Cotton fabric coated with graphene-based silver nanoparticles: synthesis, modification, and antibacterial activity

In this study, reduced graphene oxide with immobilized silver nanoparticles cotton fabric (Ag/rGO/cotton) was produced by the dip-coating cotton in silver immobilizing onto graphene oxide (Ag/GO) suspension to prepared Ag/GO/cotton material followed by the addition of vitamin C (VC) as an environmentally friendly reducing agent. The characteristics of Ag/GO and modified cotton were investigated by Fourier transform infrared spectroscopy, X-ray diffraction, Raman spectroscopy, transmission electron microscopy, scanning electron microscope, X-ray photoelectron spectroscopy, and energy-dispersive X-ray spectroscopy. Silver nanoparticles (AgNPs) were uniformly distributed on the surface of graphene oxide (GO) sheets with an average size of 10–15 nm, while the cotton surface was evenly covered by Ag/rGO. The zone of inhibition against Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli) bacteria indicated that Ag/rGO/cotton possessed the highest antibacterial activity when compared to other modified cotton. Moreover, the Ag/rGO/cotton also exhibited effective hydrophobicity with a wetting angle of 103.85° ± 0.75°, which supported the prevention of bacterial infection and adherent on the cotton surface. To confirm the low cytotoxic property of Ag/rGO/cotton for human use, the cell viability of HepG2, A549, and Hek293 cell lines were evaluated when contacted with the material, while the low amount of leached Ag+ from Ag/rGO/cotton was under the accepted limit. All results of the study confirmed that Ag/rGO/cotton possesses significant potential for several antibacterial applications such as protective equipment.


Introduction
Cotton is a natural cellulosic fiber with structures consisting of a cellulose polymer with repeated glucose residues that make it breathable, soft, and biodegradable, making it useful in many industries (Gao et al. 2021;Lam et al. 2019). Thanks to a hydrophilic structure, cotton shows a superior adsorption capacity of 24-27 times its mass (Hosseini Ravandi and Valizadeh 2011). However, the hydrophilic property of cotton fabric can generate a humid environment and promote the growth of bacteria that cause unpleasant odors, allergies, and infections for consumers (Shahid-ul-Islam and Butola 2019). These drawbacks of the cotton fabric have been studied to resolve and ensure the best safety for the users. Moreover, the manufacturing development leads to an increasingly polluted environment with the strong growth of pathogenic organisms present in the soil, water, air, or on the surface of any tools or equipment. These cause many progressive diseases such as infections, respiratory infections, and skin diseases. The user's health may be threatened if the cotton layer is not resistant to bacteria (Pullangott et al. 2021;Zhong et al. 2020). Microbes or germs growing on the face have a harmful influence not only on the materials but also on the user. As a result, the use of antibacterial materials is deemed significant attention in the medical area to reduce the bacterial population and lessen pathogenic illnesses produced by face mask textile materials since its bacterial resistance boosts product quality and durability (Pullangott et al. 2021;Zhong et al. 2020). Therefore, antibacterial materials have been studied and developed, which are confirmed to be essential in various fields such as food storage, clothes, wastewater treatment, medical devices, etc. (Huang et al. 2020).
In nanotechnology, silver nanoparticles (AgNPs) have been widely used, especially in the antibacterial field, to reduce the usage of antibiotics as not being necessary. Nevertheless, AgNPs have many disadvantages such as agglomeration due to the high surface energy, which adversely affects the antibacterial activity (Guzmán et al. 2019). Furthermore, AgNPs can cause several health problems for humans such as organ toxicity, argyria, neurological symptoms, etc. (AshaRani et al. 2009). Therefore, stabilizer agents are needed to control the size, shape, and cytotoxicity of AgNPs. On the other hand, graphene oxide (GO) has been known as a potential material for the combination with AgNPs to synthesize stable nanocomposites without compromising the antibacterial property. Thanks to the presence of many oxygen-functionalized groups, GO can be easily dispersed in water and forms different functional groups on fabric surfaces simultaneously (Chauhan et al. 2020). Therefore, the synthesized silver immobilized on graphene oxide (Ag/GO) was reported to overcome the disadvantages of both AgNPs and GO, which can prevent agglomeration and enhance antibacterial resistance as opposed to pre-materials (Mariadoss et al. 2020;Shao et al. 2015). Previous research coated Ag/GO onto cotton fabric (Ag/GO/cotton) to prevent bacteria growth (Diep et al. 2020). However, Ag/GO/cotton is hydrophilic, which endows favorable conditions for the growth of bacteria. Besides, Ag/GO/cotton can stick to wounds during treatment, so they would destroy newly formed cells and create slow-healing wounds (Khalid et al. 2017;Zeng et al. 2018).
Nowadays, there have been many methods for material modification such as chemical vapor deposition, sol-gel, and coating methods, which may possess toxicity, use toxic chemicals pathway and also affect Ag/GO antibacterial activity Lu et al. 2019). Thus, the use of a chemical reduction method (usually with d-glucose, vitamin C, etc.) for treating the Ag/GO is necessary, affirming the simple and low-cost performance due to the direct reduction of the existing substrate without any toxic reagents (Tang and Yan 2017). As a result, the cotton can be treated with silver immobilized on reduced graphene oxide (Ag/rGO) with a moderate hydrophobicity. This can prevent the bacteria come into contact with cotton.
In this study, Ag/GO was synthesized by the insitu method before using for the dip-coating process to form Ag/GO/cotton and chemically reduced to Ag/ rGO/cotton by vitamin C (VC). The effect of reduction conditions including VC:Ag/GO mass ratio, reducing temperature, and reducing time on the antibacterial activity of the cotton was also investigated. The bioactivities of cotton samples were evaluated not only to determine the antibacterial property against the Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli) bacteria via agar disk diffusion test, but also to assess the cytotoxic feature with HepG2, A549 cancer cell lines, and Hek 293 human normal cell line. The durability of the cotton was also investigated under different conditions such as colorfastness to washing at 40 °C, colorfastness to alkaline, acidic of perspiration, and hot pressing at 150 °C.

Synthesis of Ag/GO
Graphene oxide (GO) was prepared via an improved Hummers' method (Dat et al. 2021(Dat et al. , 2022. Ag/GO was synthesized by an in-situ method with d-glucose as a reducing agent. 0.5 g of GO was dispersed in 100 mL of distilled water to obtain solution A. Then, the preparation of solution B was carried out by dissolving 0.5 g of AgNO 3 in 80 mL of distilled water, as well NH 4 OH was added slowly to the solution until a dark brown precipitate disappeared and obtained a clear solution. After that, solution B was added to solution A and the mixture was ultrasonicated for about 10 min. Then, 20 mL d-glucose was added to the prepared mixture and stirred for 1 h at 60 °C. After that, this mixture was cooled down to room temperature and rinsed with distilled water. Finally, Ag/GO was collected after drying at 50 °C.

Fabrication of Ag/GO/cotton
Ag/GO/cotton was produced by the dip-coating method (Diep et al. 2020). First, the cotton fabric (40 × 40 cm 2 ) was cleaned with 50 ml NaOH 1 M solution and sonicated for 30 min. It was then rinsed with the distilled water until pH 7 and dried until constant weight. Finally, the cotton was dipped quickly into Ag/GO solution at room temperature (25 °C) as shown in Fig. 1. The concentrations of GO suspension were varied to 160, 280, 400, 520, and 640 mg/L. For each concentration, the number of dips was changed to 1,2,3,4,5,6,7,8,9, and 10 times, respectively.

Fabrication of Ag/rGO/cotton
The obtained Ag/GO/cotton was reduced by the chemical reduction method with eco-friendly reducing agent VC to synthesize Ag/rGO/cotton as shown in Fig. 1. Briefly, after dipping the cotton fabric into Ag/GO solution with different VC:Ag/GO mass ratios, the fabric, and VC solution were sealed in an autoclave with survey temperature and time. During the reaction process, the temperature was monitored and maintained by the furnace controller. Finally, the Ag/rGO/cotton was washed and dried at 60 °C. The reducing conditions were analyzed such as: VC:Ag/ GO mass ratios (0:1, 1:1, 2:1, 3:1, 4:1, and 5:1), Fig. 1 Preparation of Ag/GO/cotton and Ag/rGO/cotton reducing temperature (100, 120, 140, 160, 180, and 200 °C), and reducing time (20, 40, 60, 80, 100, and 120 min).

Characterization
Fourier-transform infrared spectroscopy (FTIR) (Alpha-E, Bruker Optik GmbH, Ettlingen, Germany) was used for determining functional groups on the surface of Ag/GO at wavenumber in the range of 4000 to 500 cm −1 . X-ray diffraction (XRD) was used to observe the structure of Ag/GO by CuKα radiation (λ = 0.154 nm) in the scope of 5°-80° (D2 Phaser, Brucker, Germany). Raman spectra were performed using a LabRam micro-Raman spectrometer with a wavelength of 632 nm (He-Ne-laser). Transmission electron microscopy (TEM) (S-4800, Hitachi, Japan) was determined at an accelerating voltage of 100 kV. A scanning electron microscope (SEM) (Hitachi S-4800, Japan) was used to reflect the observation of the surface structures of the material. X-ray photoelectron spectroscopy (XPS) was carried out using the Quantum 2000 Scanning ESC Microprobe, Physical Electronics (Chungnam National University, South Korea) with a beam of monochromatic Al Kα X-ray. Energy-dispersive X-ray spectroscopy (EDS) (Jeol-JMS 6490, Japan) was also used to investigate the composition of elements C, O, and Ag in the material. Static water contact angle (OCA-20, DataPhysics-Germany, scale 0.7-4.5) was used for investigating the water wettability of Ag/GO/cotton and Ag/rGO/cotton.

Antibacterial activity
The antibacterial activity of cotton and Ag nanomaterials were determined via the diameter zone of inhibition against three strains of Gram-positive (S. aureus) and Gram-negative (P. aeruginosa, E.coli) bacteria were selected to evaluate their antibacterial ability. First, bacteria were cultivated and proliferated in the Mueller-Hinton Agar medium. Then, the Ag/GO/cotton and Ag/rGO/cotton (1 × 1 cm 2 ) were placed on the surface of the jelly discs and incubated at 37 °C. After 24 h, the antibacterial ability of the cotton is evaluated by measuring the bacterial inhibition zone (Balouiri et al. 2016).
The impact of Ag/GO/cotton, Ag/rGO/cotton, and pristine cotton on bacterial morphology was examined by SEM. The fabric was treated with fixed E. coli density and incubated for 24 h at 37 °C. The bacterial cells were centrifuged and then fixed within modified Karnovsky's fixative (2% glutaraldehyde (v/v) and 2% paraformaldehyde (v/v) in 0.05 M sodium cacodylate buffer pH 7.2) for 2 h at 4 °C followed by post-fixing in 1% osmium tetroxide in 0.05 M sodium cacodylate buffer (pH 7.2) and washing with sterile PBS and dehydrating in a series of ethanol (20, 40, 60, 80, 95, 100, 100, and 100% for 10 min each). The samples were treated with hexamethyldisilazane and dried overnight and then analyzed on a SEM.
The release of Ag + from Ag/rGO/cotton was measured by inductively coupled plasma-mass spectrometry (7700 × ICP-MS, Agilent Technologies) with a detection limit of 0.0005 ppb. 300 mg Ag/rGO/cotton were incubated in 50 mL solution with different pH (6, 7, 8, 9, and 10) and different exposure time (20, 40, 60, 80, 100, and 120 h) at pH 7. The materials were then removed from the solution and the concentration of Ag + ions in the solution was determined by ICP-MS. Prior to the analysis, samples were acidified using 0.1 N HNO 3 to ensure the availability of soluble Ag + ions.

Cytotoxicity assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cell viability of HepG2, A549 cancerous cells, and Hek 293 human normal cell line by exposing this cell to Ag/GO at different concentrations (12.5, 25, and 50 μg/mL). Briefly, the cells were seeded at a density of 3 × 10 4 cells/well and incubated with Ag/rGO at 37 °C. Subsequently, the treated and non-treated cells were fixed by adding 10 μL of MTT solution (5 mg/ mL) to each well after 24 h incubation. The plate was incubated for 4 h at 37 °C in a CO 2 environment, followed by removing the medium, washing with PBS, and adding 100 μL of dimethyl sulfoxide (DMSO) per well. The cell viability was evaluated by recording the intensity of formazan crystals at 540 nm using an enzyme-linked immuno-absorbent assay (ELISA) plate reader.

Durability tests of Ag/GO/cotton and Ag/rGO/cotton
The durability of the fabric (Ag/GO/cotton and Ag/ rGO/cotton) was assessed under various conditions such as colorfastness to washing at 40 °C, colorfastness to alkali, to the acidity of perspiration, and to hot pressing at 150 °C. The color change of the cotton samples was evaluated by comparison to the corresponding grayscale after the samples were held at standard conditions for four hours.

Characterization of Ag/GO
The functional groups of GO and Ag/GO were identified through FTIR spectra as shown in Fig. 2a respectively. In addition, the 1637 cm −1 peak was attributed to aromatic bending vibration of unsaturated C = C of the non-oxidized graphitic domain (Kumar and Chandra Srivastava 2018). As for Ag/ GO, it had a similar pattern of FTIR spectrum to GO but the intensities of diffraction peaks declined due to the linkage between AgNPs and GO sheets (Zheng et al. 2012). Figure 2b shows the XRD pattern of the Ag/GO. Accordingly, four 2θ values of Ag/GO were observed at 38°, 44°, 64°, and 77°, representing characteristic diffraction peaks of AgNPs and well-matched with standard peaks in JCPDS file No. 04-0783 (Dat et al. 2021(Dat et al. , 2022. The diffraction peak at 2θ = 9.5° was recorded, which reflects the (002) plane of GO (Shao et al. 2015). In Ag/GO, this peak disappeared due to the AgNPs formulated on the GO surface, preventing the restacking of GO sheets.
As shown in Fig. 2c, Raman spectra of GO and Ag/ GO are characterized by two bands: D and G, which were observed at approximately 1330 and 1595 cm -1 , respectively. The D band is assigned to the breathing mode of K-point phonons with A 1g symmetry, and the G band is ascribed to the tangential stretching mode of the E 2g phonon of sp 2 carbon atoms corresponding to the defects in the graphitic structure and sp 2 bonding carbon in GO structure (Dat et al. 2021(Dat et al. , 2022. After anchoring AgNPs onto the GO surface, the D and G band values increase as a result of chemical bond formation and intercalation of the GO surface and AgNPs (Pimenta et al. 2007).
The TEM images were used to analyze the morphologies of GO and Ag/GO as shown in Fig. 3a, b, respectively. The results demonstrated that sphericallike AgNPs were uniformly distributed on the GO nanosheets-dark thin films with an average size of 10-15 nm. The results suggested that the addition of GO as a stability agent, was vitally crucial as it played an important role in stabilizing the formation of AgNPs onto GO, preventing the agglomeration of those particles.
According to the EDS spectrum in Fig. 3c, the percentage of C, O, and Ag of the material were 23.06, 23.40, and 54.54 wt.%, respectively. This result did not change too much compared to the precursor ratio (1:1 AgNO 3 :GO mass ratio). There were peaks at 3 to 4 keV indicating the presence of Ag, and two small impurity peaks at 1 and 2.1 keV possibly corresponding to Na and Au in the analyzed equipment, respectively (Kumari et al. 2020). Besides, the SEM images were used to investigate the morphological surface of GO and Ag/GO as shown in Fig. 3d-f. It was indicated that, as opposed to the form of GO sheets having a lamellar structure that was bonded closely on the surface of Ag/GO, while there were many folds with rougher surfaces than that of GO due to AgNPs evenly distributed on the GO sheets. The images revealed the presence of an ultrafine layer, which forms a wavelike structure when they were stacked together. Consequently, the results affirm the uniform presence of Ag on the GO structure. The TEM images were used to analyze the morphologies of GO and Ag/GO as shown in Fig. 3a, b, respectively. The results demonstrated that spherical-like AgNPs were uniformly distributed on the GO nanosheets-dark thin films with an average size of 10-15 nm. The results suggested that the addition of GO as a stability agent, was vitally crucial as it played an important role in stabilizing the formation of AgNPs onto GO, preventing the agglomeration of those particles (Shao et al. 2015).

Characterization and antibacterial activity of Ag/GO/ cotton
The influence of concentration and number of dips on the mass loss of Ag/GO is shown in Fig. 4a. The test results showed that when the Ag/GO concentration increased, the amount of material attached to the cotton increased. After 10 dip coatings, the amount of material adhering to the cotton still tended to increase in the concentration range of 160 to 400 mg/L. Meanwhile, the mass of cotton fabric virtually remained the same after 6 times of dipping coating at a concentration of 520 mg/L, indicating that raising the concentration of Ag/GO suspension would reduce the number of dips, thereby decreasing the period of fabrication time. When the Ag/GO concentration was raised to 640 mg/L, the quantity of Ag/GO clinging to the fabric began to develop slowly by the third dipcoating, and by the fifth dip, the amount of Ag/GO clinging to the cotton had increased insignificantly. Based on the foregoing findings, 640 mg/L was the appropriate concentration for the Ag/GO dip-coating  Fig. 4b, c presents the comparison of antibacterial activity on Ag/GO/cotton samples fabricated after dip-coated 10, 5, and 3 times. Test results revealed that the antibacterial capacity of all samples after different dip coatings times was identical. This could be explained by the fact that the quantity of Ag/ GO in samples after a dip-coated 10, 5, and 3 times was not substantially different. Therefore, the number of suitable dip coating was determined to be 3 times at an Ag/GO suspension concentration of 640 mg/L.
The surface morphology of the Ag/GO/cotton samples was analyzed by SEM images. Before Ag/ GO dip-coating, the surface of the fabric had an interlocking fiber structure (Fig. 5a, b) and the surface of the fibers was smooth (Fig. 5c). When dipped with Ag/GO material, the surface of the fabrics still maintained the original fiber structure, suggesting dipcoating with Ag/GO did not affect the fabric structure (Fig. 5d, e). However, it is interesting to observe that the surface of each fabric changed significantly. The fiber surface became rougher due to being covered by Ag/GO sheets. More importantly, the presence of small white spots is uniformly distributed on the surface of the fabric fibers, indicating the presence of AgNPs, as shown in Fig. 5f.

Characterization and antibacterial activity of Ag/ rGO/cotton
The effect of reducing conditions on the antibacterial activity of Ag/rGO/cotton is depicted in Fig. 6. When increasing the reducing temperature from 100 to 200 °C, the mass loss gradually increased. The cotton samples after being fabricated at 100 and 120 °C, the mass loss increased by 5.20 and 5.89%, respectively. It could be seen that 140 °C was the appropriate temperature for GO to be reduced to rGO with a mass loss of 7.08%. However, the cotton samples after reduction at a temperature of 160 °C onwards appeared material peeling on the cotton surface with the mass loss also increasing, corresponding to the values of 7.32, 9.47, and 12.77%, respectively. When the reducing temperature was higher than 200 °C, the cotton sample began to show signs of decomposition. It is worth noticing that not only GO was reduced to rGO, but the cotton was also decomposed leading to an increase in the mass loss of the cotton at high temperatures. Besides, the antibacterial results of the Ag/rGO/cotton samples at the reducing temperature investigation in Fig. 6a showed that in both agar plates, samples reduced at 140 °C exhibited the best antibacterial ability. Although the inhibitory zone of the samples reduced at 160, 180, and 200 °C did not change significantly, there was material peeling on the cotton surface of these samples. Therefore, the cotton fabric reduced to 140 °C was suitable and had a higher antibacterial ability than the remaining samples.
The effect of the VC:Ag/GO ratio on the mass loss in the reduction process was investigated and illustrated in Fig. 6b. The results revealed that the mass loss went up rapidly when the ratio between Ag/GO on the fabric surface and the reducing agent increased. When increasing the mass ratio of VC:Ag/ GO from 0:1 to 5:1, the mass loss increased sharply from 5.49 to 6.76%, respectively. When the mass ratio of VC:Ag/GO increased from 2:1 to 5:1, the mass loss increased insignificantly with values of 6.81 to 7.06%, respectively. According to the results, the ratio of 1:1 was exactly adequate for reducing GO to rGO; however, if the VC:Ag/GO mass ratio continued to rise, the mass loss would not change significantly, while the use of excess residual chemical would increase the cost of the synthesis process. Therefore, the mass ratio of VC:Ag/GO 1:1 was chosen to conduct the following experiments. The antibacterial results against two bacteria strains of the Ag/rGO/cotton samples for reducing agent ratio investigation in Fig. 6b showed  , c), and Ag/rGO/cotton (c, f) samples. Antibacterial activity of cotton samples against E. coli using inhibition zone method (g). The contact angle measurement results of Ag/GO/cotton (h) and Ag/rGO/cotton (i) that in both agar plates, the samples were prepared in the condition of reducing agent ratio 1:1 to 5:1 exhibited a minor antibacterial zone difference and were larger than the sample reduced at 0:1 ratio.
The influence of reducing time on the mass loss in the reduction process is shown in Fig. 6c. When the reducing time increased from 20 to 40 min, the mass loss increased from 6.23 to 7.08% respectively. When the reaction time increased from 60 to 100 min, mass loss increased from 7.62 to 7.93%, respectively, these values increased but did not differ significantly. Even after 100 min, it was inadequate to convert all GO to rGO, thus the reaction time needs to be increased. However, when the reducing time was 120 min, the mass loss peaked at 8.45%. As a result, Ag/rGO/cotton sample prepared at 120 min has the largest inhibition zone out of the others. After evaluating the antibacterial capacity of Ag/rGO/cotton at different reduction conditions, the sample with the highest antibacterial ability was reduced at a temperature of 140 °C, VC:Ag/GO mass ratio of 1:1, and reaction time of 120 min.
The antibacterial activity was re-evaluated on E. coli bacteria. In Fig. 7g, the cotton coated with Ag/ GO and Ag/rGO showed much better efficiencies than rGO and GO samples, which confirmed that AgNPs enhanced the ability to inhibit the growth of bacteria. Moreover, SEM images of the bacterial cells were recorded, to observe the change in the morphology of the bacteria after exposure to Ag/GO, Ag/rGO, and pristine cotton samples. As shown in Fig. 7a, d, bacterial cells of E. coli were viable and had a smooth and integral cell wall structure when adsorbed and adhered onto the pristine cotton surface. When bacterial cells were exposed to Ag/GO/cotton, they experienced extensive cell lysis and severely damaged surface, indicating cell death (Fig. 7b, e). In contrast, fragmentation of the cells was not noticed after interaction with Ag/rGO/cotton (Fig. 7c, f). For better illustration, water contact angles of Ag/GO and Ag/ rGO cotton were investigated. The spherical droplet was repelled and could not penetrate into the treated cotton after reducing GO to rGO, which could be due to its hydrophobic property. The water contact angles of Ag/GO and Ag/rGO cotton as shown in  Fig. 7h, i, respectively, also confirmed the hydrophobic property. The left and right contact angles were: 103.1° and 104.6°, respectively, less than 150°, which showed an effective hydrophobicity of the Ag/rGO/ cotton. As a result, the cotton became hydrophobic and prevented bacteria from adhering to the cotton surface, which is consistent with the previous study .
Besides, Ag/rGO/cotton showed somewhat greater bactericidal activity than Ag/GO/cotton, albeit the difference was not significant. This could be explained by the fact that when in direct cell contact, highly conductive rGO mediated stronger oxidative stress than lower conductive GO (Kumar et al. 2021;Liu et al. 2011). Although rGO was not as well dispersed in water as GO, in this study Ag/rGO/cotton was only partially reduced, so Ag/rGO clinging to the fabric surface did not completely lose the water absorption properties of cotton fabrics, thereby allowing to contact with bacteria to cause physical damage to cell membranes and oxidative stress. The results of this antibacterial evaluation were also in good agreement with the results of a previous study published on the antibacterial effectiveness of fabric dipped in the following order: rGO-Ag > Ag > GO > Ag-GO (Farouk et al. 2020). The SEM images of Ag/GO and Ag/rGO cotton are illustrated in Fig. 8. The results showed that there were no significant differences between Ag/ GO and Ag/rGO cotton, indicating over the reduction process did not affect the fabric structure or surface.
The distribution of C, O, and Ag elements of the Ag/rGO/cotton sample after surface modification was presented in Fig. 9a-d. The results showed that the material was still evenly distributed on the surface of the cotton fabric and did not affect by the reduction process. After reduction, the % mass of elements altered, with the % O decreasing from 37.65 to 35.22%, proving the oxygen-containing functional groups in the structure of GO had been partially reduced as shown in Fig. 9e. While the % Ag did not change significantly, which could be explained by the fact that the Ag/rGO has bonded strongly with the fabric surface, hence there was less Ag loss during the reduction process.  Fig. 10 a XPS spectra of pristine, Ag/GO/cotton, Ag/rGO/cotton; C1s core-level XPS spectrum of b Ag/GO/cotton, c Ag/rGO/cotton; d Ag 3d core-level XPS spectrum of Ag/rGO/cotton; O1s core-level XPS spectrum of e Ag/GO/cotton, f Ag/rGO/cotton XPS spectra of Ag/GO/cotton and Ag/rGO/cotton are presented in Fig. 10. In Fig. 10a, it is evident that the intensity of O1s in Ag/rGO/cotton showed lower than that of Ag/GO/cotton while the height of C1s peak likely remained in both two samples. Besides, two new peaks can be seen in 368.3 and 374.2 eV corresponding to Ag 3d 5/2 and Ag 3d 3/2 , respectively. In Fig. 10b, c, the C1s spectra of two cotton samples have the appearance at 284.1, 286.4, and 287.5 eV, which match with characteristics of the bonds of C-C, C-O, and C = O, respectively (Dat et al. 2021(Dat et al. , 2022. In addition, it is obvious that in Ag/rGO/cotton the intensity of two peaks at 286.4 and 287.5 eV was lower than these peaks of Ag/GO/cotton due to the decreasing content of C-O, and C = O after reduction by chemical reduction. To further demonstrate the reduction of GO to rGO, the deconvolution of O1s of Ag/GO/cotton and its reduced derivative is provided. As demonstrated in Fig. 10e, f, absorption peak locates at 529.5, 531.8, and 533.6 eV, corresponding to the bonds of C = O, C-O, and O-C = O, indicates the grafting of Ag/GO active layers onto the cotton fabric. After being reduced with VC, their witnesses a decrease in the intensity of O-C and O-C = O, validating that Ag/rGO has been. Furthermore, it is noteworthy that the absorption peak intensity of O = C increases. During the reduction of VC, a small portion of Ag/GO sheets have been peeled off from the fabrics, creating an exposed area with the cotton fabric. As a result, the X-ray, emitted from the source, has scanned this exposed region when conducting the XPS analysis of the reduced cotton fabrics, giving out an increased intensity of O = C deconvolution peaks due to the sheer presence of the fibers in the sample. This result confirms the efficiency of the method that improves the hydrophobicity of cotton fabric. The Ag connected to cotton fabric is shown in Fig. 10d which is found at 368.3 eV (Ag 3d 5/2 ) and 374.2 eV (Ag 3d 3/2 ) (Dat et al. 2021(Dat et al. , 2022. The presence of these peaks proves that AgNPs are successfully coated on the cotton fabric and after reducing, the intensity of these peaks are likely indistinguishable. Together with the aforementioned results, Ag/rGO/cotton fabric was successfully prepared.
Cytotoxicity and silver ion release from Ag/rGO/ cotton It was evident that the nano-scale of AgNPs not only exhibited antibacterial activity but also posed cytotoxicity toward human cell lines. The cell viabilities of HepG2, A549, and Hek 293, when exposed to various Ag/rGO concentrations, are summarized in Table 1. Even when a large dose of Ag/rGO of 50 µg/mL was used, more than half of the cells for both normal and cancer cell line remain. The obtained results show that AgNPs show low toxicity toward both normal and cancer cell lines. To further validate the actual toxicity of the nanocomposite, the leaching of Ag + was assessed.
As demonstrated in Fig. 11a, the release of Ag + was generally facilitated by the pH of the media, which could inhibit the growth of human cell lines (Table 1). It is noteworthy that the peak release of Ag + at pH 10 was much lower than the studied concentration, revealing that the leached Ag + posed little threat to the human cell line. Another factor that should be taken into account is the release of Ag + in long-term exposure. The release of Ag + after a total of 5 days is monitored. As can be seen from Fig. 11b, the graph saw an increase in the leaching rate of Ag + when the material was submerged in media from 20 to 120 h, while the value showed equilibrium values between 40 to 80 h. It could be explained that the surface of the nanomaterial was covered by subvalent Ag with a low oxidation state. The leftover Ag was partially oxidized after the primary Ag had been released and become fully oxidized, resulting in the production of a second species with a higher oxidation state (Molleman and Hiemstra 2017). Although the Ag + concentration leached after 5 days was significantly higher, it was still lower than the studied cytotoxicity concentration, implying that long-term exposure to the nanocomposite should pose a little impact on humans. Moreover, due to the hydrophobic property as investigated in Fig. 7h, i, which may reduce the full contact of AgNPs and medium and hence less amount of Ag + present in the solution. The results also confirmed the small amount of AgNPs released into the media, which was below the acceptable limit (Silver in Drinking-Water Background Document for Development of WHO Guidelines for Drinking-Water Quality 2003), due to the combination of rGO as a stabilizer (Mariadoss et al. 2020;Shao et al. 2015).

Durability evaluation
The adhesion of Ag/GO and Ag/rGO sheets to the cotton was evaluated by determining the colorfastness  Table 2. The analysis results showed that under acidic, alkaline, and high-temperature conditions, the adhesion of Ag/GO and Ag/rGO to the cotton was quite high. When hot washing with soap, the adhesion of Ag/GO and Ag/rGO to the cotton decreased, but still managed to reach the level of 2-3 on the scale of 1-5. However, the hydrophobic character of Ag/rGO/ cotton, which reduces interaction with water molecules and so helps to prevent washout throughout the process, may account for the much greater strength of Ag/rGO/cotton. These findings demonstrate that increasing the hydrophobicity of the outer cotton also supports enhancing the adhesion strength of Ag/rGO to the cloth.
Fabrication and antibacterial mechanisms of Ag/rGO/ cotton Ag/rGO/cotton was produced by dipping cotton into Ag/GO suspension. Then, the oxygen-containing functional groups on the GO surface were reduced to rGO by chemical means. In particular, VC was used as a non-toxic and environmentally friendly reducing agent. This method gave the product a relatively high-water resistance and the cotton after modification retained its antibacterial effect. In GO there was Fig. 12 Ag/rGO/cotton fabrication mechanism a conjugate system p-π, π-π from the O of the -OH and -C = O groups, making the H of the OH group attached to the double-bonded C very mobile. The mechanism of Ag/GO reduction by VC is shown in Fig. 12. Initially, the five-ring electron recovery chain of VC increased the acidity of the β and γ-hydroxyl groups, leading to the dissociation of the two protons to form an anion (HOAO-). Then, the anion underwent reactivation on the epoxy and diol groups of GO, yielding the intermediate product. The intermediate was reduced and the VC was oxidized to dehydroascorbic acid (Zhang et al. 2010). The Ag/rGO nanocomposite material was evenly coated on the stable cotton fabric, increasing the chance of bonding between the bacterial cell and the rGO sheet with silver attached to it. The sharp rGO plate lacerates the cell wall by physical interaction. At the same time, AgNPs release Ag + ions, which are attached strongly with thiol groups (− SH) of enzymes and proteins on the cell surface by electrostatic attraction, destroying the function of cell membranes and cell walls in selective permeation of intracellular and extracellular substances. Inside the bacterial cell, AgNPs and Ag + ions played a role in the formation of reactive oxygen species (ROS) due to oxygen imbalance. In addition, Ag + bound to the DNA of microbial cells and inhibited the replication function, stopping and preventing the growth of bacteria. To get further insights into the bactericidal mechanism of Ag/rGO nanocomposite material, the interaction process is illustrated in Fig. 13 and divided into four stages: (1) rGO plates with a large specific surface area helped to capture bacteria; (2) Formation of ROS free radicals, inhibiting and destroying bacterial cell membranes; (3) The sharp edges of rGO penetrated the membrane, damaging and cutting the cell membrane for Ag + to enter the cell; (4) Ag + ions bound to DNA, the enzyme inhibited replication, disrupted metabolism inside cells, killed bacteria.

Conclusions
In this study, Ag/GO/cotton was prepared by dipcoating cotton in Ag/GO solution followed by green chemical reduction to produce Ag/rGO/cotton. The results showed that the 1:1 VC:Ag/GO mass ratio, the reducing temperature of 140 °C, and the reducing time of 120 min for Ag/rGO/cotton production also showed a higher antibacterial effect against S. aureus, P. aeruginona, and E. coli bacteria. The hydrophobicity of Ag/rGO/cotton was observed with a wetting angle of 103.85° ± 0.75°, which prevented the adsorption of water droplets containing the density of bacteria and hence weakened the interaction between bacteria and cotton. Moreover, the Ag/GO also showed not only potential selective bioactivities to combat pathogenic bacteria and two cancerous HepG2 and A549 cell lines, but also good biocompatibility with Hek 293 cell lines. The small amount of Ag + leaching from Ag/rGO/cotton indicated that AgNPs had strong stability property in higher pH media due to the existence of GO as a capping agent, thereby reducing the excess dissolution of AgNPs, lowering the cytotoxicity of AgNPs in a hazardous environment, and keeping the material below the safety limits for human use. In addition, given the relevance of technology and personal protective equipment, the Fig. 13 Antibacterial mechanism of Ag/rGO/cotton current research provides a realistic, practical, and potential method for device development for medical applications.