International Journal of Biological Macromolecules 78 (2015) 249–256
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Functionalization of medical cotton by direct incorporation of silver nanoparticles Hossam E. Emam a,∗ , N.H. Saleh b , Khaled S. Nagy c , M.K. Zahran b a b c
Textile Research Division, National Research Centre, Dokki, Cairo 12311, Egypt Chemistry Department, Faculty of Science, Helwan University, Ain-Helwan, Cairo 11795, Egypt Food Engineering and Packaging Department, Agricultural Research Center, 9 Cairo University St., Giza, Egypt
a r t i c l e
i n f o
Article history: Received 11 March 2015 Received in revised form 2 April 2015 Accepted 12 April 2015 Available online 20 April 2015 Keywords: Medical cotton AgNPs Functionalization Coloration Absorbency Antibacterial action
a b s t r a c t Medical cotton is usually used to clean skin, pack wounds and in other surgical tasks. Such important usages make imparting the antibacterial property to medical cotton is so essential research. The current research focuses on functionalization of medical cotton by direct incorporation of silver nanoparticles (AgNPs) in two-step process namely, pre-alkalization followed by sorption. Decorative color and antibacterial action were accomplished for medical cotton after in situ incorporation of AgNPs without using any other external reducing agent. AgNPs were produced due to the reduction action of alcoholic and aldehydic groups of cotton’s skeletal blocks. Cotton fibers were acquired a decorative color attributed to surface plasmon resonance of AgNPs. The treated cotton was characterized by using electron microscope. Results showed that Ag0 with size distribution of 0–160 nm was formed in the cotton fibers and their size majority (70%) was less than 80 nm. The reduction of Ag+ to Ag0 was confirmed by measuring the carboxylic and aldehydic contents. The treated cotton exhibited excellent antibacterial action at low silver contents. The absorbency of cotton was not affected by treatment. The produced medical cotton could be used to safe cleaning of wounds without getting any microbial infections. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Medical cotton is produced from full bleached and sterilized cotton. It has a wide range of usage as it stored especially as stock medical and cosmetics’ supplies. Medical cotton is made in several forms, such as balls, pads, roll and fabrics. One common use for this type of cotton is as a vehicle for makeup remover, which can be poured onto it and then swabbed across the face. So it is produced commonly in the form of bandages, medical swabs, and other similar products; and because it is sterilized, it can also be used to pack wounds and in other surgical tasks, and thus it is so called medical cotton. Due to many processes occurred for the production of medical textiles; medical cotton is containing more function groups compared to raw cotton. Ionic (Ag+ ) and metallic (Ag0 ) forms of silver can be applied in the cellulose to impart the antibacterial property [1–4]. Sorption process is the common method to insert Ag+ in cellulose through the ion exchange interaction between Ag+ and H+ of carboxyl groups of cellulose [5–9]. Incorporation of Ag0 onto cellulose
∗ Corresponding author. Tel.: +20 201008002487. E-mail address: [email protected] (H.E. Emam). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.018 0141-8130/© 2015 Elsevier B.V. All rights reserved.
is mainly occurred by deposition, which could be takes place via two different procedures. Ag+ is reduced to Ag0 followed by deposition on cellulose in the first procedure [3,4,10–12] while in the second one; Ag+ is absorbed on cellulose followed by in situ reduction [13–15]. Application of metallic nano-silver in cellulose is more preferable due to high surface area to volume ratio and the lower release of Ag+ ions from silver nanoparticels compared to silver ion salt [16] leads to the higher adhesion between metallic nano-silver and cellulose. Moreover, silver in its nano-metallic form has two important characters; color related to surface plasmon resonance absorption (SPR) and antibacterial actions [3,4,9,17–22]. Shape and size of nanosilver are two factors play an essential role in its SPR and then consequently influence their optical properties [23–26]. Since medical cotton is a material which comes in direct contact with the human body, its quality is very important and should satisfy the required pharmaceutical parameters. In the present work, AgNPs was introduced directly into medical cotton roll to impart antibacterial property and acquire a decorate color without any external reducers. Direct incorporation of AgNPs was carried out in two simple steps process namely, pre-alkalization using sodium hydroxide and submerging in AgNO3 . Cotton itself as cellulosic material was used as reducing agent for Ag+ and stabilizing agent
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for the produced Ag0 by its polymeric structure. UV/vis absorbance spectroscopy, scanning electron microscope, energy dispersive Xray spectroscopy and X-ray diffraction were all used to characterize the incorporation process. The colorimetric measurements (color strength and color space), aldehydic content and carboxylic content were measured to the treated cotton. The biological action of the cotton before and after treatment was estimated using disk diffusion method against two types of bacteria and two types of fungi.
2. Experimental 2.1. Materials and chemicals Absorbent medical cotton was provided by Chemical & Medical Industries Co., the Sixth Artificial region, 6th of October City, Egypt. The cotton fibers were used as received without further treatment. Silver nitrate (99.5%, from Sisco Reserch Laboratories PVT, Mumbai, India), Sodium hydroxide (96% from El Nasr Pharmaceutical chemicals Co., Cairo, Egypt), nitric acid (55%, from the Egyptian company for chemicals and pharmaceuticals, 10th of Ramadan, Egypt), boric acid (from El Gomhouria Co., Cairo, Egypt), hydrochloric acid (35.5%, from El Gomhouria Co., Cairo, Egypt), methylene blue MS dye (C16 H18 N3 SCl·xH2 O, 70% from s.d. Fine-Chem Ltd, Mumbai, India), potassium hydroxide (from El Nasr Pharmaceutical chemicals Co., Cairo, Egypt), bromophenol blue as pH indicator (from s.d. Fine-Chem. Ltd., Mumbai, India). Hydroxyl amine hydrochloride (98%, from Alpha Chemica, Mumbai, India) and absolute ethanol (99%, from El Nasr Pharmaceutical chemicals Co., Cairo, Egypt) were all analytical reagent grade and used without further purification.
3.2. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) The top view of untreated and Ag-treated cotton fibers surfaces were characterized by High resolution scanning electron microscopy (HR-SEM). Fiber samples were located on copper coated carbon tap double face, and then coated by the gold layer by evaporization of gold in argon atmosphere using sputter coater (S150 A, Edwards, UK). The surfaces of samples were scanned using high resolution scanning electron microscopy (HR-SEM Quanta FEG 250 with field emission technique, Philips, Netherland). Energy dispersive X-ray spectroscopy (EDX) analysis unit (EDAX AMETEK analyzer, materials analysis division, Philips, Netherland) conducts with the scanning electron microscope was used to characterize the surface chemical structures of cotton fibers after Ag treatment. 3.3. Color measurements The colorimetric data of the treated cotton fibers was recorded using a spectrophotometer with pulsed xenon lamps as light source (UltraScan Pro, Hunter Lab, USA), using 10◦ observer with illuminant of D65, d/2 viewing geometry and 2 mm measurement area. Color measurement data are lightness (L) from black to white, a* is a redness/greenish ratio and b* is yellowish/bluish ratio [27]. The corresponding color strength value (K/S) was assessed at 430 nm wavelength (430 ) by applying the Kubelka Munk [28] (Eq. (1)). The absorbance of treated cotton fibers were measured by the same instrument at 430 . (1 − R)2 K = S 2R
(1)
where R is the decimal fraction of the reflection of the cotton fibers, K is the absorption coefficient and S is the scattering coefficient. 3.4. Biological activity
2.2. Procedure The incorporation of Ag in medical cotton fibers was carried out in two steps process namely, pre-alkalization and sorption. In the pre-alkalization step, definite amount from medical cotton fibers was soaked in different concentrations of NaOH (0.01 N and 0.1 N) using 1/50 materials to liquor ratio (M/L) at room temperature for different time durations (5, 30 and 60 min). After certain time, fibers were taken out, gently squeezed and were then transferred to the sorption bath. In the second step, the wet alkalized fibers were fully immersed in AgNO3 solution with different concentrations (20–400 mg/L) using the same liquor ratio. The reaction mixture was agitated and its temperature was adjusted to RT, 50 ◦ C and 70 ◦ C. At the end of reaction time, treated fibers were removed and rinsed two times by tap water at RT using the same liquor ratio of treatment for neutralization, and then dried in oven at 70 ◦ C for 1 h, prior to further analysis. The residual solutions were kept to measure the absorbance spectra.
Antimicrobial activity of the cotton fibers was tested qualitatively against bacteria and fungi using a modified Kirby–Bauer disk diffusion method [29]. Two types of both bacteria and fungal were used in the test and they namely, Escherichia coli as gram −ve bacteria, Staphylococcus aureus as gram +ve bacteria, Aspergillus’s flavous grows as filamentous fungi and Candida albicans grows as yeast. Briefly, 100 L of the tested bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/mL for bacteria or 105 cells/mL for fungi [30]. A 100 L of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Plates inoculated with E. coli and S. aureus at 35–37 ◦ C for 24–48 h, with C. albicans at 30 ◦ C for 24–48 h and with A. flavous at 25 ◦ C for 48 h. The diameters of the inhibition zones were measured in millimeters with slipping calipers of the National Committee for Clinical Laboratory Standards [31]. The average width for zone of inhibition along a streak on either side of the tested fibers was calculated using Eq. (2). Blank sample with a diameter 1 cm was impregnated 10 L of tested concentration of the stock solutions. T −D 2
3. Measurements
W=
3.1. UV–visible spectra
where W is the width of clear zone of inhibition in mm, T is the total diameter of tested fiber and clear zone in mm, and D is the diameter of the tested fiber in mm.
At the end of treatment process, the cotton fibers were removed and the absorbance of supernatant solutions was measured. The UV–vis absorption spectra were used to measure the extinction of the residual solutions using a multi-channel spectrophotometer (T80 UV/VIS, cell length = 10 mm, PG Instruments Ltd, Japan) at the wavelength range of 250–600 nm.
(2)
3.5. Absorbency of fibers A 1 g of the dried cotton fibers was accurately weighed and compressed to form a disk with thickness of 3 mm to get taut surface
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of fiber with free of wrinkles. The fiber disk was used to measure the absorbency of fibers using the standard method for absorbency of textile [32]. The buret was placed in an appropriate location in the lab, and then the fiber disk was located 9.5 ± 1.0 mm below the tip of the buret. A one drop of distilled water was allowed to fall on the fiber disk and the stopwatch was started immediately. The time required for the drop of water to completely disappear and appear only as a dull wet spot was measured. If the water drop disappears immediately, the time records as “zero” and when the elapsed time exceeds 60 s, it records as “60+ s”. The time of disappearance of water drop was tested at five different locations and the average was considered. 4. Chemical analyses 4.1. Detection of silver content Silver in the 0.2 g treated dried cotton fiber was extracted by 20 mL of 15 wt% nitric acid for 2 h at 80 ◦ C. The Ag concentrations in the extracted solution was analyzed with Contraa 700 flame atomic absorption spectrophotometer (AAS, Analytik, Jena, Germany) equipped a silver lamp at 328.1 nm wavelength. The extracted Ag from fibers was calculated according to Eq. (3). C ×V W (1 − MC/100)
A−B × 100 A
4.3. Aldehydic content The aldehydic content in the medical cotton fibers was measured based on oxime reaction [34] as follows: a 50 g of hydroxyl amine hydrochloride was dissolved in 100 mL distilled water and dilute to one liter using ethanol, then the pH of solution was adjusted to 3.4 with ethanolic potassium hydroxide. A 0.2 g of cotton fiber was immersed in 30 mL of the hydroxyl amine solution, mixed thoroughly and allowed to stand at room temperature for 30 min. A blank solution without sample was performed. Solutions were titrated with 0.25 N ethanolic KOH till the greenish yellow end point, using bromophenol blue as indicator. The percentage of aldehydic content was calculated according to Eq. (6). CHO% =
where CHO is the aldehydic content in cotton fibers (%), B is the KOH volume consumed in titration of blank solution (mL); S is the KOH volume consumed in titration of sample solution (mL); W is the weight of dried treated fibers (g); MC is the moisture content in dried treated fibers (%).
(4)
Medical cotton are sterilized fabrics as discussed previously, used for cleaning skin, pack wounds and in other surgical tasks, however, it does not have any antimicrobial activity. Thus, our target is acquiring an additional function as antibacterial actions 2500
4.2. Carboxyl content
A
RT
50 °C
70 °C
2000
1500
1000
500
(5)
where COOH is the carboxyl content (mmol/g), C1 is the concentration of methylene blue in the blank (sample without fibers) (mg/L), C2 is the concentration of methylene blue in the samples (in the
0 50
0
100
150
200
250
300
350
400
450
AgNO3 in Solution (mg/L) 150
Ag in Fiber (mg/Kg)
Carboxylic group contents of cotton fibers before and after Agtreatments were measured using the methylene blue dye method [33]. The method can be described briefly as follows: three different solutions of 300 mg/L aqueous methylene blue, borate buffer solution with pH 8.5 and 0.1 M HCl were prepared and namely, solutions A, B and C respectively. Equal volume of 25 mL from solutions A and B were added to a 0.17 g of cotton fiber in a 50 mL bottle followed by shaking for 20 h at room temperature. A 2.5 mL of that solution mixture was added to a 5 mL of solution C and total volume was completed to 50 mL by distilled water. The absorbance of solutions were measured using multi-channel Spectrophotometer (T80 UV/VIS, d = 10 mm, PG Instruments Ltd, Japan) at wavelength of 664.5 nm (max of methylene blue). A stock solution of 100 mg/L methylene blue was used to prepare calibration solutions at pH 8.5 in the range of 0.5–10 mg/L. Using the slope of calibration curve, the carboxyl content was calculated from Eq. (5). (C1 − C2 ) × 0.00313 W (1 − MC/100)
(6)
5. Results and discussion
where MC is the moisture content (%), A is the initial condition weight (g) and B is the weight of oven dried fiber (g).
COOH =
(B − S) × 100 W (1 − MC/100)
(3)
where Ag is the silver content in cotton fibers (g/kg), C is the silver concentration (mg/L) in extracted solution; V is the volume of extracted solution (0.02 L); W is the weight of dried treated fibers (g); MC is the moisture content in dried treated fibers (%). The moisture contents in the untreated and treated cotton fibers were measured as follows: a 1 g fiber was weighed accurately up to four digital numbers and then dried at 105 ◦ C for ca. 4 h. The dried samples were reweighed up to fixing weight and then the moisture contents were calculated according to Eq. (4). The obtained moisture contents were 5.8% for untreated cotton fibers. MC =
presence of fibers) (mg/L), W is the weight of cotton fiber samples (g) and MC is moisture content (%).
Ag in Fiber (mg/Kg)
Ag =
251
B
RT
50 °C
70 °C
100
50
0 0
20
40
60
AgNO3 in Solution (mg/L) Fig. 1. Ag content in cotton fibers as function of silver nitrate concentration in solution (A), magnified at low concentration (B).
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to medical cotton by the in situ incorporation of AgNPs. The additional function helps medical cotton for peaceful cleaning of injuries/wounds and for the perfect usage in surgical tasks without getting any microbial infections. Two steps process was carried out for direct incorporation of Ag in the medical cotton fibers namely, pre-alkalization and sorption. Pre-alkalization step was used to swell the fibers to facilitate the Ag diffusion into fibers in the sorption step [35]. It is known that, pKa of COOH in cellulose equals to 4.0 [36]. Sorption of silver ions on cellulose is an ion-exchange reaction between Ag+ with COO− of cotton [5,37]. So, sorption capacity of Ag is largely influenced by the pH of solution. Pre-alkalization process of cotton fibers increases the sorption capacity due to two factors; fully dissociation of COOH for cellulose and increasing the affinity to Ag as a result of the swelling of cellulose. In the sorption step, Ag+ was absorbed firstly on cellulose from AgNO3 by ion-exchange with COO− of cotton. Reduction of Ag+ to Ag0 was carried along with the ion-exchanging process or thereafter. The reduction was occurred by reducing groups of cellulose
mainly aldehydic and alcoholic groups and it was catalyzed by alkalinity and heat [7,21,38,39]. Additionally, the tendency of Ag to auto-catalytic reduction, as the reduction rate was accelerated after forming the first nuclei of Ag0 [40,41].
5.1. Silver content In Fig. 1, the silver content in the cotton fibers at the different temperatures was shown plotted against the realistic content of Ag in solution. The Ag content in cotton fibers was ranged between 48.7 and 2391.5 mg/kg and it was observed increased continuously with increasing AgNO3 in solution. At the lower concentration (20 mg/L), Ag content was observed almost the same at different temperatures. At 70 ◦ C, Ag in fibers was very high and reached 2391.5 mg/kg, but it was lower more than 20 times to record 113.4 mg/kg by using 400 mg/L AgNO3 . Except with using 400 mg/L, Ag content in fibers was not changed dramatically from RT to 50 ◦ C. In conclusion, it can be say that, the Ag content in fibers
Fig. 2. Absorbance spectra of residual solutions after treatment of cotton fiber with AgNO3 at different conditions: (A) 0.01 N NaOH, (B) 0.1 N NaOH, (C) immersion in AgNO3 for 1 h at room temperature, (D) immersion in AgNO3 for 1 h at 50 ◦ C, (E) immersion in AgNO3 for 1 h at 70 ◦ C.
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Absorbance (at 420 nm)
1.6
RT
50 °C
5.3. Color measurements
70 °C
1.2
0.8
0.4
0 0
50
100
150
200
250
300
253
350
400
450
AgNO3 Concentration (mg/L) Fig. 3. Absorbance of Ag–cotton fibers at wavelength of 430 nm as function of silver nitrate concentration in solution.
was follow the order of at 70 ◦ C 50 ◦ C ≥ RT. This is may account for the greater Ag deposition in fibers at 70 ◦ C.
5.2. UV–visible absorption spectra Depending on their size and morphological structure, AgNPs exhibit surface plasmon band resonance (SPR) as known formerly [17–20]. Thus, at the end of sorption step, the absorbance spectra of residual solution for silver were measured by using spectrophotometer. Effect of NaOH concentrations (0.01 and 0.1 N) and temperature (RT, 50 ◦ C and 70 ◦ C) as function of reaction time was studied and showed in Fig. 2. An absorbance peak was observed in the wavelength range of yellow color (410–435 nm), which is in agreement with visual observation of solution color. The absorbance peak is corresponding to the SPR of spherical AgNPs [17,21,42]. The peak of Ag+ , at range of 250–300 nm [43], was not recorded in all experiment. The absorbance intensity was showed higher two times with using 0.1 N comparing to 0.01 N of NaOH concentrations. This is reflecting the role of alkalinity in the reduction of Ag+ besides their function in swelling of cotton fibers to penetrate AgNPs [22]. At different reaction temperatures, changing in Ag+ concentration is accompanied by observable change in the absorbance intensity. By apply 70 ◦ C, the absorbance intensity is a directly proportional with Ag+ concentration. But this trend was not seen at lower temperature, which could be related to the heterogeneous process of Ag+ reduction as a result of insufficient heating to activate the reduction process. This is explained the important role of temperature in activation the reduction process of Ag+ . Absorbance of treated cotton fibers was measured by Ultrasacn pro spectrophotometer at maximum peak (max ). It was found that the max equal to 430 nm which is characteristic for the SPR of spherical AgNPs and this is compatible with the results of absorbance spectra for residual solution. The absorbance data obtained for treated cotton fibers is comparable to that reported for cotton fabrics after in situ deposition of AgNPs using citrate [44]. Fig. 3 declares that, at max , the absorbance of Ag-fibers was increased with temperature and Ag+ concentration and reached 1.4 by using 400 mg/L at 70 ◦ C. This is an indication for that the amount of AgNPs in the cotton fabrics was largely affected by reaction temperature and Ag+ concentration. The absorbance intensity is directly proportional to the AgNPs content in fibers, as the higher absorbance intensity, the higher AgNPs content and this was achieved at 70 ◦ C.
The color of cotton fibers was changed after removed from the impregnation bath. Furthermore, this was quantified through measurements of color coordinates and color strength of the dried fibers after treatment and recorded in Table 1. The strength of color (K/S) was not considerably changed up to 50 ◦ C. But, by raising temperature to 70 ◦ C, the K/S was grown up from zero to 10.32 with increment the Ag+ concentration from zero to 400 mg/L. The lightness (L*) of fibers was decreased with increasing both of the Ag+ concentration and the temperature from RT to 70 ◦ C, while values of a* and b* were increased. L* was recorded 95.93 (untreated) and 76.87 (RT), 60.77 (50 ◦ C) and 37.03 (70 ◦ C) for fibers treated with 400 mg/L. At the same experimental conditions, the value of a* was changed from 0.02 to 4.17, 6.20 and 14.89, while, the values of b* were 2.50, 3.40, 10.82 and 19.66 respectively. From these data, color of fibers was changed from white through creamy to yellowish red after Ag-treatment and the color became more deeply by increasing the concentrations of Ag+ . 5.4. SEM images, EDX spectrum and size distribution Scanning electron microscope (SEM) was used to notify the change in morphology of cotton fibers surface caused by incorporation of AgNPs. In Fig. 3, SEM images show the smooth surface of cotton fabrics before treatment (Fig. 3a and b). After Ag-treatment, the Ag clearly with intense amounts on the cotton surface and some aggregates consisting of smaller particles were observable. The shape of Ag particles showed here was similar to that obtained previously on cellulose fiber/fabrics [10,22,45]. The presence of Ag particles on the cotton fibers were further confirmed by EDX, as signal of Ag was appeared in Fig. 3e. The amount of Ag was recorded ca. 0.98% from the sample in two different places, but this value cannot be considered because it depends on some factors such like dispersion homogeneity and the area which electron beam incident on. Size of Ag particles in the fibers was measured by software program using SEM photos. Fig. 3f confirms that the Ag particles were in nano-dimension and their size distributions were ranged from 0 to 160 nm. The majority of particles were located in 0–80 nm (ca. 70%). Size distribution of Ag particles in cotton fibers was lower than that recorded previously [9,45]. The fact of AgNPs presence on the cotton fibers explained and supported UV–vis absorption spectra and color results for cotton fibers (Figs. 2 and 4 and Table 1). 5.5. Carboxylic and aldehydic content The reduction of Ag+ to Ag0 by cotton fibers was initially detected by color change of solution and fiber to yellow and was later confirmed by scanning electron microscopic. The reduction was carried out by the reducing end group (aldehydic e.g. CHO and alcoholic groups e.g. CH2 OH) of cotton. The reaction between silver and cotton is two-faced reaction, comprised a reduction reaction for Ag+ contrary oxidation reaction for cotton. When Ag+ is transformed to Ag0 , jointly CH2 OH and/or CHO are oxidized to COOH [21]. Therefore, the carboxyl (COOH) and aldehyde (CHO) contents were both measured for cotton fibers before and after treatment and the data was recorded in Table 2. For untreated medical cotton, CHO content was 15.82% and COOH content recorded 20.76 mmol/kg. After Ag treatment, CHO content decreased while COOH content increased by increasing both of AgNO3 concentration and temperature, confirming the reduction of Ag+ to Ag0 by functional groups of cotton. At room temperature (RT), CHO and COOH contents were not changed obviously, however AgNO3 concentration may be accounted for the insufficient temperature to activate the reduction process [7,38,39,43]. By increasing the
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Fig. 4. Scanning electron microscope photos for (A and B) untreated at different magnifications, (C and D) Ag–cotton fibers at different magnifications, (E) EDX analysis for Ag–cotton fibers and (F) size distribution of AgNPs on cotton fibers.
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Table 1 Color data of treated cotton as function of silver content. Sample
AgNO3 conc. (mg/L) 0
Ag content (mg/kg)
L*
a*
b*
K/S (at = 430 nm)
0
95.93
0.02
2.5
0.01
RT
20 50 100 200 400
48.7 50.2 59.6 81.4 113.4
91.93 89.65 85.56 81.29 76.87
1.38 1.21 2.93 2.26 4.17
1.93 2.90 3.00 4.43 3.40
0.00 0.05 0.10 0.16 0.28
50 ◦ C
20 50 100 200 400
59.5 48.5 101.4 181.1 1576.0
76.11 81.84 77.88 77.73 60.77
6.63 5.16 5.77 6.74 6.20
13.60 9.53 14.83 11.41 10.82
0.49 0.26 0.49 0.41 1.62
70 ◦ C
20 50 100 200 400
66.8 147.4 1144.8 1779.7 2391.5
74.79 62.20 60.46 40.42 37.03
7.46 9.94 12.47 14.01 14.98
17.29 17.86 20.28 18.84 19.66
0.69 1.68 2.05 7.75 10.32
Untreated
Table 2 Carboxylic and aldehydic contents of treated cotton fibers. Sample Untreated
AgNO3 conc. (mg/L) 0
Ag content (mg/kg)
Carboxyl content (mmol/kg)
Aldehyde content (%)
0
20.76
15.82
RT
20 50 100 200 400
48.7 50.2 59.6 81.4 113.4
19.42 23.81 23.09 23.66 22.76
14.54 15.56 13.45 13.48 13.35
50 ◦ C
20 50 100 200 400
59.5 48.5 101.4 181.1 1576.0
21.50 27.44 27.55 32.76 34.30
15.67 13.48 10.21 10.35 9.46
70 ◦ C
20 50 100 200 400
66.8 147.4 1144.8 1779.7 2391.5
22.02 26.25 26.35 39.76 46.80
12.51 10.82 9.44 7.65 7.28
reaction temperature to 50 ◦ C, the decrement in CHO content and the increment in COOH content were observed clearly as function of AgNO3 concentration. As COOH and CHO contents were estimated to be 34.30 mmol/kg and 9.46%, respectively by treatment with 400 mg/L AgNO3 . The percent of CHO content was much diminished to 7.28% and the COOH content was much augmented to 46.80 mmol/kg when the temperature was raised to 70 ◦ C. The considerable variation in the contents of CHO and COOH by rising the temperature, explains the role of heating in the stimulation of redox reaction between Ag and cotton.
5.6. Absorbency Medical cotton is a soft mass important to clean skin and pack wounds and its high absorbency is a significant sign. Thus, to be used in the medical field, the absorbency property for medical cotton supposed to be not decreased significantly. The absorbency of medical cotton fibers was measured by using the standard method for water droplet disappear [32]. Results showed that the original medical cotton was absorbed water droplet and completely wetted by water immediately, due to the presence of numerous freely hydroxyl groups on the surface of medical cotton. The same behavior was obviously recorded for Ag treated cotton sample, as water droplet was easily adsorbed and vanished immediately on its surface at zero time, however Ag content. This indicates to that the incorporation of AgNPs into medical cotton did not change
absorbency property of cotton, making the Ag treated medical cotton is more appropriate to use in the treatment and packing of wounds.
5.7. Antimicrobial activity Natural fibers/fabrics can provide an excellent environment for growing the microorganisms due to their ability to retain moisture. On the other hand, medical cotton was commonly used to pack the wounds and in other surgical tasks, thus it is so important to be having antimicrobial action. The antimicrobial property of the untreated and treated cotton fibers was tested qualitatively against bacteria and fungi using disk diffusion method. E. coli as gram −ve and S. aureus as gram +ve were used as bacteria, while A. flavous and C. albicans were used as fungi. Three samples different in Ag content were used in the antimicrobial test. Regardless Ag concentration on the fibers, antifungal action was not shown for Ag-medical cotton fibers. Table 3 shows the antibacterial activity results and the untreated medical fibers were not exhibited any antibacterial action. But, the antibacterial activity was observed for Ag-medical cotton fibers due to the leached of Ag ions from fibers surface to surrounding. The inhibition of bacterial colonies was depended on the Ag concentration on the fibers and bacterial type. At lower Ag content (66.8 mg/kg), no antibacterial action was notified against E. coli (0 mm inhibition zone), while it was observed against S. aureus (12 mm inhibition zone). By increasing the Ag
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Table 3 Biological activities of treated cotton fibers at 70 ◦ C. Sample
AgNO3 conc. (mg/L)
Ag content (mg/kg)
Antibacterial action Inhibition zone diameter (mm) Escherichia coli (gram −ve)
Untreated 1 2 3
0.0 20 100 400
0.0
0.0
66.8 1144.8 2391.5
0.0 14 13
content on fibers, the difference in diameter of inhibition zone was not significant, but it was observed for both types of bacteria strains. The results of antibacterial presented here for medical cotton fibers, is better than that recorded in the literature for cotton fibers treated with AgNPs [3,4,13]. 6. Conclusions Functional medical cotton with an excellent antibacterial property and with a yellowish decorative color was effectively obtained by direct incorporation of AgNPs. The AgNPs incorporation into cotton was carried out without using any external reducer, in twostep process namely, pre-alkalization and sorption. The reducing groups of cotton were used to reduce Ag+ to Ag0 , and the results of COOH and CHO contents confirmed the reduction process. AgNPs was shown on the cotton surface by SEM and their size was ranged between 0 and 160 nm. Color data declare that yellow colored cotton was obtained with different color shades as function of Ag content. Excellent antibacterial property was recorded against the common pathogenic bacteria: E. coli as gram −ve and S. aureus as gram +ve. The absorbency for cotton was not absolutely changed by Ag treatment. The acquired properties due to treatment, lead medical cotton is more appropriate for safe cleaning of wounds or in surgical tasks without getting any microbial infections. References [1] N. Sachinvala, D.V. Parikh, P. Sawhney, S. Chang, J. Mirzawa, W. Jarrett, B. Joiner, Polym. Adv. Technol. 18 (8) (2007) 620–628. [2] M. Bajpai, P. Gupta, S.K. Bajpai, Fib. Polym. 11 (1) (2010) 8–13. [3] M.H. El-Rafie, H.B. Ahmed, M.K. Zahran, Carbohydr. Polym. 107 (2014) 174–181. [4] M.K. Zahran, H.B. Ahmed, M.H. El-Rafie, Carbohydr. Polym. 108 (2014) 145–152. [5] T. Saito, A. Isogai, Carbohydr. Polym. 61 (2) (2005) 183–190. ˇ ´ N. Radic, ´ M.B. Obradovic, ´ S. Dimitrijevic, ´ M.M. Kuraica, P. Skundri ´ [6] M. Kostic, c, Chem. Ind. Chem. Eng. Q. 14 (4) (2008) 219–221. [7] S. Ifuku, M. Tsuji, M. Morimoto, H. Saimoto, H. Yano, Biomacromolecules 10 (9) (2009) 2714–2717. [8] C.I. Su, C.C. Peng, Y.C. Lu, Text. Res. J. 79 (16) (2009) 1486–1501. ˇ [9] H.E. Emam, A.P. Manian, B. Siroká, H. Duelli, B. Redl, A. Pipal, T. Bechtold, J. Clean. Prod. 39 (2013) 17–23. [10] H.J. Lee, S.Y. Yeo, S.H. Jeong, J. Mater. Sci. 38 (10) (2003) 2199–2204. ˇ ´ Z. Saponji ´ V. Vodnik, B. Potkonjak, P.N.J. Jovanˆcic, ´ M. Radetic, ´ Carbohydr. [11] V. Ilic, c, Polym. 78 (3) (2009) 564–569. [12] S. Ravindra, Y. Murali Mohan, N. Narayana Reddy, K. Mohana Raju, Colloids Surf. A: Physicochem. Eng. Aspects 367 (23) (2010) 31–40. [13] T. Maneerung, S. Tokura, R. Rujiravanit, Carbohydr. Polym. 72 (1) (2008) 43–51. [14] L.C.S. Maria, A.L.C. Santos, P.C. Oliveira, A.S.S. Valle, H.S. Barud, Y. Messaddeq, S.J.L. Ribeiro, Ciênc. Technol. 20 (1) (2010) 72–77.
Staphylococcus aureus (gram +ve) 0.0 12 13 12
[15] R. Tankhiwale, S.K. Bajpai, Colloids Surf. B: Biointerfaces 69 (2) (2009) 164–168. [16] J. Delattre, M. Height, R. Volpe, Comments of the Silver Nanotechnology Working Group for Review by the FIFRA Scientific Advisory Panel, 2009. [17] A.A. Hebeish, M.H. El-Rafie, F.A. Abdel-Mohdy, E.S. Abdel-Halim, H.E. Emam, Carbohydr. Polym. 82 (2010) 933–941. [18] M.H. El-Rafie, H.B. Ahmed, M.K. Zahran, Facile Precursor for Synthesis of Silver Nanoparticles Using Alkali Treated Maize Starch. International Scholarly Research Notices ID 702396, 2014, 12 pp. [19] M.K. Zahran, H.B. Ahmed, M.H. El-Rafie, Carbohydr. Polym. 111 (2014) 10–17. [20] M.K. Zahran, H.B. Ahmed, M.H. El-Rafie, Carbohydr. Polym. 111 (2014) 971–978. [21] H.E. Emam, S. Mowafi, H.M. Mashaly, M. Rehan, Carbohydr. Polym. 110 (2014) 148–155. [22] H.E. Emam, M.K. El-Bisi, Cellulose 21 (6) (2014) 4219–4230. [23] M.A. El-Sayed, Acc. Chem. Res. 34 (4) (2001) 257–264. [24] S. Eustis, Gold and silver nanoparticles: characterization of their interesting optical properties and the mechanism of their photochemical formation (PhD thesis), Georgia Institute of Technology, College of Science, 2006. [25] L.A. Dick, A.D. McFarland, C.L. Haynes, D.R.P. Van, J. Phys. Chem. B 106 (2002) 853–860. [26] L. Qiaoling, C. Yahong, in: Y. Orhan (Ed.), Nanorods 8: Preparation and Characterization of Gold Nanorods, InTech, 2012, ISBN 978-953-51-0209-0, p. 1. [27] AATCC Evaluation Procedure 6-2003; “Instrumental Color Measurement”, AATCC Technical Manual, vol. 83, 2008, pp. 384–389. [28] J.M. Menter, K.L. Hatch, Curr. Prob. Dermatol. 31 (2003) 50–63. [29] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (4) (1966) 493–496. [30] M.A. Pfaller, L. Burmeister, M.A. Bartlett, M.G. Rinaldi, J. Clin. Microbiol. 26 (1988) 1437–1441. [31] National Committee for Clinical Laboratory Standards, Performance 41 (1993). [32] AATCC Test Method 79-2007: Absorbency of Textiles, AATCC Technical Manual, vol. 83, 2008, pp. 99–100. [33] B. Klemm, B. Philipp, T. Heinze, U. Heinze, W. Wagenknecht, Comprehensive Cellulose Chemistry, vol. 2, Wiley-VCH, Weinheim, Germany, 1998, pp. 236. [34] O.S. Veelaert, D. Wit, K.F. Gotlieb, R. Verb, Carbohydr. Polym. 33 (1997) 153–162. ˇ ˇ ´ B. Siroká, [35] J. Sirok y, T. Bechtold, Woven Fabrics Book – Chapter 7: Alkali Treatments of Woven Lyocell Fabrics, 2012, ISBN 978-953-51-0607-4, http://dx.doi. org/10.5772/2594 [36] N.A. Bagrovskaya, T.A. Nikiforova, V.A. Kozlov, Russ. J. Gen. Chem. 72 (3) (2002) 345–348. [37] Z.R. Yue, W. Jiang, L. Wang, H. Toghiani, S.D. Gardner, Jr.C.U. Pittman, Carbon 37 (10) (1999) 1607–1618. [38] N.E. Kotelnikova, V.N. Demidov, G. Wegener, E. Windeisen, V.P. Kotelnikov, Cell. Chem. Technol. 237 (3–4) (2003) 225–238. [39] J. Cai, S. Kimura, M. Wada, S. Kuga, Biomacromolecules 10 (1) (2009) 87–94. [40] T. Rabilloud, L. Vuillard, C. Gilly, J.J. Lawrence, Cell. Mol. Biol. (Noisy-le-Grand, France) 40 (1) (1994) 57–75. [41] M. Harada, E. Katagiri, Langmuir 26 (23) (2010) 17896–17905. [42] B. Harekrishna, Kr. Dipak, G.P. Bhui, P.S. Sahoo, P. Santanu, M. Ajay, Colloids Surf. A: Physicochem. Eng. Aspects 348 (2009) 212–216. [43] P.K. Khanna, V.S. Subbarao, Mater. Lett. 57 (15) (2003) 2242–2245. [44] M. Rehan, H. Mashaly, S. Mowafi, A. Abou El Kheir, H.E. Emam, Dyes Pigments 118 (2015) 9–17. [45] Y.L. Wang, Y.Z. Wan, X.H. Dong, G.X. Cheng, H.M. Tao, T.Y. Wen, Carbon 36 (11) (1998) 1567–1571.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Functionalization of medical cotton by direct incorporation of silver nanoparticles Hossam E. Emam a,∗ , N.H. Saleh b , Khaled S. Nagy c , M.K. Zahran b a b c
Textile Research Division, National Research Centre, Dokki, Cairo 12311, Egypt Chemistry Department, Faculty of Science, Helwan University, Ain-Helwan, Cairo 11795, Egypt Food Engineering and Packaging Department, Agricultural Research Center, 9 Cairo University St., Giza, Egypt
a r t i c l e
i n f o
Article history: Received 11 March 2015 Received in revised form 2 April 2015 Accepted 12 April 2015 Available online 20 April 2015 Keywords: Medical cotton AgNPs Functionalization Coloration Absorbency Antibacterial action
a b s t r a c t Medical cotton is usually used to clean skin, pack wounds and in other surgical tasks. Such important usages make imparting the antibacterial property to medical cotton is so essential research. The current research focuses on functionalization of medical cotton by direct incorporation of silver nanoparticles (AgNPs) in two-step process namely, pre-alkalization followed by sorption. Decorative color and antibacterial action were accomplished for medical cotton after in situ incorporation of AgNPs without using any other external reducing agent. AgNPs were produced due to the reduction action of alcoholic and aldehydic groups of cotton’s skeletal blocks. Cotton fibers were acquired a decorative color attributed to surface plasmon resonance of AgNPs. The treated cotton was characterized by using electron microscope. Results showed that Ag0 with size distribution of 0–160 nm was formed in the cotton fibers and their size majority (70%) was less than 80 nm. The reduction of Ag+ to Ag0 was confirmed by measuring the carboxylic and aldehydic contents. The treated cotton exhibited excellent antibacterial action at low silver contents. The absorbency of cotton was not affected by treatment. The produced medical cotton could be used to safe cleaning of wounds without getting any microbial infections. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Medical cotton is produced from full bleached and sterilized cotton. It has a wide range of usage as it stored especially as stock medical and cosmetics’ supplies. Medical cotton is made in several forms, such as balls, pads, roll and fabrics. One common use for this type of cotton is as a vehicle for makeup remover, which can be poured onto it and then swabbed across the face. So it is produced commonly in the form of bandages, medical swabs, and other similar products; and because it is sterilized, it can also be used to pack wounds and in other surgical tasks, and thus it is so called medical cotton. Due to many processes occurred for the production of medical textiles; medical cotton is containing more function groups compared to raw cotton. Ionic (Ag+ ) and metallic (Ag0 ) forms of silver can be applied in the cellulose to impart the antibacterial property [1–4]. Sorption process is the common method to insert Ag+ in cellulose through the ion exchange interaction between Ag+ and H+ of carboxyl groups of cellulose [5–9]. Incorporation of Ag0 onto cellulose
∗ Corresponding author. Tel.: +20 201008002487. E-mail address: [email protected] (H.E. Emam). http://dx.doi.org/10.1016/j.ijbiomac.2015.04.018 0141-8130/© 2015 Elsevier B.V. All rights reserved.
is mainly occurred by deposition, which could be takes place via two different procedures. Ag+ is reduced to Ag0 followed by deposition on cellulose in the first procedure [3,4,10–12] while in the second one; Ag+ is absorbed on cellulose followed by in situ reduction [13–15]. Application of metallic nano-silver in cellulose is more preferable due to high surface area to volume ratio and the lower release of Ag+ ions from silver nanoparticels compared to silver ion salt [16] leads to the higher adhesion between metallic nano-silver and cellulose. Moreover, silver in its nano-metallic form has two important characters; color related to surface plasmon resonance absorption (SPR) and antibacterial actions [3,4,9,17–22]. Shape and size of nanosilver are two factors play an essential role in its SPR and then consequently influence their optical properties [23–26]. Since medical cotton is a material which comes in direct contact with the human body, its quality is very important and should satisfy the required pharmaceutical parameters. In the present work, AgNPs was introduced directly into medical cotton roll to impart antibacterial property and acquire a decorate color without any external reducers. Direct incorporation of AgNPs was carried out in two simple steps process namely, pre-alkalization using sodium hydroxide and submerging in AgNO3 . Cotton itself as cellulosic material was used as reducing agent for Ag+ and stabilizing agent
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for the produced Ag0 by its polymeric structure. UV/vis absorbance spectroscopy, scanning electron microscope, energy dispersive Xray spectroscopy and X-ray diffraction were all used to characterize the incorporation process. The colorimetric measurements (color strength and color space), aldehydic content and carboxylic content were measured to the treated cotton. The biological action of the cotton before and after treatment was estimated using disk diffusion method against two types of bacteria and two types of fungi.
2. Experimental 2.1. Materials and chemicals Absorbent medical cotton was provided by Chemical & Medical Industries Co., the Sixth Artificial region, 6th of October City, Egypt. The cotton fibers were used as received without further treatment. Silver nitrate (99.5%, from Sisco Reserch Laboratories PVT, Mumbai, India), Sodium hydroxide (96% from El Nasr Pharmaceutical chemicals Co., Cairo, Egypt), nitric acid (55%, from the Egyptian company for chemicals and pharmaceuticals, 10th of Ramadan, Egypt), boric acid (from El Gomhouria Co., Cairo, Egypt), hydrochloric acid (35.5%, from El Gomhouria Co., Cairo, Egypt), methylene blue MS dye (C16 H18 N3 SCl·xH2 O, 70% from s.d. Fine-Chem Ltd, Mumbai, India), potassium hydroxide (from El Nasr Pharmaceutical chemicals Co., Cairo, Egypt), bromophenol blue as pH indicator (from s.d. Fine-Chem. Ltd., Mumbai, India). Hydroxyl amine hydrochloride (98%, from Alpha Chemica, Mumbai, India) and absolute ethanol (99%, from El Nasr Pharmaceutical chemicals Co., Cairo, Egypt) were all analytical reagent grade and used without further purification.
3.2. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) The top view of untreated and Ag-treated cotton fibers surfaces were characterized by High resolution scanning electron microscopy (HR-SEM). Fiber samples were located on copper coated carbon tap double face, and then coated by the gold layer by evaporization of gold in argon atmosphere using sputter coater (S150 A, Edwards, UK). The surfaces of samples were scanned using high resolution scanning electron microscopy (HR-SEM Quanta FEG 250 with field emission technique, Philips, Netherland). Energy dispersive X-ray spectroscopy (EDX) analysis unit (EDAX AMETEK analyzer, materials analysis division, Philips, Netherland) conducts with the scanning electron microscope was used to characterize the surface chemical structures of cotton fibers after Ag treatment. 3.3. Color measurements The colorimetric data of the treated cotton fibers was recorded using a spectrophotometer with pulsed xenon lamps as light source (UltraScan Pro, Hunter Lab, USA), using 10◦ observer with illuminant of D65, d/2 viewing geometry and 2 mm measurement area. Color measurement data are lightness (L) from black to white, a* is a redness/greenish ratio and b* is yellowish/bluish ratio [27]. The corresponding color strength value (K/S) was assessed at 430 nm wavelength (430 ) by applying the Kubelka Munk [28] (Eq. (1)). The absorbance of treated cotton fibers were measured by the same instrument at 430 . (1 − R)2 K = S 2R
(1)
where R is the decimal fraction of the reflection of the cotton fibers, K is the absorption coefficient and S is the scattering coefficient. 3.4. Biological activity
2.2. Procedure The incorporation of Ag in medical cotton fibers was carried out in two steps process namely, pre-alkalization and sorption. In the pre-alkalization step, definite amount from medical cotton fibers was soaked in different concentrations of NaOH (0.01 N and 0.1 N) using 1/50 materials to liquor ratio (M/L) at room temperature for different time durations (5, 30 and 60 min). After certain time, fibers were taken out, gently squeezed and were then transferred to the sorption bath. In the second step, the wet alkalized fibers were fully immersed in AgNO3 solution with different concentrations (20–400 mg/L) using the same liquor ratio. The reaction mixture was agitated and its temperature was adjusted to RT, 50 ◦ C and 70 ◦ C. At the end of reaction time, treated fibers were removed and rinsed two times by tap water at RT using the same liquor ratio of treatment for neutralization, and then dried in oven at 70 ◦ C for 1 h, prior to further analysis. The residual solutions were kept to measure the absorbance spectra.
Antimicrobial activity of the cotton fibers was tested qualitatively against bacteria and fungi using a modified Kirby–Bauer disk diffusion method [29]. Two types of both bacteria and fungal were used in the test and they namely, Escherichia coli as gram −ve bacteria, Staphylococcus aureus as gram +ve bacteria, Aspergillus’s flavous grows as filamentous fungi and Candida albicans grows as yeast. Briefly, 100 L of the tested bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/mL for bacteria or 105 cells/mL for fungi [30]. A 100 L of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained. Plates inoculated with E. coli and S. aureus at 35–37 ◦ C for 24–48 h, with C. albicans at 30 ◦ C for 24–48 h and with A. flavous at 25 ◦ C for 48 h. The diameters of the inhibition zones were measured in millimeters with slipping calipers of the National Committee for Clinical Laboratory Standards [31]. The average width for zone of inhibition along a streak on either side of the tested fibers was calculated using Eq. (2). Blank sample with a diameter 1 cm was impregnated 10 L of tested concentration of the stock solutions. T −D 2
3. Measurements
W=
3.1. UV–visible spectra
where W is the width of clear zone of inhibition in mm, T is the total diameter of tested fiber and clear zone in mm, and D is the diameter of the tested fiber in mm.
At the end of treatment process, the cotton fibers were removed and the absorbance of supernatant solutions was measured. The UV–vis absorption spectra were used to measure the extinction of the residual solutions using a multi-channel spectrophotometer (T80 UV/VIS, cell length = 10 mm, PG Instruments Ltd, Japan) at the wavelength range of 250–600 nm.
(2)
3.5. Absorbency of fibers A 1 g of the dried cotton fibers was accurately weighed and compressed to form a disk with thickness of 3 mm to get taut surface
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of fiber with free of wrinkles. The fiber disk was used to measure the absorbency of fibers using the standard method for absorbency of textile [32]. The buret was placed in an appropriate location in the lab, and then the fiber disk was located 9.5 ± 1.0 mm below the tip of the buret. A one drop of distilled water was allowed to fall on the fiber disk and the stopwatch was started immediately. The time required for the drop of water to completely disappear and appear only as a dull wet spot was measured. If the water drop disappears immediately, the time records as “zero” and when the elapsed time exceeds 60 s, it records as “60+ s”. The time of disappearance of water drop was tested at five different locations and the average was considered. 4. Chemical analyses 4.1. Detection of silver content Silver in the 0.2 g treated dried cotton fiber was extracted by 20 mL of 15 wt% nitric acid for 2 h at 80 ◦ C. The Ag concentrations in the extracted solution was analyzed with Contraa 700 flame atomic absorption spectrophotometer (AAS, Analytik, Jena, Germany) equipped a silver lamp at 328.1 nm wavelength. The extracted Ag from fibers was calculated according to Eq. (3). C ×V W (1 − MC/100)
A−B × 100 A
4.3. Aldehydic content The aldehydic content in the medical cotton fibers was measured based on oxime reaction [34] as follows: a 50 g of hydroxyl amine hydrochloride was dissolved in 100 mL distilled water and dilute to one liter using ethanol, then the pH of solution was adjusted to 3.4 with ethanolic potassium hydroxide. A 0.2 g of cotton fiber was immersed in 30 mL of the hydroxyl amine solution, mixed thoroughly and allowed to stand at room temperature for 30 min. A blank solution without sample was performed. Solutions were titrated with 0.25 N ethanolic KOH till the greenish yellow end point, using bromophenol blue as indicator. The percentage of aldehydic content was calculated according to Eq. (6). CHO% =
where CHO is the aldehydic content in cotton fibers (%), B is the KOH volume consumed in titration of blank solution (mL); S is the KOH volume consumed in titration of sample solution (mL); W is the weight of dried treated fibers (g); MC is the moisture content in dried treated fibers (%).
(4)
Medical cotton are sterilized fabrics as discussed previously, used for cleaning skin, pack wounds and in other surgical tasks, however, it does not have any antimicrobial activity. Thus, our target is acquiring an additional function as antibacterial actions 2500
4.2. Carboxyl content
A
RT
50 °C
70 °C
2000
1500
1000
500
(5)
where COOH is the carboxyl content (mmol/g), C1 is the concentration of methylene blue in the blank (sample without fibers) (mg/L), C2 is the concentration of methylene blue in the samples (in the
0 50
0
100
150
200
250
300
350
400
450
AgNO3 in Solution (mg/L) 150
Ag in Fiber (mg/Kg)
Carboxylic group contents of cotton fibers before and after Agtreatments were measured using the methylene blue dye method [33]. The method can be described briefly as follows: three different solutions of 300 mg/L aqueous methylene blue, borate buffer solution with pH 8.5 and 0.1 M HCl were prepared and namely, solutions A, B and C respectively. Equal volume of 25 mL from solutions A and B were added to a 0.17 g of cotton fiber in a 50 mL bottle followed by shaking for 20 h at room temperature. A 2.5 mL of that solution mixture was added to a 5 mL of solution C and total volume was completed to 50 mL by distilled water. The absorbance of solutions were measured using multi-channel Spectrophotometer (T80 UV/VIS, d = 10 mm, PG Instruments Ltd, Japan) at wavelength of 664.5 nm (max of methylene blue). A stock solution of 100 mg/L methylene blue was used to prepare calibration solutions at pH 8.5 in the range of 0.5–10 mg/L. Using the slope of calibration curve, the carboxyl content was calculated from Eq. (5). (C1 − C2 ) × 0.00313 W (1 − MC/100)
(6)
5. Results and discussion
where MC is the moisture content (%), A is the initial condition weight (g) and B is the weight of oven dried fiber (g).
COOH =
(B − S) × 100 W (1 − MC/100)
(3)
where Ag is the silver content in cotton fibers (g/kg), C is the silver concentration (mg/L) in extracted solution; V is the volume of extracted solution (0.02 L); W is the weight of dried treated fibers (g); MC is the moisture content in dried treated fibers (%). The moisture contents in the untreated and treated cotton fibers were measured as follows: a 1 g fiber was weighed accurately up to four digital numbers and then dried at 105 ◦ C for ca. 4 h. The dried samples were reweighed up to fixing weight and then the moisture contents were calculated according to Eq. (4). The obtained moisture contents were 5.8% for untreated cotton fibers. MC =
presence of fibers) (mg/L), W is the weight of cotton fiber samples (g) and MC is moisture content (%).
Ag in Fiber (mg/Kg)
Ag =
251
B
RT
50 °C
70 °C
100
50
0 0
20
40
60
AgNO3 in Solution (mg/L) Fig. 1. Ag content in cotton fibers as function of silver nitrate concentration in solution (A), magnified at low concentration (B).
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to medical cotton by the in situ incorporation of AgNPs. The additional function helps medical cotton for peaceful cleaning of injuries/wounds and for the perfect usage in surgical tasks without getting any microbial infections. Two steps process was carried out for direct incorporation of Ag in the medical cotton fibers namely, pre-alkalization and sorption. Pre-alkalization step was used to swell the fibers to facilitate the Ag diffusion into fibers in the sorption step [35]. It is known that, pKa of COOH in cellulose equals to 4.0 [36]. Sorption of silver ions on cellulose is an ion-exchange reaction between Ag+ with COO− of cotton [5,37]. So, sorption capacity of Ag is largely influenced by the pH of solution. Pre-alkalization process of cotton fibers increases the sorption capacity due to two factors; fully dissociation of COOH for cellulose and increasing the affinity to Ag as a result of the swelling of cellulose. In the sorption step, Ag+ was absorbed firstly on cellulose from AgNO3 by ion-exchange with COO− of cotton. Reduction of Ag+ to Ag0 was carried along with the ion-exchanging process or thereafter. The reduction was occurred by reducing groups of cellulose
mainly aldehydic and alcoholic groups and it was catalyzed by alkalinity and heat [7,21,38,39]. Additionally, the tendency of Ag to auto-catalytic reduction, as the reduction rate was accelerated after forming the first nuclei of Ag0 [40,41].
5.1. Silver content In Fig. 1, the silver content in the cotton fibers at the different temperatures was shown plotted against the realistic content of Ag in solution. The Ag content in cotton fibers was ranged between 48.7 and 2391.5 mg/kg and it was observed increased continuously with increasing AgNO3 in solution. At the lower concentration (20 mg/L), Ag content was observed almost the same at different temperatures. At 70 ◦ C, Ag in fibers was very high and reached 2391.5 mg/kg, but it was lower more than 20 times to record 113.4 mg/kg by using 400 mg/L AgNO3 . Except with using 400 mg/L, Ag content in fibers was not changed dramatically from RT to 50 ◦ C. In conclusion, it can be say that, the Ag content in fibers
Fig. 2. Absorbance spectra of residual solutions after treatment of cotton fiber with AgNO3 at different conditions: (A) 0.01 N NaOH, (B) 0.1 N NaOH, (C) immersion in AgNO3 for 1 h at room temperature, (D) immersion in AgNO3 for 1 h at 50 ◦ C, (E) immersion in AgNO3 for 1 h at 70 ◦ C.
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Absorbance (at 420 nm)
1.6
RT
50 °C
5.3. Color measurements
70 °C
1.2
0.8
0.4
0 0
50
100
150
200
250
300
253
350
400
450
AgNO3 Concentration (mg/L) Fig. 3. Absorbance of Ag–cotton fibers at wavelength of 430 nm as function of silver nitrate concentration in solution.
was follow the order of at 70 ◦ C 50 ◦ C ≥ RT. This is may account for the greater Ag deposition in fibers at 70 ◦ C.
5.2. UV–visible absorption spectra Depending on their size and morphological structure, AgNPs exhibit surface plasmon band resonance (SPR) as known formerly [17–20]. Thus, at the end of sorption step, the absorbance spectra of residual solution for silver were measured by using spectrophotometer. Effect of NaOH concentrations (0.01 and 0.1 N) and temperature (RT, 50 ◦ C and 70 ◦ C) as function of reaction time was studied and showed in Fig. 2. An absorbance peak was observed in the wavelength range of yellow color (410–435 nm), which is in agreement with visual observation of solution color. The absorbance peak is corresponding to the SPR of spherical AgNPs [17,21,42]. The peak of Ag+ , at range of 250–300 nm [43], was not recorded in all experiment. The absorbance intensity was showed higher two times with using 0.1 N comparing to 0.01 N of NaOH concentrations. This is reflecting the role of alkalinity in the reduction of Ag+ besides their function in swelling of cotton fibers to penetrate AgNPs [22]. At different reaction temperatures, changing in Ag+ concentration is accompanied by observable change in the absorbance intensity. By apply 70 ◦ C, the absorbance intensity is a directly proportional with Ag+ concentration. But this trend was not seen at lower temperature, which could be related to the heterogeneous process of Ag+ reduction as a result of insufficient heating to activate the reduction process. This is explained the important role of temperature in activation the reduction process of Ag+ . Absorbance of treated cotton fibers was measured by Ultrasacn pro spectrophotometer at maximum peak (max ). It was found that the max equal to 430 nm which is characteristic for the SPR of spherical AgNPs and this is compatible with the results of absorbance spectra for residual solution. The absorbance data obtained for treated cotton fibers is comparable to that reported for cotton fabrics after in situ deposition of AgNPs using citrate [44]. Fig. 3 declares that, at max , the absorbance of Ag-fibers was increased with temperature and Ag+ concentration and reached 1.4 by using 400 mg/L at 70 ◦ C. This is an indication for that the amount of AgNPs in the cotton fabrics was largely affected by reaction temperature and Ag+ concentration. The absorbance intensity is directly proportional to the AgNPs content in fibers, as the higher absorbance intensity, the higher AgNPs content and this was achieved at 70 ◦ C.
The color of cotton fibers was changed after removed from the impregnation bath. Furthermore, this was quantified through measurements of color coordinates and color strength of the dried fibers after treatment and recorded in Table 1. The strength of color (K/S) was not considerably changed up to 50 ◦ C. But, by raising temperature to 70 ◦ C, the K/S was grown up from zero to 10.32 with increment the Ag+ concentration from zero to 400 mg/L. The lightness (L*) of fibers was decreased with increasing both of the Ag+ concentration and the temperature from RT to 70 ◦ C, while values of a* and b* were increased. L* was recorded 95.93 (untreated) and 76.87 (RT), 60.77 (50 ◦ C) and 37.03 (70 ◦ C) for fibers treated with 400 mg/L. At the same experimental conditions, the value of a* was changed from 0.02 to 4.17, 6.20 and 14.89, while, the values of b* were 2.50, 3.40, 10.82 and 19.66 respectively. From these data, color of fibers was changed from white through creamy to yellowish red after Ag-treatment and the color became more deeply by increasing the concentrations of Ag+ . 5.4. SEM images, EDX spectrum and size distribution Scanning electron microscope (SEM) was used to notify the change in morphology of cotton fibers surface caused by incorporation of AgNPs. In Fig. 3, SEM images show the smooth surface of cotton fabrics before treatment (Fig. 3a and b). After Ag-treatment, the Ag clearly with intense amounts on the cotton surface and some aggregates consisting of smaller particles were observable. The shape of Ag particles showed here was similar to that obtained previously on cellulose fiber/fabrics [10,22,45]. The presence of Ag particles on the cotton fibers were further confirmed by EDX, as signal of Ag was appeared in Fig. 3e. The amount of Ag was recorded ca. 0.98% from the sample in two different places, but this value cannot be considered because it depends on some factors such like dispersion homogeneity and the area which electron beam incident on. Size of Ag particles in the fibers was measured by software program using SEM photos. Fig. 3f confirms that the Ag particles were in nano-dimension and their size distributions were ranged from 0 to 160 nm. The majority of particles were located in 0–80 nm (ca. 70%). Size distribution of Ag particles in cotton fibers was lower than that recorded previously [9,45]. The fact of AgNPs presence on the cotton fibers explained and supported UV–vis absorption spectra and color results for cotton fibers (Figs. 2 and 4 and Table 1). 5.5. Carboxylic and aldehydic content The reduction of Ag+ to Ag0 by cotton fibers was initially detected by color change of solution and fiber to yellow and was later confirmed by scanning electron microscopic. The reduction was carried out by the reducing end group (aldehydic e.g. CHO and alcoholic groups e.g. CH2 OH) of cotton. The reaction between silver and cotton is two-faced reaction, comprised a reduction reaction for Ag+ contrary oxidation reaction for cotton. When Ag+ is transformed to Ag0 , jointly CH2 OH and/or CHO are oxidized to COOH [21]. Therefore, the carboxyl (COOH) and aldehyde (CHO) contents were both measured for cotton fibers before and after treatment and the data was recorded in Table 2. For untreated medical cotton, CHO content was 15.82% and COOH content recorded 20.76 mmol/kg. After Ag treatment, CHO content decreased while COOH content increased by increasing both of AgNO3 concentration and temperature, confirming the reduction of Ag+ to Ag0 by functional groups of cotton. At room temperature (RT), CHO and COOH contents were not changed obviously, however AgNO3 concentration may be accounted for the insufficient temperature to activate the reduction process [7,38,39,43]. By increasing the
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Fig. 4. Scanning electron microscope photos for (A and B) untreated at different magnifications, (C and D) Ag–cotton fibers at different magnifications, (E) EDX analysis for Ag–cotton fibers and (F) size distribution of AgNPs on cotton fibers.
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Table 1 Color data of treated cotton as function of silver content. Sample
AgNO3 conc. (mg/L) 0
Ag content (mg/kg)
L*
a*
b*
K/S (at = 430 nm)
0
95.93
0.02
2.5
0.01
RT
20 50 100 200 400
48.7 50.2 59.6 81.4 113.4
91.93 89.65 85.56 81.29 76.87
1.38 1.21 2.93 2.26 4.17
1.93 2.90 3.00 4.43 3.40
0.00 0.05 0.10 0.16 0.28
50 ◦ C
20 50 100 200 400
59.5 48.5 101.4 181.1 1576.0
76.11 81.84 77.88 77.73 60.77
6.63 5.16 5.77 6.74 6.20
13.60 9.53 14.83 11.41 10.82
0.49 0.26 0.49 0.41 1.62
70 ◦ C
20 50 100 200 400
66.8 147.4 1144.8 1779.7 2391.5
74.79 62.20 60.46 40.42 37.03
7.46 9.94 12.47 14.01 14.98
17.29 17.86 20.28 18.84 19.66
0.69 1.68 2.05 7.75 10.32
Untreated
Table 2 Carboxylic and aldehydic contents of treated cotton fibers. Sample Untreated
AgNO3 conc. (mg/L) 0
Ag content (mg/kg)
Carboxyl content (mmol/kg)
Aldehyde content (%)
0
20.76
15.82
RT
20 50 100 200 400
48.7 50.2 59.6 81.4 113.4
19.42 23.81 23.09 23.66 22.76
14.54 15.56 13.45 13.48 13.35
50 ◦ C
20 50 100 200 400
59.5 48.5 101.4 181.1 1576.0
21.50 27.44 27.55 32.76 34.30
15.67 13.48 10.21 10.35 9.46
70 ◦ C
20 50 100 200 400
66.8 147.4 1144.8 1779.7 2391.5
22.02 26.25 26.35 39.76 46.80
12.51 10.82 9.44 7.65 7.28
reaction temperature to 50 ◦ C, the decrement in CHO content and the increment in COOH content were observed clearly as function of AgNO3 concentration. As COOH and CHO contents were estimated to be 34.30 mmol/kg and 9.46%, respectively by treatment with 400 mg/L AgNO3 . The percent of CHO content was much diminished to 7.28% and the COOH content was much augmented to 46.80 mmol/kg when the temperature was raised to 70 ◦ C. The considerable variation in the contents of CHO and COOH by rising the temperature, explains the role of heating in the stimulation of redox reaction between Ag and cotton.
5.6. Absorbency Medical cotton is a soft mass important to clean skin and pack wounds and its high absorbency is a significant sign. Thus, to be used in the medical field, the absorbency property for medical cotton supposed to be not decreased significantly. The absorbency of medical cotton fibers was measured by using the standard method for water droplet disappear [32]. Results showed that the original medical cotton was absorbed water droplet and completely wetted by water immediately, due to the presence of numerous freely hydroxyl groups on the surface of medical cotton. The same behavior was obviously recorded for Ag treated cotton sample, as water droplet was easily adsorbed and vanished immediately on its surface at zero time, however Ag content. This indicates to that the incorporation of AgNPs into medical cotton did not change
absorbency property of cotton, making the Ag treated medical cotton is more appropriate to use in the treatment and packing of wounds.
5.7. Antimicrobial activity Natural fibers/fabrics can provide an excellent environment for growing the microorganisms due to their ability to retain moisture. On the other hand, medical cotton was commonly used to pack the wounds and in other surgical tasks, thus it is so important to be having antimicrobial action. The antimicrobial property of the untreated and treated cotton fibers was tested qualitatively against bacteria and fungi using disk diffusion method. E. coli as gram −ve and S. aureus as gram +ve were used as bacteria, while A. flavous and C. albicans were used as fungi. Three samples different in Ag content were used in the antimicrobial test. Regardless Ag concentration on the fibers, antifungal action was not shown for Ag-medical cotton fibers. Table 3 shows the antibacterial activity results and the untreated medical fibers were not exhibited any antibacterial action. But, the antibacterial activity was observed for Ag-medical cotton fibers due to the leached of Ag ions from fibers surface to surrounding. The inhibition of bacterial colonies was depended on the Ag concentration on the fibers and bacterial type. At lower Ag content (66.8 mg/kg), no antibacterial action was notified against E. coli (0 mm inhibition zone), while it was observed against S. aureus (12 mm inhibition zone). By increasing the Ag
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Table 3 Biological activities of treated cotton fibers at 70 ◦ C. Sample
AgNO3 conc. (mg/L)
Ag content (mg/kg)
Antibacterial action Inhibition zone diameter (mm) Escherichia coli (gram −ve)
Untreated 1 2 3
0.0 20 100 400
0.0
0.0
66.8 1144.8 2391.5
0.0 14 13
content on fibers, the difference in diameter of inhibition zone was not significant, but it was observed for both types of bacteria strains. The results of antibacterial presented here for medical cotton fibers, is better than that recorded in the literature for cotton fibers treated with AgNPs [3,4,13]. 6. Conclusions Functional medical cotton with an excellent antibacterial property and with a yellowish decorative color was effectively obtained by direct incorporation of AgNPs. The AgNPs incorporation into cotton was carried out without using any external reducer, in twostep process namely, pre-alkalization and sorption. The reducing groups of cotton were used to reduce Ag+ to Ag0 , and the results of COOH and CHO contents confirmed the reduction process. AgNPs was shown on the cotton surface by SEM and their size was ranged between 0 and 160 nm. Color data declare that yellow colored cotton was obtained with different color shades as function of Ag content. Excellent antibacterial property was recorded against the common pathogenic bacteria: E. coli as gram −ve and S. aureus as gram +ve. The absorbency for cotton was not absolutely changed by Ag treatment. The acquired properties due to treatment, lead medical cotton is more appropriate for safe cleaning of wounds or in surgical tasks without getting any microbial infections. References [1] N. Sachinvala, D.V. Parikh, P. Sawhney, S. Chang, J. Mirzawa, W. Jarrett, B. Joiner, Polym. Adv. Technol. 18 (8) (2007) 620–628. [2] M. Bajpai, P. Gupta, S.K. Bajpai, Fib. Polym. 11 (1) (2010) 8–13. [3] M.H. El-Rafie, H.B. Ahmed, M.K. Zahran, Carbohydr. Polym. 107 (2014) 174–181. [4] M.K. Zahran, H.B. Ahmed, M.H. El-Rafie, Carbohydr. Polym. 108 (2014) 145–152. [5] T. Saito, A. Isogai, Carbohydr. Polym. 61 (2) (2005) 183–190. ˇ ´ N. Radic, ´ M.B. Obradovic, ´ S. Dimitrijevic, ´ M.M. Kuraica, P. Skundri ´ [6] M. Kostic, c, Chem. Ind. Chem. Eng. Q. 14 (4) (2008) 219–221. [7] S. Ifuku, M. Tsuji, M. Morimoto, H. Saimoto, H. Yano, Biomacromolecules 10 (9) (2009) 2714–2717. [8] C.I. Su, C.C. Peng, Y.C. Lu, Text. Res. J. 79 (16) (2009) 1486–1501. ˇ [9] H.E. Emam, A.P. Manian, B. Siroká, H. Duelli, B. Redl, A. Pipal, T. Bechtold, J. Clean. Prod. 39 (2013) 17–23. [10] H.J. Lee, S.Y. Yeo, S.H. Jeong, J. Mater. Sci. 38 (10) (2003) 2199–2204. ˇ ´ Z. Saponji ´ V. Vodnik, B. Potkonjak, P.N.J. Jovanˆcic, ´ M. Radetic, ´ Carbohydr. [11] V. Ilic, c, Polym. 78 (3) (2009) 564–569. [12] S. Ravindra, Y. Murali Mohan, N. Narayana Reddy, K. Mohana Raju, Colloids Surf. A: Physicochem. Eng. Aspects 367 (23) (2010) 31–40. [13] T. Maneerung, S. Tokura, R. Rujiravanit, Carbohydr. Polym. 72 (1) (2008) 43–51. [14] L.C.S. Maria, A.L.C. Santos, P.C. Oliveira, A.S.S. Valle, H.S. Barud, Y. Messaddeq, S.J.L. Ribeiro, Ciênc. Technol. 20 (1) (2010) 72–77.
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