Development of antimicrobial cotton fabrics by decoration with silver nanoparticles and activated carbon composite

Nanometer sized particulates demonstrate significant potential in various biomedical applications due to their large surface-to-volume ratio and exceptional physicochemical, electronic and mechanical properties. Additionally, the number of microbial infectious disease outbreaks has increased tremendously over the past decade, greatly impacting public health worldwide. In this article, we evaluate the antimicrobial activity of a cotton fabric (CF) impregnated with silver nanoparticles (AgNPs) and hemp hurd activated carbon (HHAC) (HHAC@AgNPs) composite (CF-HHAC@AgNPs). Field emission scanning electron microscopy and x-ray diffraction spectra of the CF-HHAC@AgNPs material revealed the presence of AgNPs and HHAC on the cotton fabric. Moreover, CF-HHAC@AgNPs shows excellent antimicrobial efficacy against Gram-positive and Gram-negative pathogens. The obtained results show that an HHAC@AgNPs-CF material can be prepared. It has an antimicrobial activity that suggests its potential as an inhibitory agent in various biomedical applications.


Introduction
Cotton fabrics (CFs), one of the most important cellulose fiber materials, are highly popular due to their outstanding properties such as fabric comfort, bio-degradation, softness and hygroscopic properties [1,2].Consequently, they have been widely utilized as healthcare and medical textiles for cloth face masks, bedclothes, underclothes, socks, non-implantable medical textile materials and hygienic products [2].However, they can create suitable environment for microbial growth under certain humidity and temperature conditions, causing foul odors and even negative health effects to human body [3][4][5][6].Microbial pathogens living on CFs surfaces may provide unpleasant odors, color degradation, allergic responses, deterioration of the textiles, and even potential health risks.Additionally, microorganisms can live for extended periods of time on CF surfaces, resulting in microbial contamination, enhancing the chance of microbial infections in humans [7,8].Therefore, the need for antimicrobial textiles has attracted attention from a wide range of researchers to address the abovementioned disadvantages of CFs.Currently, nanostructured materials with unique physical and chemical properties, with various size and shape-dependent properties, have been intensively used as antimicrobial agents [9][10][11][12].Silver nanoparticles (AgNPs) are effective and have great potential in biomedical applications as antimicrobial and antiviral agents with low toxicity.They have gained much attention during the past few years [9][10][11][13][14][15].Additionally, AgNPs with large surface areas can increase their contact with microbial pathogens, thus, maximizing their antimicrobial effects [16].This work was undertaken by applying hydrothermally prepared AgNP colloidal solutions to CFs and then further compositing them with activated carbon materials (AC@AgNPs) as a finishing process to impart antimicrobial activity.AC is a highly microporous amorphous carbonaceous material with both a large specific surface area and porosity, resulting in good adsorption and retention properties.The application of textile materials such as masks, air filters and deodorizing fabrics depended on the adsorption of hydrophobic activated carbon structure as well as advantages in biocompatibility and non-toxicity [17,18].AC is useful as the most popular and common adsorbent for air pollution control and wastewater treatment, as well as catalyst for energy storage and conversion [19][20][21].In addition, AC has a porous structure to hold and collect small molecules including nanoparticles although it did not have any antimicrobial effects on the tested microbial species.However, combining it with AgNPs to boost both compounds' potential antimicrobial efficacy against all microbial species was significantly enhanced [20].It could reduce the bioavailability of AgNPs through the complexion of surfaces, obstructing their solubility capacity, and releasing damaging silver ions into the growth media [21].Moreover, the combination with AgNPs could significantly decrease the expression of anti-apoptotic genes and pro-inflammatory cytokines compared to individual material treatments [20].Various raw materials, such as agricultural and forest waste biomass, coal, petroleum residues, and bones have been reported as AC precursors [20][21][22].Hemp hurd (HH), which accounts for about 70%-80% of hemp stalk [23], was used to prepare AC (HHAC) with antimicrobial properties.This was done by steam activation at a carbonization temperature of 400 °C.Finally, the antibacterial action of the prepared HHAC@AgNPs composite coated CF (CF-HHAC@AgNPs) was tested against two types of bacteria, Grampositive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E.coli) using three different techniques.These included qualitative determinations by a zone of inhibition (ZOI) method and quantitatively as the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods.The study details and the effects of surface modifications of the CFs on the production of multifunctional properties are of great benefit for both basic and applied research.

Preparation of hemp hurd activated carbon (HHAC)
AC was produced in a two-step process.First, dry hemp hurd, an agricultural waste, was pyrolyzed in a hightemperature furnace at 400 °C for 60 min.Following carbonization, char samples were physically treated with water vapor in an activation furnace at 850 °C for 30 min.After that, the samples were cooled naturally and stored in a closed container until further use.

Preparation of AgNPs
In an experiment, 5 ml of a 0.13 M glucose solution were added to 15 ml of a 0.01[0.5]M AgNO 3 solution.After that, 5 ml of a 0.02 M PVP solution were added and stirred.Then, 15 ml of a 0.04 M NaCl solution were added dropwise to the above solution and stirred continuously.The mixed solution was put in a Teflon-lined stainless steel autoclave and treated at 160 °C for 22 h.Finally, a precipitate was collected by centrifugation and washed with distilled water followed by isopropanol.The products were dispersed in isopropanol for further use [24].

Preparation of HHAC@AgNPs coated cotton fabric
Coated cotton fabrics were prepared using a dip-coating method to place AC and AgNPs over the fabric (CF-HHAC@AgNPs).AC and PVA, in a 9:1 ratio, were mixed in NMP and then added to solutions containing 4, 16, 36 and 64 g ml m of AgNPs.The mixtures were mixed using a magnetic stirrer at 80 °C for 1 h.Upon homogeneous distribution, the CFs were dipped in one of the solutions for 30 s and dried for 24 h, shown in figure 1.This process was repeated twice.Finally, the treated CFs samples were referenced as CF-HHAC@AgNPs (x), where x 4, 16, 36 = and m 64 , g ml m the AgNP concentration.The CF-HHAC@AgNPs were further characterized and evaluated for their antimicrobial activities.

Characterization
The synthesized samples were examined using UV-vis spectrophotometry (UV-vis), Field Emission Scanning Electron Microscopy (FESEM) and x-ray diffraction (XRD) spectroscopy.UV-vis, using a Shimadzu UV-1800 spectrophotometer, was performed to analyze the localized surface plasmon resonance in the 300-800 nm wavelength range at a resolution of 1 nm.For the XRD technique, the samples were scanned using 2θ angles from 15 to 80°and a step size of 0.2°.FESEM microscopy, using a model LEO 1450 VP, was done to observe the morphology and distribution of silver nanoparticles and activated carbon on the fabric.

Antimicrobial assays
Antimicrobial activities of CF-HHAC@AgNPs were investigated against two different pathogenic microorganisms, a Gram-negative E. coli and a Gram-positive, S. aureus.Fresh bacteria were pre-cultured in NB medium overnight at 37 °C to achieve an initial optical density (OD 600 nm) of 0.5 (equal to 10 8 CFU/ml).The samples of CF-HHAC@AgNPs used in antimicrobial tests were cut into 10 mm discs and sterilized under UV radiation for 2 h.After sterilization, the samples were used in antimicrobial assays.The antimicrobial activities were performed in the biosafety laboratory level 2.

Zones of inhibition (ZOI)
Fresh bacteria were transferred onto NA plates and gently spread with sterile cotton swabs over their surfaces.Then, samples of CF-HHAC@AgNPs were swabbed on the surface of NA sheets onto which the pathogenic bacteria were spread.The NA plates were incubated at 37 °C to permit better diffusion to the surfaces of bacterial cells.After 18 h of incubation, zones of inhibition around the discs were observed and their widths were measured using Vernier calipers.

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The MIC of the test sample was determined by the broth microdilution method.In brief, different test samples were prepared at 512, 256, 128, 64, 32, and 16 μg ml −1 in a sterile NB liquid medium.Fresh bacteria tested E. coli and S. aureus 5 μl, OD 0.2 (equal to 10 7 CFU/ml) were added to each 24-well cell culture plate.The prepared samples were added one by one to each well.The control was defined by a well without nanoparticles and the samples were incubated at 37 °C for 18 h.After the incubation, the concentration of the solution converted to turbidity was measured with a microplate reader at 600 nm.The MIC was defined as the lowest concentration that inhibited microbial growth.To determine the MBC of the sample, all wells without bacterial growth were spread on NA plates and incubated for 18 h at 37 °C.Agar plates without bacterial growth were used to define the MBC.

Structural and morphological characteristics
The morphologies of the original AgNPs, HHAC and cotton fabric were investigated in FESEM micrographs, shown in figure 2. As shown in figure 2(a), the prepared AgNPs display various morphologies, including spherical, cubic, triangular, polygon slab-like, rod-like and wire-like forms.The observed AgNPs have various particle sizes, with an average diameter of around 216 nm.As mentioned above, this work utilized a PVP in the synthesis process of AgNPs.It has been reported as one of the best polymeric stabilizers to stabilize and prevent the aggregation of AgNPs [25,26].It introduces large enough repulsive forces between individual particles to counterbalance the attractive van der Waals dispersion force [27,28].Therefore, the obtained AgNPs with different sizes and shapes of particles (see figure 2) may cause the structural transition during hydrothermal treatment [29,30].The presence of AgNPs was confirmed by observing the color of the aqueous solution.A color change was observed from a colorless solution of AgNO 3 to a gray colloidal solution of AgNPs, as seen in the insets of figure 2(b).This is in agreement with previous reports [24].UV-vis measurements were also made to investigate the formation of AgNPs (figure 2(b)).The UV-vis absorption spectra of colloidal AgNPs in figure 2(b) show an absorbance peak centered at around 432 nm, which is attributed to excitation of surface plasmon resonance (SPR) in the AgNPs, in agreement with previous reports [31,32].This suggests a reduction of AgNO 3 to AgNPs.The SPR is due to collective oscillation of free electrons of synthesized AgNPs in resonance with incident light.Since SPR absorbance relies heavily on the particle size, shape, and concentration, as well as the environment in which the nanoparticles are formed [24], the absorbance peaks in each of the previous works were observed at different absorbed energy positions [31,32].Figure 2(c) shows an FESEM image of pure hemp hurd activated carbon (HHAC).It can be clearly seen that HHAC exhibits irregular, rough and coarse surfaces with cracks and crevices, as well as well-developed pores.It is suggested that these characteristic properties could have resulted from doping with AgNPs and subsequent formation of HHAC@AgNPs composites.Before the compositing process, the CFs are white.An FESEM image of the CFs surfaces is given in figure 2(d).The original CFs surfaces had a very smooth longitudinal fibril structure with no contaminating particles on its surfaces.Additionally, it reveals the micron-sized pore network structure on CFs.
Significant differences in the CF surface morphology were observed after treatment with HHAC@AgNPs.FESEM images of CF-HHAC@AgNPs (4), CF-HHAC@AgNPs (16), CF-HHAC@AgNPs (36), and CF-HHAC@AgNPs(64) are shown in figures 3(a)-(d), respectively.An image (inset in each figure) of CFs shows the changed color, from white to black, after treatment with HHAC@AgNPs.The FESEM images clearly show that the surface morphology of the CFs becomes rough in all samples.The surfaces of CFs, as well as pores or voids within the fabric, were filled with HHAC@AgNPs particles.The energy dispersive x-ray spectroscopy (EDS) elemental analysis was also performed to investigate the bulk atomic composition of the major elements in each CF-HHAC@AgNPs sample.This was done to provide additional information on particle mapping and elemental analysis of the CF-HHAC@AgNPs.The inset tables in figures 3(a)-(d) confirms the presence of AgNPs and HHAC in all samples.However, few AgNPs were observed on the surfaces and in the pores of CFs, especially for the CF-HHAC@AgNPs (4), CF-HHAC@AgNPs (16), and CF-HHAC@AgNPs (36) samples.EDS also confirmed the presence of a small amount of AgNPs in each sample.However, the CF-HHAC@AgNPs (64) sample, which has highest concentration of AgNPs (x 64  4(a)).These are characteristic of the cellulose I crystalline form of CFs [33,34], in agreement with previous reports [35].The XRD pattern of the pure HHAC shows large broad diffraction peaks at 2q angles of  200), ( 220) and (311) reflection lines of the face-centered cubic phase of AgNPs, respectively, (No.JCPDS 00-004-0783 standard data).After treating CFs with HHAC@AgNPs, the XRD spectra shown in figures 4(d)-(g) were obtained for CF-HHAC@AgNPs(4), CF-HHAC@AgNPs( 16), CF-HHAC@AgNPs(36), and CF-HHAC@AgNPs(64) samples, respectively.The characteristic peaks due to AC and metallic AgNPs are present in all the CF-HHAC@AgNPs samples, but with very low intensity after deposition on CFs.The characteristic peaks for AgNPs at 2 38.1 q =  and 44.5  can be clearly observed, while at 2 64.2 q =  and 77.6  they are barely visible for the CF-HHAC@AgNPs(16), CF-HHAC@AgNPs(36), and CF-HHAC@AgNPs(64) samples.However, at low AgNPs concentrations, especially for HHAC@AgNPs(4) sample, peaks for AgNPs at 2 38.1 q =  and 44.5  are faintly observed, while at 2 64.2 q =  and 77.6  they are not found, in agreement with FESEM observations.The low and absent characteristic diffraction peaks are likely attributable to the small particle size and may also be due to the low sensitivity of XRD toward AgNPs detection on organic matter, corresponding to FESEM imagery (figure 3).

Antimicrobial properties
The CF-HHAC@AgNPs products containing AgNPs were tested for antimicrobial activity against the pathogenic microorganisms.E. coli (figure 5(a)) represents Gram-negative bacteria whereas S. aureus (figure 5(b)) represents Gram-positive bacteria.It has been widely used in antibacterial experiments since it is a pathogenic microorganism that causes disease in humans.Both bacteria often cause superficial infections, such as wound infections or urinary tract infections [36].The ZOI, a qualitative method, was first used for screening.This method was performed by placing each sample in contact with NA plates containing bacterial cells, shown in figure 5. Cotton fabric containing standard antibiotic (streptomycin 1 mg ml ) was used as positive control while untreated cotton fabric was used as negative control for each experimental set.After incubation, the zones of inhibition were determined by measuring the diameter of the zone with no bacterial growth.Untreated CFs did not present any antimicrobial activity against either E. coli or S. aureus bacteria.However, ZOIs due to antimicrobial activity can be observed for treated CF or CF-HHAC@AgNPs (x) products, where x 4, 16, 36 = and 64 g ml m (see figure 5).The results revealed that the CF-HHAC@AgNPs (64) sample demonstrated the greatest antimicrobial efficacy against both the tested strains (see figure 5).ZOI diameters of ∼13.83 and ∼12.83 mm were found against S. aureus and E. coli strains, respectively.The ZOI of S. aureus was larger than that of E. coli, indicating that the AgNPs contained in CF-HHAC@AgNPs had more effective biocidal properties against S. aureus, consistent with previous studies [37,38], probably due to differences in the cell membrane structure of E. coli and S. aureus [39].The ZOI diameter provides some indication of the potency of the antimicrobial activity of the treated CF products, but it cannot be used as a quantification method.The quantitative methods, MIC MBC provide values of antimicrobial activity based on the reduction of planktonic bacterial cell growth, as shown in figure 6.The calculated MIC was determined to be between 16 and 512 g ml m against both Gram-negative E. coli and Gram-positive S. aureus bacteria, as shown in table 1 and figure 6.The MIC was found to be 32 g ml m for both bacteria.The concentration of synthesized AgNPs that can completely inhibit any visible bacterial colony growth is the MBC value (figure 6), which was approximately about 128 g ml m and 512 g ml m for E. coli and S. aureus, respectively (table 1 and figure 6).
AgNPs and AgNP based composite materials have been shown effective agents for inhibition of a wide range of antibiotic resistant strains, including Gram-negative and Gram-positive bacteria [40][41][42][43][44].They act in a manner similar to the antimicrobial agents utilized for the treatment of bacterial infections.The development of CF-HHAC@AgNPs materials was suitable for a wide range of applications in the textile industry, healthcare industry and biomedical applications, including the development of masks or clothing with the ability to inhibit odor, and dressings for wound healing.For future research, this is the first step towards biomedical applications.Those agents have four different mechanisms of action.First, they may interfere with bacterial cell membrane synthesis.Another mechanism is inhibition of protein synthesis.A third is interference with nucleic acid synthesis, and while the fourth is inhibition of metabolic pathways [41].In the first mode, AgNPs attach to the surfaces of bacterial cell membranes and disturb their functions, such as permeability and respiration [45].Additionally, smaller AgNPs having a larger surface area provide better contact and greater interaction with microorganisms than larger AgNPs [46].The next mode involves AgNP penetration into bacteria through the cell walls possibly to interact with sulfur-and phosphorus-containing compounds, such as deoxy ribonucleic acid (DNA), which in turn negatively affects cell viability.Moreover, AgNPs can inactivate enzymes and generate hydrogen peroxide causing bacterial cell death [47,48].In the final mode, AgNPs have an outermost electronic configuration of 4d 10 , 5s 1 and cannot hold an extra electron.Consequently, silver ions (Ag + ), which are positively charged and much smaller than neutral AgNPs, make an additional contribution to the bactericidal effect [49].The mechanism of inhibition by Ag + on microorganisms causes DNA to lose its replication capability so that cellular proteins become inactivated upon Ag + treatment [45,50,51] higher concentrations of Ag+ have been shown to interact with cytoplasmic components and nucleic acids of bacteria [49,52].Moreover, the production of reactive oxygen species (ROS) in the vicinity of bacteria and inside cause oxidative stress.Higher intracellular ROS production resulted in even higher oxidative damage caused by AgNPs.Interactions between AgNPs with DNA, respiratory enzymes and cell membranes lead to cell apoptosis [53].

Summary
Antimicrobial cotton fabrics were successfully fabricated by decorating cotton fibers with silver nanoparticles and an activated carbon composite (CF-HHAC@AgNPs).FESEM and XRD spectra of the CF-HHAC@AgNPs material reveal the presence of AgNPs and an AC composite on the CFs.The obtained results show that AgNPs@AC composites were successfully synthesized via a stirring and attached to the surfaces of cotton fabric.The prepared CF-HHAC@AgNPs show excellent antimicrobial efficacy against Gram-positive and Gramnegative pathogens.The findings in this study are useful in active food packaging and biomedical applications.

Figure 1 .
Figure 1.Schematic diagram of the preparation and analysis CF-AgNPs@AC.

Figure 2 .
Figure 2. (a) FESEM images of synthesized AgNPs, the histograms show size distribution of AgNPs (b) UV-visible spectrum of the purified AgNP colloidal solution, dispersed in ethanol after purification, (c) FESEM images of HHAC and (d) untreated cotton fabric.The insets in (b) shows an image of AgNP powder and its colloidal form.
g ml = m ), was covered with AgNPs, as indicated by the brighter aspect of the CFs, which was then confirmed by higher magnification and EDS analysis (figures 3(e) and (f)).The XRD technique was again used to investigate the structure of treated CFs, CF-HHAC@AgNPs, as shown in figure 4. In this figure, in a comparison with original CFs, HHAC and AgNPs (the spectra in figures 4(a)-(c), respectively), typical CFs peaks have three characteristic diffraction peaks at 17 , ~ 22 ~ and 26 ~(figure

Figure 6 .
Figure 6.Antimicrobial activity of CF-HHAC@AgNPs (64) samples against E. coli (upper row) and S. aureus (lower row) strains.(A) MIC test shows the NB liquid medium turbidity assays of bacteria suspensions after incubation with the CF-HHAC@AgNPs(64) sample for 18 h at 37 °C, and (B) MBC test shows colonies of the bacteria suspensions taken from the MIC test tubes and incubated for another 18 h on an NA plate.