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Structure and properties of silk fibroin grafted carboxylic cotton fabric via amide covalent modification
Accepted Manuscript Title: Structure and properties of silk fibroin grafted carboxylic cotton fabric via amide covalent modification Author: Yunhui Xu Dingding Chen Zhaofang Du Jifeng Li Yunxia Wang Zhen Yang Fengxia Peng PII: DOI: Reference:
S0144-8617(16)31456-4 http://dx.doi.org/doi:10.1016/j.carbpol.2016.12.071 CARP 11878
To appear in: Received date: Revised date: Accepted date:
19-9-2016 3-12-2016 31-12-2016
Please cite this article as: Xu, Yunhui., Chen, Dingding., Du, Zhaofang., Li, Jifeng., Wang, Yunxia., Yang, Zhen., & Peng, Fengxia., Structure and properties of silk fibroin grafted carboxylic cotton fabric via amide covalent modification.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.12.071
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Structure and properties of silk fibroin grafted carboxylic cotton fabric via amide covalent modification Yunhui Xu a,*, Dingding Chen b, Zhaofang Du a, Jifeng Li a, Yunxia Wang a, Zhen Yang a, Fengxia Peng a a
College of Light-Textile Engineering and Art, Anhui Agricultural University, Hefei, Anhui 230036, China Biotechnology Center, Anhui Agricultural University, Hefei, Anhui 230036, China
b
* Corresponding author. Tel.: +86 551 6578 6459; fax: +86 551 6578 6331. E-mail address: [email protected] (Y. Xu).
Research highlights 1. HNO3/H3PO4–NaNO2 oxidized cotton fabrics were modified by silk fibroin crosslink. 2. Silk fibroin was grafted on carboxylic cotton fabric via the C–N amido bond. 3. Textile properties of silk fibroin-grafted oxidized cotton fabrics improved. 4. Cactus flavonoid-treated grafted cotton fabric showed high antimicrobial activity.
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Abstract A novel eco-friendly production of silk fibroin-grafted carboxylic cotton fabrics without using any crosslinking agents was developed via the reaction of silk fibroin with oxidized cotton. The effect of reaction parameters on mechanical properties of oxidized fabrics and graft add-on of silk fibroin in grafted fabrics was examined. The results showed that appropriate oxidation time of HNO3/H3PO4–NaNO2 mixture and grafting time of fibroin were 45 min and 2 h respectively. FTIR analysis of grafted sample indicated that the C–N amido bond was generated through the reaction between primary amines in silk fibroin and carboxyl groups in oxidized cotton, which was further confirmed by XPS spectra. The grafted fabrics were also evaluated for physical properties like tensile strength, wrinkle recovery angle, moisture regain and yellowness index. Cactus flavonoid coated on grafted fabric through treatment with flavonoid extract of cactus, such treated fabric exhibited a highly inhibitory effect against both Staphylococcus aureus and Escherichia coli bacteria.
Keywords: Carboxylic cotton fabric; Grafting; Silk fibroin; Cactus flavonoid extract; Antibacterial activity
1. Introduction
Natural cellulose products, which are polymers with glucose linked through 1,4-β-glucosidic bond, especially cotton fabrics, are highly popular for their excellent properties, such as versatility, softness, hygroscopicity, breathability, affinity to skin, biodegradability and regeneration property (Liu, Ren, & Liang, 2015; Shafei & Abou-Okeil, 2011). These products, however, have some inherent limitations such as wrinkle, shrinkage, low dye uptake, ultraviolet (UV) and microbial degradation (Hou, Zhang, & Wang, 2012; Liu et al., 2015; Zhang, Chen, Ling, & Zhang, 2009). Therefore the modification of natural cellulose products is an important method to provide those products with improved properties to overcome the above mentioned limitations. The modification usually involves introducing functional materials to the surface or interior of the natural celluloses, or grafting them on the macromolecule of the celluloses (Bashar & Khan, 2013; Simoncic & Tomsic, 2010; Tissera, Wijesena, & Nalin de Silva, 2016; Wijesena, Tissera, & Nalin de Silva, 2015; Wijesena, Tissera, Perera, Nalin de Silva, & Amaratunga, 2015). Selective oxidation, as a new modification approach to cellulose, aroused many scholars’ interest. Basing on the different oxidizing agents and oxidation conditions, the selective oxidation occurs at two adjacent secondary -3-
hydroxyls in cellulose glucose units and cleaves the C2–C3 bond of the glucopyranoside ring and introduces two aldehyde groups at C2 and C3 positions (Potthast, Schiehser, Rosenau, & Kostic, 2009; Xu, Huang, & Wang, 2013), or the oxidation reaction proceeds with the primary hydroxyl of cellulose and acquires the carboxyl group at C6 in cellulose anhydroglucose units (de Nooy, Besemer, & van Bekkum, 1995; Kumar & Yang, 2002; Xu, Liu, Liu, Tan, & Zhu, 2014). Among them, the oxidant system of HNO3/H3PO4–NaNO2 exhibits an arresting route to introduce carbonyl and carboxyl functional groups into cellulose fibers which is characterized by its quick reaction rate, high oxidation selectivity and yield, low cellulose degradation and mild reactive conditions during the oxidation process (Kumar & Yang, 2002; Xu et al., 2014). In particular, partial oxidation of cellulose by the HNO3/H3PO4–NaNO2 system not only causes new functional groups in addition to the hydroxyl groups in polymers but also retains physical property of cellulose, which fabricates the reactive cellulose polymers for further chemical modification. Recently, many researches have been devoted to the preparation, microstructure, and its application of the oxidized cellulose (Kaputskii et al., 1995; Sharma, Rajamohanan, & Varma, 2014; Wu, He, Huang, Wang, & Tang, 2012). However, less attention has been paid to the oxidized cotton fiber for the textile usage (Liu, Nishi, Tokura, & Sakairi, 2001; Xu et al., 2013). Silk fibroin is a natural biological polymer composed of 18 α-amino acids, among which Gly, Ala, and Ser are the main amino acids (Tao, Kaplan, & Omenetto, 2012). Through a degumming process, silk fibroin is purified from outer sericin of silk fibers. Regarded as a safe material for human beings and animals (Li, Liu, Li, & Wang, 2012), silk fibroin has been extensively applied in the textile, foodstuff, cosmetic, and biomedical material (Kundu et al., 2014; Lu et al., 2011). Therefore, in recent years, the efforts of possible end-users of silk fibroin as green finishing agent for clothing products (e.g. natural or man-made textiles), which present requiring properties of softness, comfort, moisture absorption, and antistatic properties, have attracted considerable attention. Various methods have been developed to fabricate fibroin treated fabrics, the modifications have been limited to integration fibroin on the cellulose textiles with crosslinking agents (Lu, 2010), plasma irradiation (Xia & Wang, 2004) or partial carboxymethylation of cellulose (Wijesena, Tissera, Perera, & Nalin de Silva, 2014). By periodate oxidation of cotton and generation dialdehyde groups in cellulose chain, the crosslinking reaction could proceed between fibroin and oxidized cotton fiber through formation of Schiff base (Lin, Yao, Chen, & Wang, 2008). In our previous study, a carboxylic cellulose fiber, characterized by a carboxyl group of C6 site in glucose of cellulose chain, was produced by selective oxidation of HNO3/H3PO4–NaNO2 system (Xu et al., 2014). It has been -4-
utilized to graft chitosan without any other reagents for antibacterial finishing of the textile (Xu, Qiu, Zhang, & Zhang, 2014). This paper presents a novel approach of silk fibroin grafted oxidized cotton fabric (SFGCF), the facile process involves conversion of the C6 primary hydroxyl in cotton glucose units into carboxyl group by selective oxidation of HNO3/H3PO4–NaNO2 and their subsequent treatment with fibroin aqueous solution in an amine formation reaction, which is the classic crosslinking reaction between the generated carboxyl groups in oxidized cotton and amino groups in silk fibroin (Scheme 1). This novel development is to crosslink silk fibroin into textile substrates for formulating advanced cellulose–fibroin based composites without using any cross-linkers. The optimum parameters of the graft reaction of fibroin on carboxylic cotton fabric were discussed. The reaction was also probed by the Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The morphology and physical properties of grafted cotton fabrics were also measured. Additionally, SFGCF has a polycationic fibroin residue, which may interact with flavonoid extract of cactus that has an acidic function, such fibroin grafted cotton can serve as a best platform for the attachment of flavonoid extract of cactus to apply in antibacterial products. Flavonoid is extracted from the succulent cladode of cactus (Opuntia dillenii Haw). Cactus flavonoid is well known to have antimicrobial, antioxidant, anti-inflammatory, and anti-tumor activities (Benayad, Martinez-Villaluenga, Frias, Gomez-Cordoves, & Es-Safi, 2014; Cai, Gu, & Tang, 2010; Sanchez et al., 2014). It is taken cactus flavonoid as antibacterial to endow fibroin grafted cotton fabric with antimicrobial performance by treating SFGCF surface in a solution of flavonoid extract from cactus. The significance of this processing is to achieve bioactive fibers cactus flavonoid-coated SFGCF, which may be used as underwear, footgear, medical textiles and packing materials. And then, the antibacterial activity of flavonoid extract of cactus complex SFGCF was also examined.
Scheme 1. Crosslinking reaction between carboxylic cotton and silk fibroin.
2. Materials and methods
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2.1. Materials
Sodium nitrite (NaNO2), nitric acid (HNO3, 68%), phosphoric acid (H3PO4, 85%), acetone, sodium carbonate, hydrochloric acid, sodium hydroxide, calcium chloride, and ethanol were purchased from Aladdin Chemical Reagent Co., USA, as analytical grades. Cactus flavonoid extract was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All chemicals were used without further purification. Woven, bleached and scoured cotton fabric (58×30 number of yarns per cm2, 121 g/m2) was purchased from Intertek Group (Shanghai, China). Bombyx mori (B. mori) silk yarn was kindly supplied by Sericulture Research Institute, Anhui Academy of Agricultural Sciences (China). Staphylococcus aureus (S. aureus, ATCC 6538) and Escherichia coli (E. coli, ATCC 8099) were obtained from Shanghai Luwei Sci. & Tech. Co., Ltd. (China). Nutrient broth and nutrient agar were purchased from Scas Ecoscience Technology Inc (China). Deionized water (18MΩ.cm) was employed throughout this work.
2.2. Methods
2.2.1. Oxidation of cotton fabrics by HNO3/H3PO4–NaNO2 system Nitric acid and phosphoric acid were mixed in the brown wide-necked bottle with a 2:1 (v/v) ratio, thereafter the dried cotton fabrics, with a liquor ratio of 1:20 (w/v), were soaked in the mixture solution. Subsequently 1.4% (w/v) of sodium nitrite was added all at once, the bottle containing mixtures was covered with a glass stopper and gently oscillated at 40 ºC for 15–300 min in the absence of light. After the oxidation, the cotton fabrics were washed thoroughly with acetone and soaked in deionized water for 3 h to remove oxidant. The resulting carboxylic fabrics were dried in the atmosphere overnight at room temperature and then kept in desiccator before grafting reactions.
2.2.2. Preparation of SF-grafted carboxylic cotton fabrics Raw B. mori silk was first degummed twice in the boiling solution containing 0.2% (w/w) Na2CO3 and 1% (w/w) degumming agent in liquor ratio of 1:100 for 1 h to remove the sericin protein. Afterwards the degummed silk yarn was dissolved in a mixture solution of calcium chloride, ethanol, and water (CaCl2:C2H5OH:H2O = 1:2:8 mole ratio) at 70 ºC for 1 h. After filtering, the resulting fibroin solution was dialyzed for 3 days against running deionized water to eliminate CaCl2 salt, smaller molecules, and some impurities via a Oso-T8340 dialysis membrane with the molecular cutoff of 3500 Da (Union Carbide Co., USA), and then the silk solution was freeze-dried. Eventually, the fibroin powder with the molecular weight of 5000–30000 (Mw) was achieved by the degradation method with -6-
aqueous hydrochloric acid reported by Nam and Park (2001). Subsequently a fibroin solution was prepared with deionized water and adjusted to about pH 6.0 using 0.1 M HCl solution. Then the grafting reaction was carried out in a flask. The above mentioned oxidized fabric (of known weight) was placed in flask containing fibroin solution (2%, w/w) for 0.5–4 h at 60 ºC maintaining fabric to liquor ratio 1:50, with constant stirring. After that, the grafted sample was washed with hot water several times and soaked in deionized water for 12 h at ambient temperature, to remove the ungrafted silk fibroin on the fabric. Finally the resulting cotton fabric was dried at 80 ºC under vacuum for 2 h to produce the silk fibroin grafted oxidized cotton fabric (SFGCF). The un-oxidized cotton fabric was also treated using the same procedure to produce the control sample. Thereafter the samples ungrafted or grafted with silk fibroin conditioned in a vacuum desiccator containing silica gel till the constant weight was reached, and then weighed on a BS210S photoelectric scale, which was termed the dry weight. The graft add-on was calculated through the following formula: Graft add-on (% w/w)
where
W1 W0 100 W0
(1)
W0 and W1 are the dry weight of ungrafted fabric and grafted fabric, respectively.
2.2.3. Carboxyl group content analysis The carboxyl groups of the oxidized cotton react with the salts of weaker acids such as calcium acetate, forming a salt of the oxidized cotton and releasing an equivalent amount of the weaker acid. On this basis as well as by the method described in our recent paper (Xu et al., 2014). The carboxyl group content in the oxidized sample was calculated as follows: Carboxyl group content (mmol/g)
where 0.1
0.1M VNaOH 1000 m (1 w / 100)
(2)
M is the concentration of NaOH, VNaOH is the volume (l) of NaOH solution used in titration after
correcting for the blank,
m is the weight of oxidized cotton sample (g), and w is the moisture content (%).
2.2.4. Fourier transform infrared spectroscopy (FTIR) analysis The FTIR spectra of fabric samples were recorded with a Nicolet 380 FTIR spectrophotometer (Thermo-Electron Corporation, USA) using transmission technique for KBr pellets by recording 50 scans in %T mode with a resolution of 4 cm–1 in the wave numbers range of 4000–400 cm−1. -7-
2.2.5. X-ray photoelectron spectroscopy (XPS) analysis XPS spectra were carried out with a XSAM-800 photoelectron spectrometer (Kratos, UK) equipped with a monochromatic Al-Kα X-ray source. The samples were recorded using X-ray anode of 180 W and high voltage of 12.0 kV operating at a working pressure of 2×10–7 Pa in 0.1 eV steps with 100 eV analyzer passing energy at the radiation (1253.6 eV). The position of the carbon peak (284.8 eV) for C1s was used to calibrate the XPS scale for all substrates. Acquired data fitting was performed using commercially available software with 100 % Gaussian curve fitting. A linear background subtraction was used for C1s, N1s, and O1s curves, and a Shirley background subtraction was used for Cu 2p3/2 (932.67 eV).
2.2.6. Scanning electron microscopy (SEM) analysis Surface morphologies of oxidized cotton fabric and grafted cotton fabric were imaged by scanning electron microscope (Hitachi X-650, Japan) operating at 5.0 kV. All samples were conductively plated with gold sputtering before imaging.
2.2.7. Measurement of fabric properties 2.2.7.1. Moisture regain. The moisture regain was determined by the vacuum desiccator method with sodium nitrite to give 65% relative humidity (RH) at 21 ± 1 ºC (Hebeish et al., 1983).
2.2.7.2. Wrinkle recovery angle (WRA). The dry wrinkle recovery angles of the cotton fabric samples were measured according to AATCC Test Method 66-1998 using a YG541B crease-recovery tester (Changzhou Textile Instrument Co. Ltd., China). The wrinkle recovery angles of specimens in 10 warp and 10 weft directions were averaged, respectively.
2.2.7.3. Mechanical properties. The tensile breaking strength of cotton fabrics were performed with a YG(B)026D-250 electronic tension tester (Ningbo Textile Instrument Factory, China), by following this usual procedure: the effective gauge length of the sample was 250 mm, extension speed was 200 mm/min, and the tensile results were measured as the mean of 10 individual fabric. The samples were conditioned in a room (20 ºC, a relative humidity of 65%) for 48 h before measurement.
2.2.7.4. Yellowness index. Cotton samples were evaluated for yellowness by determining the E-313 yellowness -8-
index using Spectraflash SF 300 (Datacolor International, USA).
2.2.8. Preparation of cactus flavonoid extract loaded SFGCF A dry preweighed piece of silk fibroin grafted carboxylic cotton fabric (SFGCF) was put in an aqueous solution of flavonoid extract from cactus, prepared by dissolving cactus flavonoid (desired concentrations 0.5%, 1%, 2% and 3%, w/w) in deionized water at 60 ºC, maintaining fabric to liquor ratio 1:50 and stirring for 2 h at the same temperature. Thereafter, the resulting yellowish-brown color of the grafted fabric indicated the coating of flavonoid extract of cactus within the fibroin polymer part of the grafted fabric. Finally the fabric was rinsed with distilled water for 5 min and dried in vacuum oven at 60 ºC for 2 h, and then conditioned in dust free chamber at ambient temperature until it gained constant weight. The flavonoid extract of cactus containing grafted carboxylic cotton fabric was designated as cactus flavonoid-SFGCF. The percentage of weight gain for the grafted samples during cactus flavonoid extract treatment was calculated with the following formula: Weight gain (% w/w)
where
W2 W1 100 W1
(3)
W1 and W2 represent the weight of grafted sample before and after cactus flavonoid extract treatment,
respectively.
2.2.9. Antibacterial testing The antibacterial activity of the treated cotton fabrics was estimated using two categories of bacterial assays, qualitative and quantitative, against S. aureus and E. coli. For the qualitative bacterial test, an agar diffusion plate method was applied. The agar plate was prepared by pouring the hot nutrient agar onto sterile Petri dishes until it solidified. One milliliter of microbial culture (1×108–5×108 cfu/ml) was distributed uniformly on each plate. The treated cotton fabric disks as well as a control cotton fabric disk (7mm diameter) were placed on the plates simultaneously. After 24 h of incubation at 37 ºC, the dimension of the inhibition zone was measured to evaluate the antibacterial properties of the samples. The quantitative antibacterial assay was carried out by a shake flask method according to GB/T 20944.3-2008 (China). The test procedure was performed as follows: a 0.75 g sample fabric was cut into small pieces of dimensions around 0.5×0.5 cm and dipped into a flask containing 70 ml of phosphate buffered saline (PBS, pH 7.2) and 5 ml of bacterial culture which had a cell concentration of 3×105–4×105 cfu/ml. The flask was placed on a rotary -9-
shaker at 150 rpm for 18 h at 24 ºC. A solution (1ml) was drawn from each sample well, diluted and distributed into an agar plate. All plates were incubated at 37 ºC for 48 h and then the colonies were counted. The percentage reduction (R, %) of bacteria was calculated by the following equation:
R(%) (C A) 100 / C where
(4)
C and A are the bacterial colonies of the control (starting cotton fabric) and flavonoid extract of cactus
treated cotton fabrics test samples, respectively.
3. Results and discussion
3.1. Reaction between silk fibroin and carboxylic cotton
The first stage of obtaining silk fibroin grafted cotton fabrics involves the formation of carboxylic cellulose by the HNO3/H3PO4–NaNO2 selective oxidation of cotton fabrics. In general, the oxidation of polysaccharide compounds by HNO3 alone is sluggish and frequently requires a catalyst (e.g. H2SO4), an initiator (e.g. NO2 or HNO2). It is clearly that H3PO4, as a weak acid catalyst, slowly hydrolyzed cellulose and consequently achieved oxycellulose in high yields (de Nooy et al., 1995; Wanleg, 1956). In our studies, the oxidation of cotton cellulose by the use of HNO3 in combination with H3PO4 and NaNO2 was investigated. In the HNO3/H3PO4–NaNO2 mediated oxidation, the C6 primary hydroxyl groups of cellulose molecule are converted into carboxyl groups (Kumar & Yang, 2002; Xu et al., 2014). The effect of HNO3/H3PO4–NaNO2 oxidation time on the carboxyl group content of oxidized cotton fabrics was shown in Fig. 1a. It can be seen that cotton fabrics oxidized by HNO3/H3PO4–NaNO2 system exhibited increase in carboxyl content ranging from 0.203 to 2.472 mmol/g during the increase of oxidation time. During the first 120 min, the carboxyl content of oxidized cotton enhanced almost linearly with relatively high rate attributed to a fast process involving the easy-to-access portion of cellulose. The cotton fabrics oxidized with prolonged oxidation time and up to 300 min had higher carboxyl group content, but that the oxidation rate of cotton by HNO3/H3PO4–NaNO2 gradually diminished during the oxidation time over 180 min, owing to a retarded process thought to involve the oxidation of the inner core of the crystalline regions in cellulose. Theoretically, increasing time, temperature and oxidant concentration may increase the carboxyl content of oxidized cotton fabrics during HNO3/H3PO4–NaNO2 oxidation, and high carboxyl content may subsequently - 10 -
increase the amount of the silk fibroin grafted to cotton fabric. Consequently, the SFGCF can fix more flavonoid extract of cactus when treated with the solution of cactus flavonoid. However, the oxidation process had a significant influence on the mechanical properties of cotton fabrics. Fig. 1a and b shows the effects of the oxidation time on the breaking strength of oxidized cotton fabrics and the graft add-on of silk fibroin on SFGCF. The warp-wise and weft-wise breaking strength of oxidized fabrics decreased with increasing oxidation time. The tensile strength of oxidized cotton fabrics did not change markedly for the oxidation time in the range of 0–45 min, whereas it dramatically decreased at the oxidation time over 45 min, probably due to breaking down the crystalline structure of the original cotton cellulose (Kumar & Yang, 2002), therefore, high carboxyl content may weaken the mechanical properties of oxidized cotton fabrics. Thus, for textile applications only oxidation of HNO3/H3PO4–NaNO2 under mild conditions should be used. Additionally, it is discovered that short time in preceded oxidation yields adequate carboxyl content for the oxidized cotton fabric to ensure sufficient amount of the silk fibroin crosslinked to the oxidized cotton fabric (see Fig. 1b). To obtain such carboxyl content, cotton fabric is oxidized in HNO3/H3PO4–NaNO2 mixture at 40 ºC for 45 min. As a result of HNO3/H3PO4–NaNO2 oxidation, as shown in Fig. 5b, the surface of the oxidized fiber becomes very rough, and some narrow stripes can be easily observed, implying that the cotton fiber was suffered from corrosion of HNO3/H3PO4–NaNO2 mixture in the oxidation process. From the microscopic analysis it is clear that we attained oxidized fibers with favorable surface, that could promote the ability of coupling with fibroin for oxidized cotton fabrics. The carboxyl group content of oxycellulose reflects not only the oxidation level of the cotton fabrics but also the extent of grafted silk fibroin. It was observed that the graft add-on of fibroin on the original cotton fabric without oxidation (oxidation time = 0 min) was very low, only 0.52% in Fig. 1b, in this case silk fibroin was just deposited on the fabric surface without chemical binding. The graft add-on of fibroin on the oxidized cotton fabrics increased nearly linearly with the oxidation time until 45 min, from 0.52% to 9.64% for the graft add-on of silk fibroin. Since the chemical reaction activity of the carboxyl group is greater than the hydroxyl group of cellulose, when the cotton fabric was selectively oxidized by HNO3/H3PO4–NaNO2 system to achieve carboxylic cellulose, which facilitated the crosslink reaction between amino groups in fibroin and carboxyl groups in oxidized cotton, and finally led to an increase of graft add-on of silk fibroin in SFGCF. However, the graft add-on of incorporated fibroin decreased and became almost constant when the HNO3/H3PO4–NaNO2 oxidation time continued to increase more than 60 min. This may be due to the differences in the reaction site of the oxidation and amido bond formation in the cellulose - 11 -
(Liu et al., 2001; Xu et al., 2014). During the oxidation, the small nitrogen oxide species are able to enter the cotton fiber interior and the glucose units both inside and on the surface of the cotton fiber may be oxidized. On the other hand, a huge fibroin molecule cannot access carboxyl groups formed in small pores of the fiber, and the grafting silk fibroin occurred only on the surface of the oxidized cotton fiber. Scanning electron microscopic analysis shown in Fig. 5 supported these speculations. Fig. 2 shows the relationship between the graft add-on of fibroin introduced into oxidized cotton fabrics and the time of grafting in the fibroin solution with a concentration of 2%. There was no silk fibroin existed in oxidized fabrics at the starting reaction. The graft add-on in grafted fabrics increased rapidly from 0.5 h to 2 h, which may be attributed to increase in number of grafting sites during the initial stage of reaction due to higher amount carboxyl groups in oxidized fabrics participating the reaction. However after 2 h, the increase in graft add-on with reaction time was nearly stabilizing as the reaction between the limited carboxyl groups in oxycellulose and amino groups in fibroin may become saturated. Hence HNO3/H3PO4–NaNO2 oxidation time of 45 min and silk fibroin grafting time of 2 h were found to be optimum for preparing silk fibroin-grafted oxidized cotton fabrics (SFGCF).
(a)
2.8
600
2.4
500
2.0 1.6
400
Warp-wise strength Weft-wise strength Carboxyl content
300
1.2 0.8
200
0.4 100 0.0 0 0
30
60
90 120 150 180 210 240 270 300 Oxidation time (min)
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Carboxyl content (mmol/g)
Tensile strength (N/m)
700
(b)
10
Graft add-on (%)
8 6 4 2 0 0
30
60
90 120 150 180 210 240 270 300 Oxidation time (min)
Fig. 1. Effect of oxidation time on carboxyl group content, tensile strength of oxidized cotton fabrics (a) and graft add-on of silk fibroin on SFGCF (b).
12
Graft add-on (%)
10 8 6 4 2 0 0.0
0.5
1.0
1.5 2.0 2.5 3.0 Reaction time (h)
3.5
4.0
Fig. 2. Effect of reaction time on graft add-on of silk fibroin in fibroin-grafted oxidized cotton fabrics.
3.2. FTIR spectra analysis
Fig. 3 shows the infrared spectra of pure cotton, oxidized cotton and silk fibroin-crosslinked oxidized cotton. The absorption peaks at 1165 cm−1 and 894 cm–1 in both spectra (a) and (b) corresponded to the C–O–C dissymmetry stretching vibration and β-(1–4) stretching vibration, respectively, which indicated that the cellulose chains of cotton did not change after oxidation by HNO3/H3PO4–NaNO2 system. A new absorption band of oxycellulose visibly appeared at around 1733 cm–1 assigned to the stretching vibration of C=O double bond of the carboxyl group as well as the peak at 1032 cm–1 due to the C–O stretching of the primary hydroxyl became weak in spectrum (b) of oxidized - 13 -
cotton fabric. Compared with original cotton, a peak about 1641 cm–1 attributed to the adsorbed water in oxycellulose increased remarkably, which suggested that the carboxyl groups of oxycellulose introduced by oxidation had the stronger affinity for water molecules (Xu et al., 2014). In addition, the shift of the broad peak centred at 3348 cm–1 corresponding to O–H stretching in spectrum (a) to a higher frequency in spectrum (b) (3389 cm−1). The peaks at 1430 cm−1 (CH2 shear vibration), 1235 cm−1 (OH in plane bending), 1113 cm−1 (asymmetry ring stretching), and 700–500 cm−1 (CC stretching vibration) decreased slightly. It demonstrates that the cotton macromolecules degraded partly during the HNO3/H3PO4–NaNO2 oxidation. While the oxidized cotton fabric was treated with silk fibroin, it is interesting that the peak at 1733 cm–1 due to carbonyl groups of oxycellulose markedly weakened in spectrum (c) and hardly appeared in spectrum (d), which displayed that the carboxyl groups in oxidized cotton may have reacted with the amino groups in silk fibroin. This result could be validated by the evidence of a new characteristic peak at about 1539 cm−1 owing to C–N stretching of the grafted fabrics in spectra (c) and (d), which could be the formation of amido bond between amino groups of fibroin and carboxyl groups in oxycellulose (Xu et al., 2014), and the intensity enhanced with the increasing graft add-on of silk fibroin. Meanwhile, the peak at 1282 cm−1 for C–H deformation stretching strengthened after grafting silk fibroin, and the peak for hydrogen-bonded –OH stretching at 3389 cm−1 in spectrum (b) has shifted to 3434 cm−1 in spectra (c) and (d) on account of the contribution of the –NH stretching of fibroin in spectrum (e) after the crosslinking reaction (Amaral, Granja, & Barbosa, 2005; Lin et al., 2008). This finding has also confirmed the amide reaction between silk fibroin and oxidized cotton fabric.
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Fig. 3. FTIR spectra of original cotton fabric (a), oxidized cotton fabric (b), silk fibroin-crosslinked oxidized cotton fabric with graft add-on of 4.56% (c) and 9.64% (d), and silk fibroin (e).
3.3. XPS spectra analysis
The chemical component of the cotton surface layers was determined with X-ray photoelectron spectroscopy (XPS). The high-resolution C1s spectra and wide scan spectra of cotton samples are displayed in Fig. 4. The chemical shifts of carbon (C1s) in original cotton fabric was deconvoluted into three binding energy peaks using the peak-fitting of Gaussian curves. Considering the chemical structure of cotton, the first peak was assigned to C–C (unoxidized carbon), the second peak was due to C–O (carbon with one oxygen bond), and the third peak was attributed to C=O or O–C–O (carbon with two oxygen bonds) (Topalovic et al., 2007). The sub-peaks of cotton at 284.7 eV (C1s scan A), 286.1 eV (C1s scan B) and 287.4 eV (C1s scan C) are contributed to C–C or C–H, C–O and - 15 -
O–C–O, respectively (Fig. 4a) (Lin et al., 2008; Mitchell, Carr, Parfitt, Vickerman, & Jones, 2005). However, the sub-peak belonging to O–C–O of cotton oxidized by HNO3/H3PO4–NaNO2 mixture was shifted to a higher frequency at 288.5 eV (C1s scan D), which is contributed to C=O or O–C=O group (Fig. 4b). Furthermore, the area of the peak corresponding to C–O at round 286.1 eV was decreased from 25.49% (before oxidation) to 21.61%. All these results reveal that the carboxylic cellulose was generated from the HNO3/H3PO4–NaNO2 oxidation of cotton fabric. After silk fibroin treatment, a new binding energy peak came up at near 287.6 eV (C1s scan C) in spectra (c) and (d), which could be attributed to C–N originated from the reaction of carboxylic cotton fabric with fibroin. Meanwhile, the significant reduction in the area contribution of 288.5 eV under spectra D in (c) and (d) than that in (b) was observed, while the spectral area of 287.6 eV component in spectra (c) and (d) increased with the graft add-on of silk fibroin on SFGCF. These can be clarified that the crosslinking reaction existed between carboxyl groups in oxidized cotton fabric and amino groups of silk fibroin, which are consistent with the FTIR results discussed in the previous section. In addition, Fig. 4e shows the wide scan spectra of the cotton samples. The main elements in the oxidized cotton fabric were carbon and oxygen, as expected, no new element was introduced into the cotton sample oxidized with HNO3/H3PO4–NaNO2. However, for silk fibroin grafted oxidized cotton fabric (SFGCF), a new element of nitrogen, which is also presence in silk fibroin, was detected. XPS results reconfirmed the generation of amido bond between silk fibroin and carboxylic cotton fabric.
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Fig. 4. XPS spectra of C1s peaks for original cotton fabric (a), oxidized cotton fabric (b) and silk fibroin-grafted oxidized cotton fabric (SFGCF) with graft add-on of 4.56% (c) and 9.64% (d), and wide scan spectra of the tested samples (e).
3.4. Morphology observation
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The SEM images of surface of cotton fabric samples were shown in Fig. 5. Overall, the pure cotton fiber with 5000 times magnification exhibited a smooth surface structure (image a). This was different from the carboxylic cotton fiber, which showed many erosion lines on the surface (image b), affected by oxidation. Surprisingly, when the oxidized cotton fabric grafted with silk fibroin, the surface of grafted cotton fiber was clearly covered with a layer of fibroin (image c), which is absent in unmodified fiber substrate. This further indicates the presence of crosslinked silk fibroin on the modified cotton fabric.
a
b
c
d
Fig. 5. SEM images of original cotton fabric (a), oxidized cotton fabric (b), silk fibroin grafted oxidized cotton fabric (c) and cactus flavonoid containing grafted cotton fabric (d).
3.5. Textile properties of cotton fabric
Results in Table 1 show that the warp-wise tensile strength of silk fibroin-grafted oxidized cotton fabric (SFGCF) with optimum graft add-on of 9.64% was lower than that of original cotton fabric, but the breaking strength of the SFGCF was higher in comparison to the only oxidized fabric, consequently the silk fibroin acts as a coupling agent to bind fibrils to the fibers “body” and increases the fabrics tensile properties, which can be seen from SEM images of the oxidized cotton fabric and the SFGCF, Fig. 5. These results are different from the data obtained by Lin and - 18 -
co-workers (2008), who observed that the treatment of silk fibroin solution has a negligible effect on mechanical properties of the periodate-oxidized cotton fibers. The wrinkle recovery angle of fibroin-grafted oxidized cotton fabrics (measured with AATCC Test Method 66-1998) enhanced a lot than that of original and oxidized cotton fabrics, indicating the SFGCF has good properties of elastic deformation and crease resistance. The moisture regain significantly increased for silk fibroin grafted cotton fabrics when compared with that of ungrafted fabric samples. This enhancement in moisture regain was ascribed to the introduction of hydrophilic silk fibroin polymer in molecular structure of cotton fibers during grafting. It could be seen that the yellowness index of fibroin-grafted oxidized cotton fabrics increased, showing obvious reduce in whiteness of fabrics, which may be due to the negative influence of oxidation and fibroin coating on the surface of the fabric to certain extent.
Table 1 Effect of silk fibroin grafting on textile properties. Samples
Tensile strength (N/m)
Wrinkle recovery angle (°) (W+F)
Moisture regain (%)
Yellowness index
Original cotton fabric
658.02 ± 5.92
130.6 ± 5.64
8.13 ± 0.56
14.57 ± 1.03
Oxidized cotton fabric
581.24 ± 3.10
109.8 ± 4.38
10.82 ± 0.44
16.72 ± 0.68
SFGCF
594.39 ± 2.76
196.2 ± 5.33
14.28 ± 0.92
22.49 ± 0.58
3.6. Preparation and antibacterial activity of cactus flavonoid extract loaded cotton fabric
The silk fibroin-grafted oxidized cotton fabric was further treated with a solution of cactus flavonoid extract, where the adsorption of cactus flavonoid molecules takes place and this adsorption process can be viewed as the interaction of the polycationic fibroin residues in SFGCF with the acidic functional groups in cactus flavonoid extract (Cai et al., 2010; Liu et al., 2001; Xu et al., 2013). Finally the flavonoid extract of cactus was fixed on the SFGCF. SEM figures clearly showed the presence of cactus flavonoid on the surface of the treated SFGCF. As shown in Fig. 5d, many small grains were distributed throughout the surface of cactus flavonoid-treated grafted cotton fabric. When the treated concentration of cactus flavonoid extract was changed, the results of weight gain for grafted cotton fabrics are displayed in detail in Table 2. For following reference, the original cotton fabric (control sample) is termed sample 0. Samples 1, 2, 3 and 4 represent the treated cotton fabrics under different concentrations of cactus flavonoid extract as shown in Table 2. It shows that the weight gain of treated samples increased with the - 19 -
increasing concentration of flavonoid extract of cactus, the cactus flavonoid-SFGCF exhibited typical yellowish-brown color development and the color of treated fabrics became deeper along with the increasing weight gain of cactus flavonoid deposited on the grafted fabrics (see Fig. 6) which also indicates the coating of cactus flavonoid in the grafted cotton fabric. The qualitative antibacterial assessment of cactus flavonoid treated cotton fabrics exposed to S. aureus and E. coli are shown in Fig. 6. It is evident that there was a dense bacterial colonies around the control sample and the original cotton fabric had no inhibitory effect against both tested bacteria, while a clear inhibition zone could be seen around the treated samples. The inhibition zones against S. aureus were much larger than those against E. coli implying that the flavonoid extract of cactus has more effective antimicrobial ability against S. aureus. Moreover, the size of inhibition zones against both S. aureus and E. coli increased when contrasting sample 1 to samples 2, 3 and 4, which illustrated the tendency to enhance the antibacterial ability with the increasing weight gain of cactus flavonoid extract coated on grafted cotton fabrics. Table 2 presents the quantitative antibacterial test results of the cactus flavonoid-SFGCF samples. The treated cotton fabrics with the weight gain of over 2.86% showed good antibacterial activity against S. aureus which had a bacterial reduction rate of exceeding 91.33%, while the bacterial reduction rate of sample 1 with lower weight gain reduced to 78.04%. In case of E. coli, the bacterial reduction rates exceeded 92.80% for the treated fabrics which had higher weight gain of above 4.90%. However, the bacterial reduction rate of sample 1 with the low weight gain against E. coli was only 49.61%. With increasing the weight gain of cactus flavonoid in the fabrics, the antibacterial properties against E. coli enhanced, and the bacterial reduction rate of sample 4 against E. coli reached 96.47%. These results distinctly suggest sufficient flavonoid extracts of cactus were immobilized on such kind of fibroin-grafted oxidized cotton fabrics. The results presented in Table 2 also indicated that the cactus flavonoid extract has stronger bactericidal effects against S. aureus which is in agreement with the result of the qualitative bacterial test. Therefore, sample 4 which contains 5.39% cactus flavonoid extract is the optimal treated cotton fabric to achieve excellent antibacterial properties against both Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli. In addition, development of yellowish-brown color can be considered as limiting factor, but since the color development on the treated fabric was found to be even across the dimensions, it can be used for colored fabric. The resulted fabrics were very encouraging as far as end uses like medical textiles are concerned.
- 20 -
a
b 1
0
0 2 4
4
1
2
3
3
Fig. 6. Photographs of inhibition zone for original cotton fabric and flavonoid extract of cactus containing grafted carboxylic cotton fabrics (cactus flavonoid-SFGCF) against S. aureus (a) and E. coli (b).
Table 2 Antibacterial properties of cactus flavonoid extract containing grafted cotton fabrics (cactus flavonoid-SFGCF). Samples
0 1
Weight gain (%)
S. aureus Surviving cells (cfu/ml)
Reduction a (%)
Surviving cells (cfu/ml)
Reduction a (%)
–
–
2.40×106
–
3.83×106
–
0.5
2
1.0
3
2.0
4 a
Concentration of cactus flavonoid (%)
3.0
1.71 2.86 4.90 5.39
5.27×10
5
2.08×10
5
6.50×10
4
3.55×10
4
E. coli
78.04 91.33 97.29 98.52
1.93×10
6
49.61
9.81×10
5
74.39
2.76×10
5
92.80
1.35×10
5
96.47
Represents average value of 3 determinations, standard deviation (%) range ±0.49–1.31.
4. Conclusion
This work expanded the application of oxycellulose in the textile industry and developed a new method to fabricate silk fibroin grafted carboxylic cotton fabric. On balance of tensile properties of oxidized cotton fabrics and graft add-on of silk fibroin in grafted fabrics, the appropriate oxidation time of HNO3/H3PO4–NaNO2 system and reaction time of immersion in fibroin solution were determined as 45 min and 2 h respectively. The reaction of silk fibroin with oxidized cotton fabric resulted in a new absorption band at 1539 cm−1 in infrared spectrum and new binding energy peak at near 287.6 eV in XPS spectrum suggesting the C–N of amido bond formed between the oxidized cotton fabric and the silk fibroin. The HNO3/H3PO4–NaNO2 oxidation decreased the tensile strength of cotton fabrics, while the fibroin treatment of the oxidized fabrics enhanced the mechanical properties of grafted - 21 -
fabrics to some extent. The grafted cotton fabrics displayed improvement in crease resistance and moisture regain indicating their surfaces had the property of protein, compared with those of ungrafted cotton fabrics. The cactus flavonoid extracts immobilized grafted cotton fabrics exhibited more effective antibacterial activities against S. aureus than E. coli, and the bacterial reduction rates against the two bacteria exceeded 96% such that this novel modified fabric is suitable as medical textiles and packing materials.
Acknowledgements
Authors gratefully acknowledge the financial supports from the Scientific Research Fund for Returned Scholars of Ministry of Education of China (No. (2011)1568), Natural Science Foundation of Anhui Province of China (No. 10040606Q16), Higher Academy Natural Science Research Key Program of Anhui province of China (No. KJ2015A001), Key Program of Support Plan for Excellent Young Talents in Academies of Anhui Province of China (No. gxyqZD2016034), and Science Foundation Key Program for Youth Scholars of Anhui Agricultural University of China (No. 2009zd03).
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S0144-8617(16)31456-4 http://dx.doi.org/doi:10.1016/j.carbpol.2016.12.071 CARP 11878
To appear in: Received date: Revised date: Accepted date:
19-9-2016 3-12-2016 31-12-2016
Please cite this article as: Xu, Yunhui., Chen, Dingding., Du, Zhaofang., Li, Jifeng., Wang, Yunxia., Yang, Zhen., & Peng, Fengxia., Structure and properties of silk fibroin grafted carboxylic cotton fabric via amide covalent modification.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.12.071
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Structure and properties of silk fibroin grafted carboxylic cotton fabric via amide covalent modification Yunhui Xu a,*, Dingding Chen b, Zhaofang Du a, Jifeng Li a, Yunxia Wang a, Zhen Yang a, Fengxia Peng a a
College of Light-Textile Engineering and Art, Anhui Agricultural University, Hefei, Anhui 230036, China Biotechnology Center, Anhui Agricultural University, Hefei, Anhui 230036, China
b
* Corresponding author. Tel.: +86 551 6578 6459; fax: +86 551 6578 6331. E-mail address: [email protected] (Y. Xu).
Research highlights 1. HNO3/H3PO4–NaNO2 oxidized cotton fabrics were modified by silk fibroin crosslink. 2. Silk fibroin was grafted on carboxylic cotton fabric via the C–N amido bond. 3. Textile properties of silk fibroin-grafted oxidized cotton fabrics improved. 4. Cactus flavonoid-treated grafted cotton fabric showed high antimicrobial activity.
-2-
Abstract A novel eco-friendly production of silk fibroin-grafted carboxylic cotton fabrics without using any crosslinking agents was developed via the reaction of silk fibroin with oxidized cotton. The effect of reaction parameters on mechanical properties of oxidized fabrics and graft add-on of silk fibroin in grafted fabrics was examined. The results showed that appropriate oxidation time of HNO3/H3PO4–NaNO2 mixture and grafting time of fibroin were 45 min and 2 h respectively. FTIR analysis of grafted sample indicated that the C–N amido bond was generated through the reaction between primary amines in silk fibroin and carboxyl groups in oxidized cotton, which was further confirmed by XPS spectra. The grafted fabrics were also evaluated for physical properties like tensile strength, wrinkle recovery angle, moisture regain and yellowness index. Cactus flavonoid coated on grafted fabric through treatment with flavonoid extract of cactus, such treated fabric exhibited a highly inhibitory effect against both Staphylococcus aureus and Escherichia coli bacteria.
Keywords: Carboxylic cotton fabric; Grafting; Silk fibroin; Cactus flavonoid extract; Antibacterial activity
1. Introduction
Natural cellulose products, which are polymers with glucose linked through 1,4-β-glucosidic bond, especially cotton fabrics, are highly popular for their excellent properties, such as versatility, softness, hygroscopicity, breathability, affinity to skin, biodegradability and regeneration property (Liu, Ren, & Liang, 2015; Shafei & Abou-Okeil, 2011). These products, however, have some inherent limitations such as wrinkle, shrinkage, low dye uptake, ultraviolet (UV) and microbial degradation (Hou, Zhang, & Wang, 2012; Liu et al., 2015; Zhang, Chen, Ling, & Zhang, 2009). Therefore the modification of natural cellulose products is an important method to provide those products with improved properties to overcome the above mentioned limitations. The modification usually involves introducing functional materials to the surface or interior of the natural celluloses, or grafting them on the macromolecule of the celluloses (Bashar & Khan, 2013; Simoncic & Tomsic, 2010; Tissera, Wijesena, & Nalin de Silva, 2016; Wijesena, Tissera, & Nalin de Silva, 2015; Wijesena, Tissera, Perera, Nalin de Silva, & Amaratunga, 2015). Selective oxidation, as a new modification approach to cellulose, aroused many scholars’ interest. Basing on the different oxidizing agents and oxidation conditions, the selective oxidation occurs at two adjacent secondary -3-
hydroxyls in cellulose glucose units and cleaves the C2–C3 bond of the glucopyranoside ring and introduces two aldehyde groups at C2 and C3 positions (Potthast, Schiehser, Rosenau, & Kostic, 2009; Xu, Huang, & Wang, 2013), or the oxidation reaction proceeds with the primary hydroxyl of cellulose and acquires the carboxyl group at C6 in cellulose anhydroglucose units (de Nooy, Besemer, & van Bekkum, 1995; Kumar & Yang, 2002; Xu, Liu, Liu, Tan, & Zhu, 2014). Among them, the oxidant system of HNO3/H3PO4–NaNO2 exhibits an arresting route to introduce carbonyl and carboxyl functional groups into cellulose fibers which is characterized by its quick reaction rate, high oxidation selectivity and yield, low cellulose degradation and mild reactive conditions during the oxidation process (Kumar & Yang, 2002; Xu et al., 2014). In particular, partial oxidation of cellulose by the HNO3/H3PO4–NaNO2 system not only causes new functional groups in addition to the hydroxyl groups in polymers but also retains physical property of cellulose, which fabricates the reactive cellulose polymers for further chemical modification. Recently, many researches have been devoted to the preparation, microstructure, and its application of the oxidized cellulose (Kaputskii et al., 1995; Sharma, Rajamohanan, & Varma, 2014; Wu, He, Huang, Wang, & Tang, 2012). However, less attention has been paid to the oxidized cotton fiber for the textile usage (Liu, Nishi, Tokura, & Sakairi, 2001; Xu et al., 2013). Silk fibroin is a natural biological polymer composed of 18 α-amino acids, among which Gly, Ala, and Ser are the main amino acids (Tao, Kaplan, & Omenetto, 2012). Through a degumming process, silk fibroin is purified from outer sericin of silk fibers. Regarded as a safe material for human beings and animals (Li, Liu, Li, & Wang, 2012), silk fibroin has been extensively applied in the textile, foodstuff, cosmetic, and biomedical material (Kundu et al., 2014; Lu et al., 2011). Therefore, in recent years, the efforts of possible end-users of silk fibroin as green finishing agent for clothing products (e.g. natural or man-made textiles), which present requiring properties of softness, comfort, moisture absorption, and antistatic properties, have attracted considerable attention. Various methods have been developed to fabricate fibroin treated fabrics, the modifications have been limited to integration fibroin on the cellulose textiles with crosslinking agents (Lu, 2010), plasma irradiation (Xia & Wang, 2004) or partial carboxymethylation of cellulose (Wijesena, Tissera, Perera, & Nalin de Silva, 2014). By periodate oxidation of cotton and generation dialdehyde groups in cellulose chain, the crosslinking reaction could proceed between fibroin and oxidized cotton fiber through formation of Schiff base (Lin, Yao, Chen, & Wang, 2008). In our previous study, a carboxylic cellulose fiber, characterized by a carboxyl group of C6 site in glucose of cellulose chain, was produced by selective oxidation of HNO3/H3PO4–NaNO2 system (Xu et al., 2014). It has been -4-
utilized to graft chitosan without any other reagents for antibacterial finishing of the textile (Xu, Qiu, Zhang, & Zhang, 2014). This paper presents a novel approach of silk fibroin grafted oxidized cotton fabric (SFGCF), the facile process involves conversion of the C6 primary hydroxyl in cotton glucose units into carboxyl group by selective oxidation of HNO3/H3PO4–NaNO2 and their subsequent treatment with fibroin aqueous solution in an amine formation reaction, which is the classic crosslinking reaction between the generated carboxyl groups in oxidized cotton and amino groups in silk fibroin (Scheme 1). This novel development is to crosslink silk fibroin into textile substrates for formulating advanced cellulose–fibroin based composites without using any cross-linkers. The optimum parameters of the graft reaction of fibroin on carboxylic cotton fabric were discussed. The reaction was also probed by the Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The morphology and physical properties of grafted cotton fabrics were also measured. Additionally, SFGCF has a polycationic fibroin residue, which may interact with flavonoid extract of cactus that has an acidic function, such fibroin grafted cotton can serve as a best platform for the attachment of flavonoid extract of cactus to apply in antibacterial products. Flavonoid is extracted from the succulent cladode of cactus (Opuntia dillenii Haw). Cactus flavonoid is well known to have antimicrobial, antioxidant, anti-inflammatory, and anti-tumor activities (Benayad, Martinez-Villaluenga, Frias, Gomez-Cordoves, & Es-Safi, 2014; Cai, Gu, & Tang, 2010; Sanchez et al., 2014). It is taken cactus flavonoid as antibacterial to endow fibroin grafted cotton fabric with antimicrobial performance by treating SFGCF surface in a solution of flavonoid extract from cactus. The significance of this processing is to achieve bioactive fibers cactus flavonoid-coated SFGCF, which may be used as underwear, footgear, medical textiles and packing materials. And then, the antibacterial activity of flavonoid extract of cactus complex SFGCF was also examined.
Scheme 1. Crosslinking reaction between carboxylic cotton and silk fibroin.
2. Materials and methods
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2.1. Materials
Sodium nitrite (NaNO2), nitric acid (HNO3, 68%), phosphoric acid (H3PO4, 85%), acetone, sodium carbonate, hydrochloric acid, sodium hydroxide, calcium chloride, and ethanol were purchased from Aladdin Chemical Reagent Co., USA, as analytical grades. Cactus flavonoid extract was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All chemicals were used without further purification. Woven, bleached and scoured cotton fabric (58×30 number of yarns per cm2, 121 g/m2) was purchased from Intertek Group (Shanghai, China). Bombyx mori (B. mori) silk yarn was kindly supplied by Sericulture Research Institute, Anhui Academy of Agricultural Sciences (China). Staphylococcus aureus (S. aureus, ATCC 6538) and Escherichia coli (E. coli, ATCC 8099) were obtained from Shanghai Luwei Sci. & Tech. Co., Ltd. (China). Nutrient broth and nutrient agar were purchased from Scas Ecoscience Technology Inc (China). Deionized water (18MΩ.cm) was employed throughout this work.
2.2. Methods
2.2.1. Oxidation of cotton fabrics by HNO3/H3PO4–NaNO2 system Nitric acid and phosphoric acid were mixed in the brown wide-necked bottle with a 2:1 (v/v) ratio, thereafter the dried cotton fabrics, with a liquor ratio of 1:20 (w/v), were soaked in the mixture solution. Subsequently 1.4% (w/v) of sodium nitrite was added all at once, the bottle containing mixtures was covered with a glass stopper and gently oscillated at 40 ºC for 15–300 min in the absence of light. After the oxidation, the cotton fabrics were washed thoroughly with acetone and soaked in deionized water for 3 h to remove oxidant. The resulting carboxylic fabrics were dried in the atmosphere overnight at room temperature and then kept in desiccator before grafting reactions.
2.2.2. Preparation of SF-grafted carboxylic cotton fabrics Raw B. mori silk was first degummed twice in the boiling solution containing 0.2% (w/w) Na2CO3 and 1% (w/w) degumming agent in liquor ratio of 1:100 for 1 h to remove the sericin protein. Afterwards the degummed silk yarn was dissolved in a mixture solution of calcium chloride, ethanol, and water (CaCl2:C2H5OH:H2O = 1:2:8 mole ratio) at 70 ºC for 1 h. After filtering, the resulting fibroin solution was dialyzed for 3 days against running deionized water to eliminate CaCl2 salt, smaller molecules, and some impurities via a Oso-T8340 dialysis membrane with the molecular cutoff of 3500 Da (Union Carbide Co., USA), and then the silk solution was freeze-dried. Eventually, the fibroin powder with the molecular weight of 5000–30000 (Mw) was achieved by the degradation method with -6-
aqueous hydrochloric acid reported by Nam and Park (2001). Subsequently a fibroin solution was prepared with deionized water and adjusted to about pH 6.0 using 0.1 M HCl solution. Then the grafting reaction was carried out in a flask. The above mentioned oxidized fabric (of known weight) was placed in flask containing fibroin solution (2%, w/w) for 0.5–4 h at 60 ºC maintaining fabric to liquor ratio 1:50, with constant stirring. After that, the grafted sample was washed with hot water several times and soaked in deionized water for 12 h at ambient temperature, to remove the ungrafted silk fibroin on the fabric. Finally the resulting cotton fabric was dried at 80 ºC under vacuum for 2 h to produce the silk fibroin grafted oxidized cotton fabric (SFGCF). The un-oxidized cotton fabric was also treated using the same procedure to produce the control sample. Thereafter the samples ungrafted or grafted with silk fibroin conditioned in a vacuum desiccator containing silica gel till the constant weight was reached, and then weighed on a BS210S photoelectric scale, which was termed the dry weight. The graft add-on was calculated through the following formula: Graft add-on (% w/w)
where
W1 W0 100 W0
(1)
W0 and W1 are the dry weight of ungrafted fabric and grafted fabric, respectively.
2.2.3. Carboxyl group content analysis The carboxyl groups of the oxidized cotton react with the salts of weaker acids such as calcium acetate, forming a salt of the oxidized cotton and releasing an equivalent amount of the weaker acid. On this basis as well as by the method described in our recent paper (Xu et al., 2014). The carboxyl group content in the oxidized sample was calculated as follows: Carboxyl group content (mmol/g)
where 0.1
0.1M VNaOH 1000 m (1 w / 100)
(2)
M is the concentration of NaOH, VNaOH is the volume (l) of NaOH solution used in titration after
correcting for the blank,
m is the weight of oxidized cotton sample (g), and w is the moisture content (%).
2.2.4. Fourier transform infrared spectroscopy (FTIR) analysis The FTIR spectra of fabric samples were recorded with a Nicolet 380 FTIR spectrophotometer (Thermo-Electron Corporation, USA) using transmission technique for KBr pellets by recording 50 scans in %T mode with a resolution of 4 cm–1 in the wave numbers range of 4000–400 cm−1. -7-
2.2.5. X-ray photoelectron spectroscopy (XPS) analysis XPS spectra were carried out with a XSAM-800 photoelectron spectrometer (Kratos, UK) equipped with a monochromatic Al-Kα X-ray source. The samples were recorded using X-ray anode of 180 W and high voltage of 12.0 kV operating at a working pressure of 2×10–7 Pa in 0.1 eV steps with 100 eV analyzer passing energy at the radiation (1253.6 eV). The position of the carbon peak (284.8 eV) for C1s was used to calibrate the XPS scale for all substrates. Acquired data fitting was performed using commercially available software with 100 % Gaussian curve fitting. A linear background subtraction was used for C1s, N1s, and O1s curves, and a Shirley background subtraction was used for Cu 2p3/2 (932.67 eV).
2.2.6. Scanning electron microscopy (SEM) analysis Surface morphologies of oxidized cotton fabric and grafted cotton fabric were imaged by scanning electron microscope (Hitachi X-650, Japan) operating at 5.0 kV. All samples were conductively plated with gold sputtering before imaging.
2.2.7. Measurement of fabric properties 2.2.7.1. Moisture regain. The moisture regain was determined by the vacuum desiccator method with sodium nitrite to give 65% relative humidity (RH) at 21 ± 1 ºC (Hebeish et al., 1983).
2.2.7.2. Wrinkle recovery angle (WRA). The dry wrinkle recovery angles of the cotton fabric samples were measured according to AATCC Test Method 66-1998 using a YG541B crease-recovery tester (Changzhou Textile Instrument Co. Ltd., China). The wrinkle recovery angles of specimens in 10 warp and 10 weft directions were averaged, respectively.
2.2.7.3. Mechanical properties. The tensile breaking strength of cotton fabrics were performed with a YG(B)026D-250 electronic tension tester (Ningbo Textile Instrument Factory, China), by following this usual procedure: the effective gauge length of the sample was 250 mm, extension speed was 200 mm/min, and the tensile results were measured as the mean of 10 individual fabric. The samples were conditioned in a room (20 ºC, a relative humidity of 65%) for 48 h before measurement.
2.2.7.4. Yellowness index. Cotton samples were evaluated for yellowness by determining the E-313 yellowness -8-
index using Spectraflash SF 300 (Datacolor International, USA).
2.2.8. Preparation of cactus flavonoid extract loaded SFGCF A dry preweighed piece of silk fibroin grafted carboxylic cotton fabric (SFGCF) was put in an aqueous solution of flavonoid extract from cactus, prepared by dissolving cactus flavonoid (desired concentrations 0.5%, 1%, 2% and 3%, w/w) in deionized water at 60 ºC, maintaining fabric to liquor ratio 1:50 and stirring for 2 h at the same temperature. Thereafter, the resulting yellowish-brown color of the grafted fabric indicated the coating of flavonoid extract of cactus within the fibroin polymer part of the grafted fabric. Finally the fabric was rinsed with distilled water for 5 min and dried in vacuum oven at 60 ºC for 2 h, and then conditioned in dust free chamber at ambient temperature until it gained constant weight. The flavonoid extract of cactus containing grafted carboxylic cotton fabric was designated as cactus flavonoid-SFGCF. The percentage of weight gain for the grafted samples during cactus flavonoid extract treatment was calculated with the following formula: Weight gain (% w/w)
where
W2 W1 100 W1
(3)
W1 and W2 represent the weight of grafted sample before and after cactus flavonoid extract treatment,
respectively.
2.2.9. Antibacterial testing The antibacterial activity of the treated cotton fabrics was estimated using two categories of bacterial assays, qualitative and quantitative, against S. aureus and E. coli. For the qualitative bacterial test, an agar diffusion plate method was applied. The agar plate was prepared by pouring the hot nutrient agar onto sterile Petri dishes until it solidified. One milliliter of microbial culture (1×108–5×108 cfu/ml) was distributed uniformly on each plate. The treated cotton fabric disks as well as a control cotton fabric disk (7mm diameter) were placed on the plates simultaneously. After 24 h of incubation at 37 ºC, the dimension of the inhibition zone was measured to evaluate the antibacterial properties of the samples. The quantitative antibacterial assay was carried out by a shake flask method according to GB/T 20944.3-2008 (China). The test procedure was performed as follows: a 0.75 g sample fabric was cut into small pieces of dimensions around 0.5×0.5 cm and dipped into a flask containing 70 ml of phosphate buffered saline (PBS, pH 7.2) and 5 ml of bacterial culture which had a cell concentration of 3×105–4×105 cfu/ml. The flask was placed on a rotary -9-
shaker at 150 rpm for 18 h at 24 ºC. A solution (1ml) was drawn from each sample well, diluted and distributed into an agar plate. All plates were incubated at 37 ºC for 48 h and then the colonies were counted. The percentage reduction (R, %) of bacteria was calculated by the following equation:
R(%) (C A) 100 / C where
(4)
C and A are the bacterial colonies of the control (starting cotton fabric) and flavonoid extract of cactus
treated cotton fabrics test samples, respectively.
3. Results and discussion
3.1. Reaction between silk fibroin and carboxylic cotton
The first stage of obtaining silk fibroin grafted cotton fabrics involves the formation of carboxylic cellulose by the HNO3/H3PO4–NaNO2 selective oxidation of cotton fabrics. In general, the oxidation of polysaccharide compounds by HNO3 alone is sluggish and frequently requires a catalyst (e.g. H2SO4), an initiator (e.g. NO2 or HNO2). It is clearly that H3PO4, as a weak acid catalyst, slowly hydrolyzed cellulose and consequently achieved oxycellulose in high yields (de Nooy et al., 1995; Wanleg, 1956). In our studies, the oxidation of cotton cellulose by the use of HNO3 in combination with H3PO4 and NaNO2 was investigated. In the HNO3/H3PO4–NaNO2 mediated oxidation, the C6 primary hydroxyl groups of cellulose molecule are converted into carboxyl groups (Kumar & Yang, 2002; Xu et al., 2014). The effect of HNO3/H3PO4–NaNO2 oxidation time on the carboxyl group content of oxidized cotton fabrics was shown in Fig. 1a. It can be seen that cotton fabrics oxidized by HNO3/H3PO4–NaNO2 system exhibited increase in carboxyl content ranging from 0.203 to 2.472 mmol/g during the increase of oxidation time. During the first 120 min, the carboxyl content of oxidized cotton enhanced almost linearly with relatively high rate attributed to a fast process involving the easy-to-access portion of cellulose. The cotton fabrics oxidized with prolonged oxidation time and up to 300 min had higher carboxyl group content, but that the oxidation rate of cotton by HNO3/H3PO4–NaNO2 gradually diminished during the oxidation time over 180 min, owing to a retarded process thought to involve the oxidation of the inner core of the crystalline regions in cellulose. Theoretically, increasing time, temperature and oxidant concentration may increase the carboxyl content of oxidized cotton fabrics during HNO3/H3PO4–NaNO2 oxidation, and high carboxyl content may subsequently - 10 -
increase the amount of the silk fibroin grafted to cotton fabric. Consequently, the SFGCF can fix more flavonoid extract of cactus when treated with the solution of cactus flavonoid. However, the oxidation process had a significant influence on the mechanical properties of cotton fabrics. Fig. 1a and b shows the effects of the oxidation time on the breaking strength of oxidized cotton fabrics and the graft add-on of silk fibroin on SFGCF. The warp-wise and weft-wise breaking strength of oxidized fabrics decreased with increasing oxidation time. The tensile strength of oxidized cotton fabrics did not change markedly for the oxidation time in the range of 0–45 min, whereas it dramatically decreased at the oxidation time over 45 min, probably due to breaking down the crystalline structure of the original cotton cellulose (Kumar & Yang, 2002), therefore, high carboxyl content may weaken the mechanical properties of oxidized cotton fabrics. Thus, for textile applications only oxidation of HNO3/H3PO4–NaNO2 under mild conditions should be used. Additionally, it is discovered that short time in preceded oxidation yields adequate carboxyl content for the oxidized cotton fabric to ensure sufficient amount of the silk fibroin crosslinked to the oxidized cotton fabric (see Fig. 1b). To obtain such carboxyl content, cotton fabric is oxidized in HNO3/H3PO4–NaNO2 mixture at 40 ºC for 45 min. As a result of HNO3/H3PO4–NaNO2 oxidation, as shown in Fig. 5b, the surface of the oxidized fiber becomes very rough, and some narrow stripes can be easily observed, implying that the cotton fiber was suffered from corrosion of HNO3/H3PO4–NaNO2 mixture in the oxidation process. From the microscopic analysis it is clear that we attained oxidized fibers with favorable surface, that could promote the ability of coupling with fibroin for oxidized cotton fabrics. The carboxyl group content of oxycellulose reflects not only the oxidation level of the cotton fabrics but also the extent of grafted silk fibroin. It was observed that the graft add-on of fibroin on the original cotton fabric without oxidation (oxidation time = 0 min) was very low, only 0.52% in Fig. 1b, in this case silk fibroin was just deposited on the fabric surface without chemical binding. The graft add-on of fibroin on the oxidized cotton fabrics increased nearly linearly with the oxidation time until 45 min, from 0.52% to 9.64% for the graft add-on of silk fibroin. Since the chemical reaction activity of the carboxyl group is greater than the hydroxyl group of cellulose, when the cotton fabric was selectively oxidized by HNO3/H3PO4–NaNO2 system to achieve carboxylic cellulose, which facilitated the crosslink reaction between amino groups in fibroin and carboxyl groups in oxidized cotton, and finally led to an increase of graft add-on of silk fibroin in SFGCF. However, the graft add-on of incorporated fibroin decreased and became almost constant when the HNO3/H3PO4–NaNO2 oxidation time continued to increase more than 60 min. This may be due to the differences in the reaction site of the oxidation and amido bond formation in the cellulose - 11 -
(Liu et al., 2001; Xu et al., 2014). During the oxidation, the small nitrogen oxide species are able to enter the cotton fiber interior and the glucose units both inside and on the surface of the cotton fiber may be oxidized. On the other hand, a huge fibroin molecule cannot access carboxyl groups formed in small pores of the fiber, and the grafting silk fibroin occurred only on the surface of the oxidized cotton fiber. Scanning electron microscopic analysis shown in Fig. 5 supported these speculations. Fig. 2 shows the relationship between the graft add-on of fibroin introduced into oxidized cotton fabrics and the time of grafting in the fibroin solution with a concentration of 2%. There was no silk fibroin existed in oxidized fabrics at the starting reaction. The graft add-on in grafted fabrics increased rapidly from 0.5 h to 2 h, which may be attributed to increase in number of grafting sites during the initial stage of reaction due to higher amount carboxyl groups in oxidized fabrics participating the reaction. However after 2 h, the increase in graft add-on with reaction time was nearly stabilizing as the reaction between the limited carboxyl groups in oxycellulose and amino groups in fibroin may become saturated. Hence HNO3/H3PO4–NaNO2 oxidation time of 45 min and silk fibroin grafting time of 2 h were found to be optimum for preparing silk fibroin-grafted oxidized cotton fabrics (SFGCF).
(a)
2.8
600
2.4
500
2.0 1.6
400
Warp-wise strength Weft-wise strength Carboxyl content
300
1.2 0.8
200
0.4 100 0.0 0 0
30
60
90 120 150 180 210 240 270 300 Oxidation time (min)
- 12 -
Carboxyl content (mmol/g)
Tensile strength (N/m)
700
(b)
10
Graft add-on (%)
8 6 4 2 0 0
30
60
90 120 150 180 210 240 270 300 Oxidation time (min)
Fig. 1. Effect of oxidation time on carboxyl group content, tensile strength of oxidized cotton fabrics (a) and graft add-on of silk fibroin on SFGCF (b).
12
Graft add-on (%)
10 8 6 4 2 0 0.0
0.5
1.0
1.5 2.0 2.5 3.0 Reaction time (h)
3.5
4.0
Fig. 2. Effect of reaction time on graft add-on of silk fibroin in fibroin-grafted oxidized cotton fabrics.
3.2. FTIR spectra analysis
Fig. 3 shows the infrared spectra of pure cotton, oxidized cotton and silk fibroin-crosslinked oxidized cotton. The absorption peaks at 1165 cm−1 and 894 cm–1 in both spectra (a) and (b) corresponded to the C–O–C dissymmetry stretching vibration and β-(1–4) stretching vibration, respectively, which indicated that the cellulose chains of cotton did not change after oxidation by HNO3/H3PO4–NaNO2 system. A new absorption band of oxycellulose visibly appeared at around 1733 cm–1 assigned to the stretching vibration of C=O double bond of the carboxyl group as well as the peak at 1032 cm–1 due to the C–O stretching of the primary hydroxyl became weak in spectrum (b) of oxidized - 13 -
cotton fabric. Compared with original cotton, a peak about 1641 cm–1 attributed to the adsorbed water in oxycellulose increased remarkably, which suggested that the carboxyl groups of oxycellulose introduced by oxidation had the stronger affinity for water molecules (Xu et al., 2014). In addition, the shift of the broad peak centred at 3348 cm–1 corresponding to O–H stretching in spectrum (a) to a higher frequency in spectrum (b) (3389 cm−1). The peaks at 1430 cm−1 (CH2 shear vibration), 1235 cm−1 (OH in plane bending), 1113 cm−1 (asymmetry ring stretching), and 700–500 cm−1 (CC stretching vibration) decreased slightly. It demonstrates that the cotton macromolecules degraded partly during the HNO3/H3PO4–NaNO2 oxidation. While the oxidized cotton fabric was treated with silk fibroin, it is interesting that the peak at 1733 cm–1 due to carbonyl groups of oxycellulose markedly weakened in spectrum (c) and hardly appeared in spectrum (d), which displayed that the carboxyl groups in oxidized cotton may have reacted with the amino groups in silk fibroin. This result could be validated by the evidence of a new characteristic peak at about 1539 cm−1 owing to C–N stretching of the grafted fabrics in spectra (c) and (d), which could be the formation of amido bond between amino groups of fibroin and carboxyl groups in oxycellulose (Xu et al., 2014), and the intensity enhanced with the increasing graft add-on of silk fibroin. Meanwhile, the peak at 1282 cm−1 for C–H deformation stretching strengthened after grafting silk fibroin, and the peak for hydrogen-bonded –OH stretching at 3389 cm−1 in spectrum (b) has shifted to 3434 cm−1 in spectra (c) and (d) on account of the contribution of the –NH stretching of fibroin in spectrum (e) after the crosslinking reaction (Amaral, Granja, & Barbosa, 2005; Lin et al., 2008). This finding has also confirmed the amide reaction between silk fibroin and oxidized cotton fabric.
- 14 -
Fig. 3. FTIR spectra of original cotton fabric (a), oxidized cotton fabric (b), silk fibroin-crosslinked oxidized cotton fabric with graft add-on of 4.56% (c) and 9.64% (d), and silk fibroin (e).
3.3. XPS spectra analysis
The chemical component of the cotton surface layers was determined with X-ray photoelectron spectroscopy (XPS). The high-resolution C1s spectra and wide scan spectra of cotton samples are displayed in Fig. 4. The chemical shifts of carbon (C1s) in original cotton fabric was deconvoluted into three binding energy peaks using the peak-fitting of Gaussian curves. Considering the chemical structure of cotton, the first peak was assigned to C–C (unoxidized carbon), the second peak was due to C–O (carbon with one oxygen bond), and the third peak was attributed to C=O or O–C–O (carbon with two oxygen bonds) (Topalovic et al., 2007). The sub-peaks of cotton at 284.7 eV (C1s scan A), 286.1 eV (C1s scan B) and 287.4 eV (C1s scan C) are contributed to C–C or C–H, C–O and - 15 -
O–C–O, respectively (Fig. 4a) (Lin et al., 2008; Mitchell, Carr, Parfitt, Vickerman, & Jones, 2005). However, the sub-peak belonging to O–C–O of cotton oxidized by HNO3/H3PO4–NaNO2 mixture was shifted to a higher frequency at 288.5 eV (C1s scan D), which is contributed to C=O or O–C=O group (Fig. 4b). Furthermore, the area of the peak corresponding to C–O at round 286.1 eV was decreased from 25.49% (before oxidation) to 21.61%. All these results reveal that the carboxylic cellulose was generated from the HNO3/H3PO4–NaNO2 oxidation of cotton fabric. After silk fibroin treatment, a new binding energy peak came up at near 287.6 eV (C1s scan C) in spectra (c) and (d), which could be attributed to C–N originated from the reaction of carboxylic cotton fabric with fibroin. Meanwhile, the significant reduction in the area contribution of 288.5 eV under spectra D in (c) and (d) than that in (b) was observed, while the spectral area of 287.6 eV component in spectra (c) and (d) increased with the graft add-on of silk fibroin on SFGCF. These can be clarified that the crosslinking reaction existed between carboxyl groups in oxidized cotton fabric and amino groups of silk fibroin, which are consistent with the FTIR results discussed in the previous section. In addition, Fig. 4e shows the wide scan spectra of the cotton samples. The main elements in the oxidized cotton fabric were carbon and oxygen, as expected, no new element was introduced into the cotton sample oxidized with HNO3/H3PO4–NaNO2. However, for silk fibroin grafted oxidized cotton fabric (SFGCF), a new element of nitrogen, which is also presence in silk fibroin, was detected. XPS results reconfirmed the generation of amido bond between silk fibroin and carboxylic cotton fabric.
- 16 -
Fig. 4. XPS spectra of C1s peaks for original cotton fabric (a), oxidized cotton fabric (b) and silk fibroin-grafted oxidized cotton fabric (SFGCF) with graft add-on of 4.56% (c) and 9.64% (d), and wide scan spectra of the tested samples (e).
3.4. Morphology observation
- 17 -
The SEM images of surface of cotton fabric samples were shown in Fig. 5. Overall, the pure cotton fiber with 5000 times magnification exhibited a smooth surface structure (image a). This was different from the carboxylic cotton fiber, which showed many erosion lines on the surface (image b), affected by oxidation. Surprisingly, when the oxidized cotton fabric grafted with silk fibroin, the surface of grafted cotton fiber was clearly covered with a layer of fibroin (image c), which is absent in unmodified fiber substrate. This further indicates the presence of crosslinked silk fibroin on the modified cotton fabric.
a
b
c
d
Fig. 5. SEM images of original cotton fabric (a), oxidized cotton fabric (b), silk fibroin grafted oxidized cotton fabric (c) and cactus flavonoid containing grafted cotton fabric (d).
3.5. Textile properties of cotton fabric
Results in Table 1 show that the warp-wise tensile strength of silk fibroin-grafted oxidized cotton fabric (SFGCF) with optimum graft add-on of 9.64% was lower than that of original cotton fabric, but the breaking strength of the SFGCF was higher in comparison to the only oxidized fabric, consequently the silk fibroin acts as a coupling agent to bind fibrils to the fibers “body” and increases the fabrics tensile properties, which can be seen from SEM images of the oxidized cotton fabric and the SFGCF, Fig. 5. These results are different from the data obtained by Lin and - 18 -
co-workers (2008), who observed that the treatment of silk fibroin solution has a negligible effect on mechanical properties of the periodate-oxidized cotton fibers. The wrinkle recovery angle of fibroin-grafted oxidized cotton fabrics (measured with AATCC Test Method 66-1998) enhanced a lot than that of original and oxidized cotton fabrics, indicating the SFGCF has good properties of elastic deformation and crease resistance. The moisture regain significantly increased for silk fibroin grafted cotton fabrics when compared with that of ungrafted fabric samples. This enhancement in moisture regain was ascribed to the introduction of hydrophilic silk fibroin polymer in molecular structure of cotton fibers during grafting. It could be seen that the yellowness index of fibroin-grafted oxidized cotton fabrics increased, showing obvious reduce in whiteness of fabrics, which may be due to the negative influence of oxidation and fibroin coating on the surface of the fabric to certain extent.
Table 1 Effect of silk fibroin grafting on textile properties. Samples
Tensile strength (N/m)
Wrinkle recovery angle (°) (W+F)
Moisture regain (%)
Yellowness index
Original cotton fabric
658.02 ± 5.92
130.6 ± 5.64
8.13 ± 0.56
14.57 ± 1.03
Oxidized cotton fabric
581.24 ± 3.10
109.8 ± 4.38
10.82 ± 0.44
16.72 ± 0.68
SFGCF
594.39 ± 2.76
196.2 ± 5.33
14.28 ± 0.92
22.49 ± 0.58
3.6. Preparation and antibacterial activity of cactus flavonoid extract loaded cotton fabric
The silk fibroin-grafted oxidized cotton fabric was further treated with a solution of cactus flavonoid extract, where the adsorption of cactus flavonoid molecules takes place and this adsorption process can be viewed as the interaction of the polycationic fibroin residues in SFGCF with the acidic functional groups in cactus flavonoid extract (Cai et al., 2010; Liu et al., 2001; Xu et al., 2013). Finally the flavonoid extract of cactus was fixed on the SFGCF. SEM figures clearly showed the presence of cactus flavonoid on the surface of the treated SFGCF. As shown in Fig. 5d, many small grains were distributed throughout the surface of cactus flavonoid-treated grafted cotton fabric. When the treated concentration of cactus flavonoid extract was changed, the results of weight gain for grafted cotton fabrics are displayed in detail in Table 2. For following reference, the original cotton fabric (control sample) is termed sample 0. Samples 1, 2, 3 and 4 represent the treated cotton fabrics under different concentrations of cactus flavonoid extract as shown in Table 2. It shows that the weight gain of treated samples increased with the - 19 -
increasing concentration of flavonoid extract of cactus, the cactus flavonoid-SFGCF exhibited typical yellowish-brown color development and the color of treated fabrics became deeper along with the increasing weight gain of cactus flavonoid deposited on the grafted fabrics (see Fig. 6) which also indicates the coating of cactus flavonoid in the grafted cotton fabric. The qualitative antibacterial assessment of cactus flavonoid treated cotton fabrics exposed to S. aureus and E. coli are shown in Fig. 6. It is evident that there was a dense bacterial colonies around the control sample and the original cotton fabric had no inhibitory effect against both tested bacteria, while a clear inhibition zone could be seen around the treated samples. The inhibition zones against S. aureus were much larger than those against E. coli implying that the flavonoid extract of cactus has more effective antimicrobial ability against S. aureus. Moreover, the size of inhibition zones against both S. aureus and E. coli increased when contrasting sample 1 to samples 2, 3 and 4, which illustrated the tendency to enhance the antibacterial ability with the increasing weight gain of cactus flavonoid extract coated on grafted cotton fabrics. Table 2 presents the quantitative antibacterial test results of the cactus flavonoid-SFGCF samples. The treated cotton fabrics with the weight gain of over 2.86% showed good antibacterial activity against S. aureus which had a bacterial reduction rate of exceeding 91.33%, while the bacterial reduction rate of sample 1 with lower weight gain reduced to 78.04%. In case of E. coli, the bacterial reduction rates exceeded 92.80% for the treated fabrics which had higher weight gain of above 4.90%. However, the bacterial reduction rate of sample 1 with the low weight gain against E. coli was only 49.61%. With increasing the weight gain of cactus flavonoid in the fabrics, the antibacterial properties against E. coli enhanced, and the bacterial reduction rate of sample 4 against E. coli reached 96.47%. These results distinctly suggest sufficient flavonoid extracts of cactus were immobilized on such kind of fibroin-grafted oxidized cotton fabrics. The results presented in Table 2 also indicated that the cactus flavonoid extract has stronger bactericidal effects against S. aureus which is in agreement with the result of the qualitative bacterial test. Therefore, sample 4 which contains 5.39% cactus flavonoid extract is the optimal treated cotton fabric to achieve excellent antibacterial properties against both Gram-positive bacteria S. aureus and Gram-negative bacteria E. coli. In addition, development of yellowish-brown color can be considered as limiting factor, but since the color development on the treated fabric was found to be even across the dimensions, it can be used for colored fabric. The resulted fabrics were very encouraging as far as end uses like medical textiles are concerned.
- 20 -
a
b 1
0
0 2 4
4
1
2
3
3
Fig. 6. Photographs of inhibition zone for original cotton fabric and flavonoid extract of cactus containing grafted carboxylic cotton fabrics (cactus flavonoid-SFGCF) against S. aureus (a) and E. coli (b).
Table 2 Antibacterial properties of cactus flavonoid extract containing grafted cotton fabrics (cactus flavonoid-SFGCF). Samples
0 1
Weight gain (%)
S. aureus Surviving cells (cfu/ml)
Reduction a (%)
Surviving cells (cfu/ml)
Reduction a (%)
–
–
2.40×106
–
3.83×106
–
0.5
2
1.0
3
2.0
4 a
Concentration of cactus flavonoid (%)
3.0
1.71 2.86 4.90 5.39
5.27×10
5
2.08×10
5
6.50×10
4
3.55×10
4
E. coli
78.04 91.33 97.29 98.52
1.93×10
6
49.61
9.81×10
5
74.39
2.76×10
5
92.80
1.35×10
5
96.47
Represents average value of 3 determinations, standard deviation (%) range ±0.49–1.31.
4. Conclusion
This work expanded the application of oxycellulose in the textile industry and developed a new method to fabricate silk fibroin grafted carboxylic cotton fabric. On balance of tensile properties of oxidized cotton fabrics and graft add-on of silk fibroin in grafted fabrics, the appropriate oxidation time of HNO3/H3PO4–NaNO2 system and reaction time of immersion in fibroin solution were determined as 45 min and 2 h respectively. The reaction of silk fibroin with oxidized cotton fabric resulted in a new absorption band at 1539 cm−1 in infrared spectrum and new binding energy peak at near 287.6 eV in XPS spectrum suggesting the C–N of amido bond formed between the oxidized cotton fabric and the silk fibroin. The HNO3/H3PO4–NaNO2 oxidation decreased the tensile strength of cotton fabrics, while the fibroin treatment of the oxidized fabrics enhanced the mechanical properties of grafted - 21 -
fabrics to some extent. The grafted cotton fabrics displayed improvement in crease resistance and moisture regain indicating their surfaces had the property of protein, compared with those of ungrafted cotton fabrics. The cactus flavonoid extracts immobilized grafted cotton fabrics exhibited more effective antibacterial activities against S. aureus than E. coli, and the bacterial reduction rates against the two bacteria exceeded 96% such that this novel modified fabric is suitable as medical textiles and packing materials.
Acknowledgements
Authors gratefully acknowledge the financial supports from the Scientific Research Fund for Returned Scholars of Ministry of Education of China (No. (2011)1568), Natural Science Foundation of Anhui Province of China (No. 10040606Q16), Higher Academy Natural Science Research Key Program of Anhui province of China (No. KJ2015A001), Key Program of Support Plan for Excellent Young Talents in Academies of Anhui Province of China (No. gxyqZD2016034), and Science Foundation Key Program for Youth Scholars of Anhui Agricultural University of China (No. 2009zd03).
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