Antimicrobial E ! ectiveness of Cellulose based Fabrics treated with Silver Nitrate Solution using Plasma Processes

In order to obtain antibacterial properties, the possibility of deposition of silver particles from silver nitrate (AgNO3) solutions by plasma deposition process using argon as a carrier gas (PDP-Ar) was explored. Hexamethyldisiloxane and acrylic acid were used as precursors and were deposited by plasma enhanced-chemical vapor deposition (PE-CVD). The processes were carried out on lyocell and modal fabrics and antimicrobial effi cacy was determined on E. coli and S. aureus using time kill assay method. The results of minimal inhibitory concentration (MIC) show that higher antimicrobial effi cacy on E. coli is exhibited by the solution of (AgNO3) in ethylene-glycol (0.066 μg/ml) rather than in absolute ethanol (0.265 μg/ml). For S. aureus, minimal inhibitory concentrations of AgNO3 solutions in both absolute ethanol and ethylene-glycol as solvents are obtained at the same value (0.132 μg/ml). Overall, the best antibacterial eff ect for both modal and lyocell samples has been achieved against E. coli using treatments with precursors (AAC and HMDSO) and AgNO3 in ethylene-glycol as solvent, with prolonged incubation time.


Introduction
Plasma technology and its application in the eld of textile technology are known as ecologically friendly processes.Textile materials exposed to plasma undergo through chemical and physical transformations in the surface layer.Using di erent gasses and reagents in the process of plasma nishing of textile materials, a variety of functional properties can be obtained.
ose include ame resistance, antistatic, hydrophobic, antibacterial properties etc. e ultimate e ect depends on many more factors such as plasma type, type of materials, plasma parameters, treatment conditions and the process used [1,2].In this study, the application of low-pressure plasma for the enhancement of surface properties of lyocell and modal bers and the achievement of antibacterial properties by deposition of silver particles was explored.Natural materials are an excellent media for the growth of microorganisms, especially in humid and warm conditions.While many synthetic bers have good resistance against microbial attack, natural bers are more easily affected.Proteins in keratin bers and carbohydrates in cellulose bers can serve as a source of food and energy for various bacteria, fungi and molds [3].Bacteria are single-celled organisms that reproduce very quickly in appropriate conditions.ey are classi ed as gram-positive and gram-negative and certain types are pathogenic and might cause serious health problems [4].Antimicrobial treatments are carried out in order to control the growth and reproduction of microorganisms.Agents can kill bacteria or fungi (biocides) or control the growth and spread of microbes (biostatics).Both prevent the spread of infection, allergic and respiratory problems as well as degradation of textile materials in the form of discolouration, staining, odors and degradation of the bers.ere are various antimicrobial agents used which di er in their chemical composition and antimicrobial activity.Quaternary ammonium compounds, polyhexamethylene biguanide, triclosan and some heavy metals can be used for biocidal purposes whereas chitosan and plant-derived bioactive agents can be used as biostatics [5].Many heavy metals such as silver, copper, zinc and cobalt have excellent antimicrobial properties at very low concentrations but silver is the most commonly used in the eld of textile industry [6].
It can be used as a metal, salt and nanoparticle [7].Single mechanism of antimicrobial action is not fully understood, but some mechanisms have been proposed that suggest that silver ion reacts with DNA and RNA molecules within the cell wall of microorganisms and prevents the replication.In addition, a reaction of silver with thiol groups (-SH) inside the cell is assumed which causes protein deactivation and reduction of enzyme activity.
is causes changes in metabolism, prevents propagation and ultimately leads to the destruction of microorganism [8][9][10].Also, it is proposed that silver acts by binding to key functional groups of enzymes.Bacterial plasma or cytoplasmic membrane are an important target site for silver ion, which causes the release of K + ions from bacteria.Silver ions inhibit cell division and damage the cell envelope and content of bacteria [11].Antimicrobial textiles are used in medical technology, health, hygiene products, home textiles, water treatment systems, as well as clothes for everyday use and personal protection [4,12].Physical sputtering and Plasma Enhanced Chemical Vapor Deposition (PE-CVD) under low-pressure plasmas are dominant techniques used in textile nishing [1].In this study, a relatively new method was used for the deposition of antibacterial agent called Direct Plasma Deposition Process (PDP) (Figure 1).

Figure 1: Reagent bottle for direct plasma deposition process
e process consists of reagent bottle with antibacterial agent, which is from one side connected to the supply of argon gas and the other side to plasma system.Argon is used as a carrier gas, which assists the transfer of reagent to the plasma system and on the textile substrate.

Textile samples and chemical agents
Plain wave lyocell (CLY) and modal (CMD) fabrics (Lenzing, Austria) were used in this study.ey were industrially prepared (desized and scoured) and weaved at the textile company Čateks, Croatia.By dissolving AgNO 3 (Sigma Aldrich) in absolute ethanol (Carlo Erba) and ethylene-glycol (Fluka Analytical), 0.1 M solutions were prepared and used for processing the fabrics in order to achieve antibacterial properties.Hexamethyldisiloxane, HMD-SO (Sigma Aldrich, ≥98.5%) and acrylic acid, AAC (Acros Organic, 99.5%) were used as precursors.Applied bacterial species were Escherichia coli ATCC 10536 (gram-negative bacteria) and Staphyloccocus aureus ATCC 6538 (gram-positive bacteria).

Plasma treatments
Treatments were done using low-pressure plasma system type NANO LF-40 kHz, by Diener.To ensure enhanced binding of silver particles on textile surface, activation process was carried out for 5 minutes using O 2 gas (purity of 99.99%, Messer) under optimized process parameters -pressure 0.34 mbar, power 300 W, frequency 40 kHz and gas ow 40 cm 3 /min.e pretreatments with HMDSO and AAC were conducted in order to enhance adsorption of silver particles onto cellulose surface using PE-CVD process.e process was conducted for 20 minutes at pressure of 0.18 mbar and power of 150 W. AgNO 3 solutions were applied on activated and pretreated samples using PDP-Ar process and argon plasma as a carrier gas for deposition.e process was carried out for 20 minutes at pressure 1.5 mbar, gas ow 40 cm 3 /min and power 150 W.

FE-Scanning electron microscopy analysis
Field emission scanning electron microscopy (FE-SEM) was conducted on untreated and plasma treated samples using Tescan SEM microscope (Mira 3 LMU).Before testing, the samples were steamed by vaporized mixture of gold and palladium under argon plasma.Micrographs were obtained using 4kx, 7kx and 10kx magni cations.

Antimicrobial effi cacy of silver nitrate solutions
Antimicrobial e cacy of AgNO 3 solutions was determined by known broth microdilution technique, which speci es minimum inhibitory concentration (MIC), i.e. the minimum concentration of antimicrobial agent that inhibits the growth of a microorganism a er a speci c time of incubation.e method is based on the dilution of the antimicrobial agent in the culture medium in microtiter plates, followed by addition of inoculum.If there is no growth of microorganism colonies, the concentration is the well represents MIC.For this purpose, a series of 12 concentrations of AgNO 3 was prepared from 4146.75 10 -3 to 2.07 10 -3 µg/ml.MICs were determined for E. coli and S. aureus a er incubation at 37 °C for 24 hours using 2,3,5-triphenyltetrazolium chloride (TTC) as a redox indicator for determination of bacteria viability.In the presence of bacteria, TTC was reduced to red coloured substance triphenyl formazan (TPF) and the colour change was directly proportional to the viable active cells.e change in colour was measured spectrophotometrically at 540 nm (Labsystems iEMS MF, type 1404, lter F6).

Antimicrobial effi cacy of treated samples
Antibacterial e cacy of treated materials was determined by time kill assay method.It is a quantitative microbiological method that determines the number of colony forming units of microorganisms a er a speci c time of incubation of samples.In this way, the rate at which a microorganism is killed as a function of time can be established.Treated samples (1cm x 1cm) were inoculated with 50 µl of microorganism suspension in sterile Petri dishes.e antibacterial e ectiveness against E. coli and S. aureus was investigated at the moment of contact of the sample with a suspension of microorganisms (t 0 ) and a er incubation time of 6 hours (t 1 ) and 18 hours (t 2 ) at 37 °C.Using sterile tweezers, inoculated samples were then transferred in test tubes, which had been pre-lled with 1 ml of saline.A er mixing with vibromixer (Vortex vibromixer Genius 3, Ika, Germany) for 30 seconds, 100 µl of the solution was transferred to an Eppendorf tube of 2 ml that had been previously lled with 900 ml of saline solution.In this way, the solution was diluted 10 times. is solution was then serially diluted and the aliquots of the dilutions series were spread-plated on an agar medium to allow for enumerating the colonies of the bacteria.Tryptic soy agar (105458 Tryptic soy agar, Merck Millipore, Germany) was used as a nutrient medium and quanti cation of the number of colony forming units of microorganisms was done a er incubation at 37 °C in the dark for 24 hours.e process is shown schematically in Figure 2.

Figure 2: Schematic representation of time kill assay method
For every concentration and time point, colonies of viable bacteria were counted and CFU/ml (colony forming units per ml) was calculated according to Equation 1: dilution factor e nal results are expressed as a percentage of inhibition in de ned time intervals.On the surface of bers treated with precursors (AAC, HMDSO) and AgNO 3 solutions, signi cant presence of precursors and silver particles over the entire ber surface is evident which was deposited a er surface activation with O 2 plasma.It con rms the e ectiveness of treatments performed using plasma processes.

Minimal inhibitory concentrations of silver nitrate solutions
Antimicrobial e ectiveness of silver nitrate on the bacterial species E. coli and S. aureus was determined.MIC values are presented graphically in Figure 4 and  For bacteria E. coli minimal inhibitory concentration of AgNO 3 solution in absolute ethanol is 0.265 µg/ml and of AgNO 3 solution in ethyleneglycol 0.066 µg/ml.According to the obtained results, higher antimicrobial e cacy on E. coli is exhibited by the solution of silver nitrate in ethylene-glycol, rather than in absolute ethanol.e MIC value of AgNO 3 solution in ethanol and in ethylene-glycol for S. aureus is 0.132 µg/ml.From MIC values, it is visible that a higher concentration of AgNO 3 solution in ethylene-glycol is needed for inhibition of S. aureus than for E. coli.Based on the obtained data it can be concluded that silver nitrate is an excellent antimicrobial agent that exhibits excellent antimicrobial e ectiveness even at very low concentrations.Although both solutions demonstrate great results, a more e ective solution for both bacterial species proved to be AgNO 3 solution in ethylene-glycol.

Antimicrobial effi cacy of treated samples
e testing results of antibacterial e ectiveness of the treated samples for bacterial species E. coli and S. aureus are presented in Figures 6-9.From the results, a positive e ect of all treated samples for tested bacterial species is visible and it grows with the incubation time.e untreated textile samples as well as those treated only with HMD-SO and AAC show slight antibacterial e ect in time.It suggests that the mentioned samples are not a sufcient nutrient for large-scale growth and propagation of tested bacterial strains in de ned time.
e best antibacterial e ect for lyocell samples against E. coli was achieved a er the treatment with AgNO 3 solution in ethylene-glycol using HMDSO as a precursor.A reduction of 75.2% of E. coli colonies a er 18 hours of incubation was achieved.A 61.8% reduction of E. coli was obtained a er 18 hours of incubation with the treatment of lyocell with AgNO 3 solution in ethylene-glycol and pretreatment with AAC.For modal samples, the highest inhibition was attained a er 18 hours of incubation a er the treatment with AgNO 3 solution in absolute ethanol both using AAC (68.2%) and HMDSO (69.1%) as precursors.In previous research [13], it was found that the application of HMDSO as a precursor enhanced the adsorption of silver particles onto cellulose providing higher antibacterial e ect.According to the Tekstilec, 2017, 60(4), 247-253 Antimicrobial Eff ectiveness of Cellulose based Fabrics treated with Silver Nitrate Solution using Plasma Processes obtained results for E. coli, both AAC and HMD-SO proved to be suitable for the application as precursors using plasma process, with HMDSO as a slightly more e cient agent.8 and 9) are not so uniform for the tested samples and inhibition is lower.e results for two treated samples that exhibit maximum inhibition (CLY/AAC, 6 hours of incubation and CMD/AAC/ AgNO 3 /EG, 18 hours of incubation) have a high degree of deviation and are inconsistent so they should be taken into account with caution and double checked.Besides them, the highest reduction of S. aureus for both lyocell and modal samples was achieved a er the treatment with AgNO 3 solution in ethylene-glycol and HMDSO as a precursor (18 hours of incubation).Feng et al. [10] conducted a study of antibacterial e ect of silver ions on E. coli and S. aureus and observed higher resistance of S. aureus than E. coli, which suggests a stronger defense system against silver ions.Because of its greater resistance, the application of higher concentration of agent should be considered.From SEM micrographs, a signi cant presence of • precursors is evident on the entire ber surface, which con rms the e ectiveness of the applied PE-CVD process.SEM micrographs also show the presence of sil- • ver particles on a treated ber surface, which suggests the applied PDP-Ar process was conducted successfully.
Based on the data for minimal inhibitory concen-• tration of AgNO 3 solutions, it can be concluded that silver is an excellent antimicrobial agent that exhibits great antimicrobial e ectiveness even at very low concentrations.
According to MIC values, AgNO • 3 solution in ethylene-glycol proved to be a slightly more effective solution for both tested bacterial species.Highest inhibition for lyocell samples against • E. coli was achieved a er the treatment with AgNO 3 solution in ethylene-glycol using HMDSO as a precursor a er 18 hours of incubation (75.2%).Highest inhibition for modal samples against • E. coli was achieved a er the treatment with AgNO 3 solution in absolute ethanol using HMDSO as a precursor a er 18 hours of incubation (69.1%).Results of antibacterial e ect against • S. aureus obtained by time kill assay are not so uniform for the tested samples and the inhibition is lower.e reason may be a higher resistance of such bacteria to silver ions.Overall, both AAC and HMDSO proved to be su-• itable for the application as precursors using PE-CVD process, with HMDSO as a slightly more efcient agent.

Figure 3 Figure 3 :
Figure3shows the obtained SEM micrographs of untreated and plasma treated lyocell and modal samples.eSEM micrographs of untreated lyocell and modal samples show smooth, clear surface of bers.

Figure 5 .
e value of the absorbance, which indicates the activity of microorganisms, in relation to a series of concentrations of tested solutions are shown.

Figure 4 :Figure 5 :
Figure 4: Graphical representation of antimicrobial e ectiveness of AgNO 3 solution in absolute ethanol (ET) and ethylene-glycol (EG) on bacteria E. coli

Figure 6 :
Figure 6: Inhibition of E. coli of treated CLY samples

Figure 7 :
Figure 7: Inhibition of E. coli of treated CMD samples e results of antibacterial e ect against S. aureus (Figures8 and 9) are not so uniform for the tested samples and inhibition is lower.e results for two treated samples that exhibit maximum inhibition (CLY/AAC, 6 hours of incubation and CMD/AAC/ AgNO 3 /EG, 18 hours of incubation) have a high degree of deviation and are inconsistent so they should be taken into account with caution and double checked.Besides them, the highest reduction of S. aureus for both lyocell and modal samples was achieved a er the treatment with AgNO 3 solution in ethylene-glycol and HMDSO as a precursor (18 hours of incubation).Feng et al.[10] conducted a study of antibacterial e ect of silver ions on E. coli and S. aureus and observed higher resistance of S. aureus than E. coli, which suggests a

Figure 8 :
Figure 8: Inhibition of S. aureus of treated CLY samples

Figure 9 :
Figure 9: Inhibition of S. aureus of treated CMD samples