC. Graphite powder (2 g) was added to 46 ml of concentrated H 2 SO 4 , followed by addition of 1 g NaNO 3 in an ice bath for 30 min. KMnO 4 (6 g) was combined with the above mixture, and the reaction temperature was maintained at 5 ℃. The mixture was continually stirred for 1 h before being heated to 35 ℃ and stirred for another 2 h. DI water (92 ml) was then added dropwise to the mixture, maintaining a high temperature at 98 ℃ for up to 20 min followed by addition of H 2 O 2 solution to terminate the oxidation procedure. The obtained product was repeatedly rinsed with DI water until the pH of the product became neutral by centrifuging at 4000 rpm. A suitable amount of solid product (graphite oxide) was mixed with 100 ml distilled water and sonicated for up to 40 min. The supernatant was then left undisturbed for a day, and the desired product obtained was graphene oxide (brown). PDADMAC solution (0.01%) was prepared by mixing 10 μl of PDADMAC solution in 100 ml of DI water. A certain amount of the graphene oxide solution was placed into a beaker and dispersed in 20 ml of PDADMAC solution and sonicated for 1 h. The mixture was kept for 24 h, undisturbed, and then centrifuged for 5-10 min at 4000 rpm. GO-PDADMAC nanocomposite solution was prepared as a resulting supernatant in this manner.

Synthesis of GO-selenium nanocomposite
Two hundred microliters of prepared GO-PDADMAC was taken into a beaker and 10 ml DI water was added to it followed by addition of different amounts of sodium selenosulfate ranging from 60 to 100 µl (2.8 to 4.7 µg of Na 2 SeSO 3 ). After this, 40 µl of 1 M HCl was added dropwise and a quick color change to bright orange ensured the formation of the nanocomposite.

Agar well diffusion assay
The agar well diffusion assay was conducted to investigate the anti-bacterial potential of the synthesized graphene oxideselenium nanocomposites against the pathogenic bacterial species, viz., Proteus vulgaris (MTCC 426), Bacillus subtilis (MTCC 441), and Staphylococcus aureus (MTCC 740). Thirty microliters of control/test sample was added in 5-mm wells on 24-h-old nutrient agar plate aseptically. Furthermore, the wells were sealed and kept for 20 min for solidification. The turbidity of bacterial test organism was adjusted with 0.5 McFarland solution. The plates were swabbed with bacterial strains and incubated at 37 °C overnight for 18 h. The anti-bacterial activity was determined by measuring the zone of inhibition (Magaldi et al. 2004;Valgas et al. 2007). Three independent experiments were performed and the average values of anti-bacterial activity were calculated. Furthermore, the minimum inhibitory concentration (MIC) of synthesized combination of graphene oxide-selenium nanocomposites exhibiting anti-bacterial activity against Staphylococcus aureus was investigated. Suspensions of the test organism were swabbed on the culture medium. Different concentrations of the nanocomposites containing varied concentrations of Na 2 SeSO 3 (2.8 to 4.7 µg) were added to the wells followed by incubation at 37 °C for 18 h. Three independent experiments were performed to determine the minimum inhibitory concentration.

Characterization
The prepared samples were characterized using a UV-vis spectrophotometer performed on the Shimadzu model UV-1900. FTIR analysis was done by using the FTIR spectrophotometer of Shimadzu Corp model 01,228. For imaging and diffraction analysis, the HR-TEM 300 kV high-resolution transmission electron microscope was employed. The triple Raman spectrometer T64000 from Horiba Scientific was used to obtain the Raman spectra. Nexsa Base was used for X-ray photoelectron spectroscopy by Thermo Fisher Scientific. Bacterial studies were done by agar well diffusion method. Figure 1a shows the UV-vis absorption spectra of GO and GO-Se nanocomposites. From UV-vis spectroscopic investigations, it is evident that the optical absorption of GO is dominated by the π-π* plasmon peak at 230 nm. The π-π* plasmon peaks rely on two sorts of conjugative impact; one is connected with nanometer-scale sp 2 groups, and the other emerges from connecting chromophore units, for example, C = C, C-O, and C = O bonds. The UV-vis retention power noticed for GO is brought about by the conjugative impact of chromophore accumulation, which impacts the π-π* plasmon peaks (Lai et al. 2012;Xiao et al. 2013). For the prepared GO-Se nanocomposites, the typical shoulder peak shifts to a shorter wavelength (blue-shift) at 269 nm compared with the GO which was attributed to the combination of Se with GO (Li et al. 2008). The absorbance at 230 nm vanishes when compared to GO, due to the combination of Se with GO. These findings also point to the fabrication of GO-Se nanocomposites being a success (Shahriary and Athawale 2014).

FTIR spectroscopy
The O-H, C = O, and C-O peaks in the FTIR spectra of GO-Se are located at 3412.17, 1744, and 1170 cm −1 , respectively. As shown in Fig. 1 b(i), hydrogen bonds between graphite and water molecules form as a result of a broad peak at 3412.17 cm −1 caused by the stretching and bending vibration of the hydroxyl group of water molecules absorbed on GO and the polar groups, particularly the surface hydroxyl groups, which explains graphene's hydrophilic property. C = O bond stretching vibrations are represented by the absorption bands at 1744 cm −1 . The peak at 2481 cm −1 can be attributed to atmospheric CO 2 (Al-Gaashani et al. 2019) . Alcohol and carboxylic acid stretch vibrations occur at 1170 and 1350 cm −1 , Fig. 1 a UV-Vis spectra of GO and GO-Se nanocomposite. b FTIR analysis of (i) GO and (ii) GO-Se nanocomposite respectively. The presence of groups containing oxygen indicates that the graphite has undergone oxidation. The peaks at 1744 and 1170 cm −1 are much weaker due to shielding effect of PDADMAC (Kochameshki et al. 2017). The FTIR spectrum of graphene oxide confirms its synthesis and also confirms that graphite was successfully oxidized. Figure 1 b(ii) shows the FT-IR spectrum of a GO-Se nanocomposite. Broad peaks at 3447 and 3360 cm −1 , which correspond to N-H and O-H bond stretching vibrations, and the bands at 3045 and 1699 cm −1 , which correspond to the C-H and C = O bond stretching vibrations, were also observed in the spectra (Park et al. 2008). The Se-C bond-type stretching vibrations appeared approximately at 958 cm −1 , confirming the selenium carbon bond's participation in the nanocomposite's synthesis.

Raman spectroscopy
As shown in Fig. 2a, the differences that occurred when GO was converted into GO-Se nanocomposite were examined using the Raman spectra of GO and GO-Se nanocomposite. The Raman spectra of graphitic materials show the G (sp 2 hybridized carbon) and D (defective sp 3 carbons bonds due to hydroxyl/epoxy bonds) bands at 1348 and 1596 cm −1 . The scattering and breathing patterns of E 2g phonon and A 1g photons of the sp 2 hybridized carbon atoms, respectively, give rise to the G and D modes (Ferrari 2007). There is a shift seen in these bands which may be due to addition of PDADMAC (Thirunavukkarasu et al. 2015). The optical E 2g phonons caused by stretching of sp 2 carbon bond pairs in chains and rings correspond to the G peak. The D peak signifies the breathing mode of the aromatic ring due to defect in the sample. While the peak at 1596 cm −1 correlates to the G band in the Raman spectra of GO, the D band is marked by the peak at 1348 cm −1 . Similar to this, the GO-Se G band and D band are seen at 1595 and 1346 cm −1 , respectively. The development of defects in graphene oxide is correlated with changes in band locations and intensities in GO-Se composite, which may be identified by the intensity ratio of D and G bands (I D /I G ) (Shar et al. 2020). The ratio I D /I G , which is nearly zero in well-organized graphene materials, is a measure of structural flaws in GO which is represented as the sp 3 /sp 2 carbon ratio. A slight increase in intensity ratio indicates the smaller degree of defects and disorders by the Se incorporation in the GO sheet (Xiao et al. 2013). This ratio was different in the GO-Se composite (0.92) than in the GO (0.96), showing that the integration of Se generated more faults and disorders in the GO sheets (Shar et al. 2020). A new Raman peak at 253 cm −1 (Fig. 2a) has also appeared, confirming the effective synthesis of GO-Se. During the oxidation process, various functional groups with sp 3 hybridized carbon and defects may be introduced along with a few layers of graphene oxide (Xiao et al. 2013). The average particle size of GO was 4.5 nm as shown in the inset of Fig. 2b. The particle size of GO-Se nanocomposite was 12.1 nm as shown in the inset of Fig. 2d. It was seen that the particle size increased after the addition of Se. On the surface of GO, dark-contrast, smaller spherical Se nanoparticles with a narrow size range are dispersed. The nanospheres were clearly constructed by huge amounts of Se nanoparticles, as seen in the inset of Fig. 2c. Furthermore, in the HRTEM image, patterns with a d-spacing of 0.36 nm correlate to the planes of Se nanoparticles (Changhe et al. 2013). Figure 2 d depicts the SAED pattern of GO-Se nanocomposite describing the noninterference of Se nanoparticles on the polycrystallinity of GO sheets. A polycrystalline structure of graphene oxide is shown by the selected area of electron diffraction (SAED) pattern (Fig. S1, Supplementary information). All of the foregoing data show that GO-Se nanocomposites were produced using a simple synthetic method (Russel et al. 2018).

XPS analysis
The XPS technique was used to examine the elemental profiling of the GO-Se composite material (Fig. 3). The presence of O, C, and Se in the nanocomposite was indicated by the binding energies of 531.47, 234.38, and 55.40 eV attributed to the characteristic peaks of O 1s, C 1s, and Se 3d as depicted in Fig. 3a-c. The spectral band of O 1s of GO-Se can be divided into two peaks at 535.90 and 531.47 eV corresponding to C-OH and C-O-C. The spectra of C 1s of GO-Se can be divided into three peaks at 288.23, 286.26, and 284.38 eV attributed to following chemically shifted groups of sp 3 carbon, and the carbon of carbonyl group (C = O), C-OH of carboxyl group, and C = C of GO. When compared to C 1s spectra of GO, GO-Se spectra exhibit a minor shift in peak position of C 1s, which is caused by the interaction of Se between GO (Ren et al. 2013). The Se peaks are deconvoluted in the Se 3d 5/2 and Se 3d 3/2 peaks with optimum binding energies of approximately 49.93 and 55.40 eV in the Se 3d spectrum (Boroumand et al. 2019). The survey spectra of GO and GO-Se samples are shown in Fig. S2, Supplementary information.

Anti-bacterial activity
The anti-bacterial activity of the GO-Se nanocomposites was examined using the agar well diffusion method for calculating the zone of inhibition against three pathogenic species of bacteria: Proteus vulgaris, Bacillus subtilis, and Staphylococcus aureus (Paul and Mittal 2012). Five groups with different concentrations of Se in GO-Se nanocomposite and a specimen blank, A (80 μl of Se precursor), B (60 μl of Se precursor), C (70 μl of Se precursor), D (90 μl of Se precursor), and E (100 μl of Se precursor) were evaluated for their anti-bacterial action (Table S2, Supplementary information). The diameters of the inhibition zones were measured, and the average value for each was computed. All of these tests were carried out in duplicates. Anti-bacterial activity was maximum in sample E exhibiting a dose-dependent increment in the anti-bacterial activity. Both Gram-positive and Gram-negative bacteria strains were clearly inhibited by the anti-bacterial activity of GO-Se nanocomposite at increasing doses. Gram-positive bacteria were more affected by the GO-Se nanocomposite than Gramnegative bacteria, which appeared to be more resistant to the prepared nanocomposite as shown in Fig. 4. The highest inhibition zone was created by sample E against Staphylococcus aureus as shown in Fig. 4c. Furthermore, determination of the minimum inhibitory concentration of the GO-Se combination as nanocomposite was performed following agar well diffusion method. MIC was obtained as sample B with 60 μl of Se precursor (Se is 350 µM and GO is 2160 µg/ml in combination as nanocomposite) against Staphylococcus aureus. The smaller nanoparticles have superior anti-bacterial action in general owing to increased surface area that results in crossing the membrane permeability barrier. The cell membrane features nanoscale holes that allow smaller nanoparticles to easily permeate the cell and access the bacteria's nuclear content (reverse osmosis) (Krishnamoorthy et al. 2012). Bacteria are classified as Gram positive or Gram negative relying on structural differences in their cell walls. There is a substantial coating of murein in the cell wall of Grampositive bacteria and a polysaccharide coat made up of teichuronic acids and teichoic. Gram-negative bacteria's cell wall, on the other hand, is very complicated with thin layers including lipopolysaccharides, lipoproteins, an outer membrane, and murein. This could mainly be the reason Gram-positive bacteria are more affected by the nanocomposite (Neto et al. 2015). There could be two possible explanations for this as represented in Fig. 5. First, Gram-negative bacteria's outer membrane contains numerous tiny channels consisting of porins that may assist impede nanoparticle entry, making them more difficult to suppress as compared to Gram-positive category of bacteria. Second, Gram-positive bacteria (spherical) are smaller than Gram-negative bacteria (rod), which may explain why they come into closer contact with the nanoparticles and exhibit more potent anti-bacterial activity. The oxidizing nature of GO can also be a cause of cellular damage in bacteria exposed to it (Krishnamoorthy et al. 2012). Graphene is a known prooxidant inducing dosedependent oxidative stress according to Ou et al. (2016). The combination of GO-Se provides a redox entity. The oxidative stress of GO may contribute to the bactericidal activity; on the other hand, selenium acts as an antioxidant to minimize the GO-mediated oxidative stress making GO-Se nanocomposite a potential prospect.

Conclusion
Graphene oxide-based nanocomposites have attracted expanded interest lately in an assortment of fields, including the counteraction of antimicrobial resistance for microbes P. vulgaris, B. subtilis, and S. aureus. Graphene oxide-selenium nanocomposite was successfully synthesized by simple dispersion method. The prepared PDAD-MAC-stabilized GO-Se nanocomposite is seen to have excellent anti-bacterial characteristics. The agar well diffusion method was used to assess the GO-Se nanocomposites' anti-bacterial efficacy against several bacterial strains. It is seen that Gram-positive bacteria show better antibacterial results than Gram-negative bacteria. The UV-vis, FTIR, Raman spectroscopy, and HRTEM confirmed the fabrication of selenium nanoparticles on GO sheets. The results indicated that the nanocomposite was made up of Se nanoparticles enwrapped within GO sheets or scattered on the GO sheets. Hence, this nanocomposite will be a promising material for broad-spectrum applications owing to its potent anti-bacterial properties.