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Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 311– 319

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

jo ur nal ho me page: www.elsev ier .com/ locate /co lsur fa

n efficient antibacterial multi-scale web of carbon fibers withsymmetrically dispersed Ag–Cu bimetal nanoparticles

hiv Singha, Harish C. Joshib, Anurag Srivastavab, Ashutosh Sharmaa,c, Nishith Vermaa,d,∗

Department of Chemical Engineering, Institute of Technology Kanpur, Kanpur 208016, IndiaDefense Materials and Stores Research & Development Establishment, Kanpur 208013, IndiaCenter for Nanosciences, Indian Institute of Technology Kanpur, Kanpur 208016, IndiaCenter for Environmental Science and Engineering, Institute of Technology Kanpur, Kanpur 208016, India

i g h l i g h t s

Synthesis of bimetals Ag and Cu Nps-dispersed carbon micro-nanofibers(ACFs/CNFs).Unique distribution of Ag and Cu Npsin the multiscale carbon web.Prepared material effectively appliedas antibacterial agents for waterpurification.Ag:Cu-ACFs/CNFs more economicaland effective compared to Ag- or Cu-ACFs/CNFs.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 24 August 2013eceived in revised form3 November 2013ccepted 21 November 2013vailable online 28 November 2013

a b s t r a c t

A multi-scale web of carbon micro-nanofibers was prepared for antibacterial applications by chemicalvapor deposition (CVD) using silver (Ag) and copper (Cu) bimetal nanoparticles (Nps). Carbon nanofibers(CNFs) were grown on activated carbon fiber (ACF) substrate using acetylene as the carbon source. TheCVD conditions were optimized such that Cu Nps catalyzed the CNF growth and were attached to the tipsof the CNFs, whereas Ag Nps remained adhered to the ACF surface. The in vitro antimicrobial capabilityof the material was evaluated against gram-negative Escherichia coli and gram-positive Staphylococcus

eywords:ctivated carbon fiberarbon nanofiberetal nanoparticle

ntibacterial agentater purification

aureus bacteria. The bimetal-grown ACFs/CNFs completely inhibited the growth of bacteria for 7 daysand performed superior to the ACFs/CNFs grown using Ag or Cu Nps alone. This way, more expensive Agis partially replaced with less expensive Cu while maintaining a superior performance. The synergisticaction of bimetal ions derived from the unique spatial segregation of Nps is likely owing to a greateropportunity for the Cu ions on the fiber tips to interact with bacteria.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

The antibacterial properties of silver (Ag) and copper (Cu)ransition metals are well established. Nanoparticles (Nps) of Agnd Cu are capable of rupturing the cell membrane when brought

∗ Corresponding author at: Department of Chemical Engineering, Indian Institutef Technology Kanpur, Kanpur 208016, India. Tel.: +91 512 2596352x7704;ax: +91 512 2590104.

E-mail addresses: nishith@iitk.ac.in, vermanishith@gmail.com (N. Verma).

927-7757/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfa.2013.11.041

in contact. The antibacterial activity of these metals is attributedto their high reactivity with sulfur and phosphorous compoundspresent in the bacterial membrane. This process denatures thebacterial nucleic acid (DNA and RNA), hampers their replication,and stops the respiration and protein synthesis of bacteria [1–4].

Several methods have been developed to produce Ag and Cu Npsin the aqueous phase, including chemical reduction using strong

reducing agents such as trisodium citrate, hydrazine hydrate,sodium borohydride, ammonia solution, and ascorbic acid [5–10].The drawback of such methods is that the Nps cannot be directlyused as antibacterial agents for water purification because of the

3 ysicochem. Eng. Aspects 443 (2014) 311– 319

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their respective metallic states (Ag/Cu). The optimum calcinationand reduction temperatures were determined by a temperatureprogram reduction (TPR) analysis. The CVD temperature and timewere set at 300 ◦C and 15 min, respectively. The flow rate of C2H2

Table 1Amounts of AgNO3 and Cu(NO3)2 used for the impregnation of different ACFs.

S. No Ag:Cu AgNO3 (g) Cu(NO3)2·3H2O (g)

1 0:1 0.714 00.00

12 S. Singh et al. / Colloids and Surfaces A: Ph

nherent risk of their entrainment in the flow. Therefore, theroduced Nps are transferred to substrates for end applications1,8,10–17]. The non-uniform distribution of Nps in the substrate,owever, limits their end-effectiveness.

Recently, a web of carbon micro-nanofibers supported Ag or Cups was developed as an antibacterial agent [18]. The multi-scale

tructure was produced by growing carbon nanofibers (CNFs) onhe activated carbon fiber (ACF) substrate by chemical vapor depo-ition (CVD) using either Ag or Cu metal Nps as the catalyst andcetylene (C2H2) as the carbon source. The growth of CNFs occurredy the tip-growth mechanism, resulting in the attachment of metalps to the tips of the fibers. Ag/Cu Nps had dual roles: as a catalyst

or the CNF growth and as an antibacterial agent. With a uniformispersion and high metal loading, the web was found to be effec-ive against Escherichia coli (E. coli) and Staphylococcus aureus (S.ureus) bacteria. In general, the amount of Ag required to preparehe material was less than that of Cu for approximately the same

agnitude of antibacterial activities.This study describes the development of a web of carbon

icro-nanofibers dispersed with both Ag and Cu Nps, applied tontibacterial applications. There are novelties in the present studyis a vis our previous study [18] on the following aspects. (1) In theresent study, a Ag and Cu bimetal-grown multi-scale web of car-on micro and nanofibers has been prepared. The previous studyocused on the multi-scale web prepared by a single metal, Ag oru only. (2) In the present study, the present material has uniquesymmetric distribution of bimetal, with Ag adhered to the surfacef the ACF substrate and Cu attached to the tips of nanofibers. In therevious material, single metal Cu or Ag was attached to the tipsf the grown fibers. (3) The method of preparation, especially CVDemperature was optimized such that Ag remained adhered to theurface and Cu moved along with the nanofibers by the tip-growthechanism. (4) The new bimetal-based material is more efficient

s antibacterial agent against E. coli and S. aureus and cost-effectivehan the single-metal-based material. Alternatively, the preparedimetal-grown web required smaller amounts of the metals thanhe web grown from the single metals for similar levels of antibac-erial activities.

. Experimental

.1. Materials

Cu(NO3)2·3H2O (purity > 99%), AgNO3 (purity > 99.9%), sodiumodecyl sulfate (SDS, purity > 99%), and the reagents used forreparing the Luria-Bertani (LB) medium, including tryptone,odium chloride (NaCl), yeast extract and agar were purchasedrom Merck, Germany. ACF derived from a phenolic resin precur-or was procured from Nippon Kynol Inc., Japan. All stock solutionsere prepared in Milli-Q water. Hydrogen (purity > 99.999%), nitro-

en (purity > 99.999%), and C2H2 (atomic absorption spectroscopyAAS) grade) were purchased from Sigma Gases, India. E. coli (K-12)nd S. aureus (RN4220) were procured indigenously.

.2. Preparation of bimetal Nps dispersed in ACFs and ACFs/CNFs

Fig. 1 is the schematic of the mechanistic steps involved inreparing the Ag–Cu bimetal-CNF. The preparation steps includehe impregnation of ACF with bimetal salts using SDS surfactant,ollowed by calcinations, reduction, and CVD. The figure also showshe optimized conditions used in the study to yield maximum and

niform growth of CNFs, with Cu attached to the tips of the CNFsnd Ag adhered to the substrate ACF.

The as-received ACF was pretreated with 5 ml of 0.03 M nitriccid in 1 L of Milli-Q water at 80 ◦C to remove any undesirable ions.

Fig. 1. Schematic of Ag:Cu-ACF/CNF preparation and its antibacterial activity.

The treated samples were oven dried for 12 h at 100 ◦C followed byvacuum drying for 12 h at 200 ◦C to remove any entrapped gasesfrom the ACFs. The ACF samples were impregnated with 140 mlaqueous solution containing Ag- and Cu nitrate. Different amountsof metal salts were used in preparing bimetal ACFs, as shown inTable 1. For reference purposes, the bimetal-impregnated ACF sam-ples were classified in terms of Ag:Cu ratio as (0:1), (1:0), (1:1), (1:3)and (3:1), depending on the relative amounts of Ag and Cu saltsused in the impregnating solutions. In our previous study, single-metal (either Ag or Cu) impregnated ACFs were prepared using theoptimized amounts of 0.714 g Ag or 13.53 g Cu salts for achievinga uniform growth of CNFs on ACFs [18]. Therefore, the nomencla-ture (0:1) refers to the ACF sample prepared using 0.714 g of Ag salt.The nomenclature (1:1) refers to 0.357 g of Ag salt and 6.765 g of Cusalt, which are 50% of their respective amounts used for impregnat-ing ACF with a single metal (Ag or Cu). Similarly, the nomenclature(1:3) refers to 0.1785 g of Ag and 10.15 g of Cu salts, which are 25%and 75% of their respective single metal salts used for impregnat-ing ACF. The concentration of SDS surfactant was optimized at 0.3%(w/w) in the impregnating solution to achieve monodispersion ofthe salts in the solution and the maximum transfer of the saltsto the ACFs without agglomeration [18]. The samples of impreg-nating solutions were collected in small borosilicate bottles beforeand after impregnation for AAS analysis to determine metal loadingin the ACFs. After impregnation, ACF samples were dried for 12 hat 100 ◦C. Next, the bimetal ACF samples were calcined at 200 ◦Cunder N2 flow (200 sccm) to convert the metal nitrates into theirrespective oxides. The calcined samples were subsequently treatedat 350 ◦C with hydrogen to convert the oxides of the metals into

2 1:0 0.000 13.523 1:1 0.357 6.7654 3:1 0.536 3.3835 1:3 0.179 10.150

S. Singh et al. / Colloids and Surfaces A: Physicoc

FC

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ig. 2. Schematic of the vertical tubular reactor used for calcinations, reduction andVD.

as maintained at a constant 30 sccm during the CVD step. Fig. 2s the schematic of the experimental setup used for calcination,eduction, and CVD. In this manuscript, the hierarchical structuresf different bimetal dispersed ACF/CNF prepared are denoted asg:Cu (1:1)-, Ag:Cu (3:1)- and Ag:Cu (1:3)-ACF/CNF for referenceurposes.

.3. Antibacterial activity test

The antibacterial tests of the prepared bimetal ACFs/CNFs wereerformed against E. coli (gram-negative) and S. aureus (gram-ositive). Fig. 3 describes the flow sheet for the preparation ofacterial culture and the antibacterial activity test. The antibac-erial activity was examined by a plate counting method using LBgar medium (recipient: 10 g tryptone, 5 g yeast extract, 15 g agar,0 g NaCl and water to 1 L). Test samples were prepared in severalonical flasks, each containing 1 ml of bacterial LB broth diluted to0 ml of sterilized Milli-Q water. To different broths, 0.1 g of bimetalg:Cu (1:1)-, Ag:Cu (3:1)-, Ag:Cu (1:3)-ACF/CNF were added. A fewroths were also prepared with the single metal impregnated ACFsr ACFs/CNFs, i.e., Ag:Cu (1:0)- and Ag:Cu (0:1)-ACF or ACF/CNFor comparison. The initial broth seeding number of bacteria was

07–108 per ml. The flasks containing the prepared materials andacterial broth were kept on an orbital shaker (speed = 120 rpm)

ncubator at 37 ◦C. Samples of 1 ml were collected from each flask

Fig. 3. Flow-chart for antibacterial analysis.

hem. Eng. Aspects 443 (2014) 311– 319 313

every 24 h for the plating. The antibacterial test of the preparedmaterials was carried out over 7 days. Each test was carried out intriplicate to ascertain the reproducibility of the results. After thereduction step (or before the CVD step), a few samples of bimetalACFs, were also tested for comparing antibacterial activities.

3. Surface characterization

The prepared bimetal Nps dispersed in ACFs and ACFs/CNFswere characterized using different analytical techniques, includ-ing AAS, TPR, N2-physisorption, broad angle X-ray diffraction(XRD), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, and Fourier transform infrared (FTIR). TheAAS (Varian AA-240, USA) analysis of the bimetal impregnatingsolution was carried out using an air-acetylene flame at wave-lengths of 324.8 nm and 328.1 nm for Cu and Ag, respectively, todetermine metal loadings on ACFs. An Autosorb-1C instrument(Quantachrome, USA) fitted with a thermal conductivity detector(TCD) was used for the TPR analysis. The Brunauer–Emmet–Teller(BET) surface area, total pore volume (Vtotal) and pore size dis-tribution (PSD) were calculated from the adsorption isothermsdetermined using the Autosorb-1C instrument with N2 as theprobe molecule. Micro-pore and meso-pore contents of the sam-ples were calculated by density functional theory (DFT) and theBarret–Joyner–Halenda (BJH) method, respectively. The XRD anal-ysis was carried out to determine crystal sizes and crystal lattices inthe prepared samples. A Cu-K� radiation (k = 1.54178 A) was usedfor the XRD patterns within a 2� range of 10–100◦ and a scan rateof 5◦ per min. The surface morphology of the prepared materialswas analyzed by field emission SEM (Supra 40 VP, Zeiss, Germany).The dispersion of Ag and Cu Nps in ACF/CNF was determined byEDX. The FTIR spectra of the materials were acquired using a Tensor27 apparatus (Bruker, Germany) in the attenuated total reflectance(ATR) mode and crystalline germanium. The resolution was set to4 cm−1 and a total of 100 scans were collected for each sample. Thesample chamber of the instrument was continuously purged withN2 gas to reduce the effect of atmospheric carbon dioxide and mois-ture. The mechanical strength of the prepared fabric material wasdetermined by the universal tensile machine (UTM). A rectangular(50 mm × 5 mm) segment of the prepared sample was subjectedto an initial strain ramp of 2.0 mm per min and a preload force of0.001 N [19]. The tests were conducted at 25 ◦C at the fixed gaugelength of 30 mm.

4. Results and discussion

4.1. Cu (II) and Ag (I) loading on the ACF

Table 2 summarizes Cu and Ag loadings in the ACFs, determinedfrom the AAS analysis. As shown in the table, Ag metal loadingon Ag:Cu (1:1)-, Ag:Cu (3:1)- and Ag:Cu (1:3)-ACFs were 90, 124and 27 mg/g, respectively, whereas Cu loading in the corresponding

ACFs were 132, 106 and 338 mg/g, respectively. Further, Ag load-ing was measured to be 162 mg/g in Ag:Cu (1:0)-ACFs, whereas Culoading was 389 mg/g in Ag:Cu (0:1)-ACFs sample. SDS significantly

Table 2AAS analysis of Ag- and Cu-loading in the impregnated bimetal ACF samples.

Ag:Cu sample Ag (mg/g) Cu (mg/g)

0:1 0 3891:0 162 01:1 90 1323:1 124 1061:3 27 338

314 S. Singh et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 311– 319

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Table 3SBET, Vtotal and PSD of prepared materials.

Sample SBET (m2/g) Vtotal (cc/g) PSD (%)

Micro Meso Macro

ACF 1507 0.69 87.40 9.11 3.50Ag:Cu(x:y)-ACF/CNF0:1 613 0.42 66.25 25.95 7.671:0 1332 0.73 81.45 12.78 5.771:1 748 0.41 85.2 8.61 6.15

Fig. 4. TPR profile of Ag:Cu (1:1) samples.

ncreased the metal loadings in the ACFs, approximately twice thanhat achieved without SDS.

.2. TPR

Fig. 4 describes the TPR profiles of Ag:Cu (1:1)-ACF calcinedor 4 h at different temperatures. As observed, reduction startedt approximately 170 ◦C in all samples. For the samples calcined at50 and 200 ◦C, a sharp reduction peak may be observed at approx-

mately 250 ◦C, which is attributed to the reduction of Ag-oxide.nother peak observed at 350 ◦C for the sample calcined at 200 ◦C

s attributed to Cu-oxide. No other sharp peaks were observed forhe samples calcined at 150, 300 and 400 ◦C. Loss in the intensityf the reduction peak was observed if the calcinations tempera-ure exceeded 200 ◦C. Based on the TPR profiles, calcination andeduction temperatures for Ag:Cu (1:1) samples were optimizedt 200 and 350 ◦C, respectively. Approximately the same tempera-ures were inferred from the TPR profiles of Ag:Cu (1:3) and (3:1)CF samples.

.3. XRD pattern

The XRD analysis of ACF and Ag:Cu (1:1)-, Ag:Cu (3:1)-, Ag:Cu1:3)-ACF samples (post-hydrogen reduction step) were carriedut at 30 ◦C. Fig. 5 describes the XRD patterns of the ACF (shownn black) and Ag:Cu (1:1)-ACF (shown in red) samples. All otherimetal samples exhibited the same characteristic peaks and are

ot included in the figure for clarity. As shown, a common peakppeared at the 2� angle of approximately 22◦ in both samples,hich is the distinctive feature of amorphous carbon with a crys-

allographic index of (0 0 2) [20]. The spectra of the bimetal ACFs

Fig. 5. XRD patterns of ACF and Ag:Cu (1:1)-ACF.

3:1 746 0.69 74.84 13.33 11.831:3 644 0.38 82.6 12.929 4.45

exhibit seven characteristic peaks at different 2�s (38.2, 43.5, 44.4,50.5, 64.5, 74.1, and 77.5◦). The peaks at 2�s = 43.3, 50.5 and 74.1◦

correspond to the crystallographic indices of (1 1 1), (2 0 0), and(2 2 0), respectively. These data confirm that the sample containedCu in its pure metallic face centered cubic (FCC) phase (JCPDS No.4-836). Furthermore, four peaks were observed at 2� angles of 38.2,44.4, 64.5 and 77.5◦, corresponding to the crystallographic indicesof (1 1 1), (2 0 0), (2 2 0) and (3 1 1), respectively. These data are cor-roborated with the FCC structure of Ag Nps (JCPDS No. 3-0921).The average crystalline sizes of Cu and Ag Nps in the Ag:Cu (1:1)-ACF samples were calculated using the Scherrer formula and werefound to vary between 20 nm (corresponding to 43.5◦) and 34 nm(corresponding to 64.5◦).

4.4. BET surface area, Vtotal and PSD

Table 3 describes the BET surface area, Vtotal and PSD of differ-ent samples, including bimetal Ag:Cu-ACF/CNF and single metalAg/Cu CNF. As observed from the table, ACF had a BET area(SBET) of approximately 1500 m2/g and a Vtotal of 0.699 cc/g. SBETdecreased in the hierarchal web because metal Nps blocked thepores of ACFs. It may also be observed from the table that Cu-ACF/CNF had the smallest SBET (613 m2/g), whereas Ag-ACFs/CNFshad the largest SBET (1332 m2/g). The bimetal samples, Ag:Cu (1:1)-,Ag:Cu (3:1)-, and Ag:Cu (1:3)-ACFs/CNFs had surface areas betweenthat of Ag:Cu (1:0)- and Ag:Cu (0:1)-ACFs/CNFs. As also observedfrom the table, the prepared materials were primarily micro-porous.

Three explanations are in order. (1) Decrease in the BET areaof ACF/CNF, following the growth of CNFs on ACF does not affectthe antibacterial properties of Ag:Cu-ACF/CNF. (2) The BET area ofACF/CNF measured using N2 as probe molecules has been shownto be underestimated by ∼40%, as the micropores of CNFs are notaccessible to N2 molecules because of the pore-diffusion limita-tion [21]. (3) The metal is required to catalyze the carbon sourcefor growing CNFs. ACF/CNF without metals can be prepared by theultra-sonication of the single-metal (Ag or Cu)-ACF/CNF only. Ultra-sonication will dislodge most of the nanoparticles from the tips,making the internal area of the grown fibers exposed to the N2probe molecule during BET area measurements. However, mostof the nanoparticles dispersed in the pores or on the surface ofthe material will not be removed. In this study, the BET analysisof the sonicated ACF/CNF was not performed because Ag and Cunanoparticles are required in the ACF/CNF to act as the antibac-terial agents. In our recent study [22], measurement of the BETarea of the sonicated ACF/CNF prepared by using single metal onlywas performed and the BET surface area was found to increase by∼40–65%.

4.5. Surface morphology

Fig. 6 describes the SEM images of Ag:Cu (1:1)-, Ag:Cu (3:1)-,and Ag:Cu (1:3)-ACFs. Almost uniform dispersion of Nps on the

S. Singh et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 311– 319 315

(b–b1)

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aiAbortofdCpiatd

Fig. 6. SEM images of (a–a1) Ag:Cu (1:1)-ACF,

CF surface may be observed for all samples. The Nps maylso be observed inside the pores. Fig. 7 shows approximateniform and dense growth of CNFs on ACF for Ag:Cu (1:1)-,g:Cu (3:1)-, and Ag:Cu (1:3)-ACFs/CNFs. The tip-growth mech-nism for Cu-ACF/CNF grown on ACF may be corroborated fromhe images, with shiny Cu Nps attached to the tip of CNFsnd Ag Nps adhered to the surface of ACFs (confirmed usingDX).

The EDX spectra of the prepared bimetal ACFs/CNFs were takent different locations on the samples. Fig. 8a–a1, b–b1 and c–c1

s the representative spectra of Ag:Cu (1:1)-, Ag:Cu (3:1)-, andg:Cu (1:3)-ACF/CNF, respectively. The spectra of the samplesefore and after sonication are shown on the left and right sidef figures, respectively. Sonication of bimetal ACF/CNF was car-ied out to dislodge the Cu Nps from the tips of the fibers ando detect Ag Nps adhered to the ACF substrate. A sonication timef 10 min using 0.05 M HNO3 solution was found to be sufficientor dislodging the Cu Nps. A few nanofibers were also removeduring the sonication. The EDX spectra confirmed the presence ofu Nps in the pre-sonicated samples and that of Ag Nps in theost-sonicated samples, as shown in the figures. Thus, the SEM

mages and EDX spectra confirmed the unique distribution of Cund Ag Nps in the bimetal-grown carbon web, with Cu at theips and Ag at the surface of the fibers. No foreign metals wereetected.

Ag:Cu (3:1)-ACF, and (c–c1) Ag:Cu (1:3)-ACF.

4.6. FTIR analysis

Fig. 9 describes the FTIR spectra of ACF and Ag:Cu (1:1)-ACF/CNFsamples before and after the adsorption of E. coli and S. aureus. Thespectra of other bimetal-grown samples exhibited similar charac-teristics and are not included here. As shown in the figure, the peaksobserved at 1555, 1031 and 692 cm−1 are assigned to N O stretch(nitro group), C N stretch (amine group) and C C stretch, respec-tively. These functional groups are inherently present in ACF. TheN O stretch appears because HNO3 was used for the pretreatmentof ACF.

In Ag:Cu (1:1)-ACF/CNF samples, the peaks observed at 1555,1031 and 692 cm−1 are assigned to N H, C N and C C stretches,respectively. During H2 reduction at high temperature, the N Hstretch replaced the N O stretch. The Ag:Cu (1:1)-ACF/CNF sam-ples exposed to bacteria exhibited a broad peak at approximately3400 cm−1, corresponding to the O H stretching of H2O, and a peakat approximately 1700–1633 cm−1, corresponding to the amidegroup of proteins or peptide molecules which originated from thesurface or intracellular fluids of E. coli and S. aureus. In addition,a few low intensity peaks observed near 1380–1200 cm−1 corre-

spond to the N H stretching of proteins, C O stretching of fattyacids, and the P O stretching of nucleic acids and lipids. These dataconfirm the adsorption of the bacteria onto Ag:Cu (1:1)-ACF/CNF[23,24].

316 S. Singh et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 311– 319

–b1) A

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Fig. 7. SEM images of (a–a1) Ag:Cu (1:1)-ACF/CNF, (b

.7. Tensile strength

As shown later, ACF/CNF impregnated with Ag and Cu Nps is potential filter for water purifiers. Such fabric materials maye wrapped over a perforated tube and used for the purificationf water under flow conditions. We carried out tests on the pre-ared materials to determine their mechanical strength. Fig. 10escribes the stress-strain behavior of ACF, Ag:Cu (1:0)-, Ag:Cu0:1)-, and Ag:Cu (1:1)-ACF/CNF samples. As observed from thegure, Ag:Cu (1:1)-ACF/CNF had largest tensile strength (approx-

mately 17.5 × 106 N/m2), nearly ten-times higher than the ACFubstrate (approximately 1.7 × 106 N/m2). Thus, the addition of theetal Nps to the ACF and/or growth of CNF on the ACF at high tem-

eratures greatly enhanced the strength of the fabrics, attributedo increase in the surface density of ACFs and strengthening of thenterfacial bonding [25]. It may also be observed that the bimetalCF/CNF had superior tensile strength compared to single metal (Cur Ag) grown ACF/CNF, which is attributed to the distribution of Agt the base and that of Cu at the tips of the web in the bimetal-repared fabrics. In the single metal-grown web, the absence ofetals at the surface of the fabric rendered the material relatively

ess strong. The earlier study also confirmed that metal Nps with

igh surface activity, high energy and smaller size were responsi-le for significant enhancement of the mechanical properties of theCF fabrics [26]. Also, Ag:Cu (0:1)-ACF/CNF had nearly four timesigher tensile strength (approximately 8.4 × 106 N/m2) than Ag:Cu

g:Cu (3:1)-ACF/CNF and (c–c1) Ag:Cu (1:3)-ACF/CNF.

(1:0)-ACF/CNF because Cu-catalyzed CNFs were longer and thickerthan the CNFs catalyzed by Ag [18].

4.8. Antibacterial activity test

Fig. 11 shows the number of colonies of E. coli grown on LB agarplates for the different ACF-based materials prepared in this studyas antibacterial agents. The data shown in the figure reveals thatAg-ACF is superior to the other ACF-based materials. As observedfrom the figure, the Ag-ACF inhibited the growth of E. coli for threedays only. However, Cu-ACF suppressed the growth for only twodays. After that, the number of colonies was uncountable. As alsoshown in the figure, bimetal Ag:Cu (1:1)- and Ag:Cu (3:1)-ACFsexhibited similar strong antibacterial effects as that of Ag-ACF andinhibited the growth of the bacteria for nearly 3 days. However,after three days, the number of colonies began to increase. ForAg:Cu (1:1)-ACF, the number of colonies was uncountable after sixdays. Bimetal Ag:Cu (1:3)-ACF was able to suppress the formationof E. coli colonies for only two days. After that time period, thenumber of colonies was uncountable. The potential advantage ofusing the bimetal Ag:Cu-ACF prepared from suitable proportionsof Ag and Cu instead of the single metal Ag-ACF is obvious (re-refer

Table 1). The amounts of Ag in Ag:Cu (1:1) and Ag:Cu (3:1) areapproximately 50% and 25% smaller, respectively, than that in Ag-ACF. Therefore, these two bimetals-based ACF can be consideredto be as effective antibacterial agents as Ag-ACF for at least three

S. Singh et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 311– 319 317

(1:1)

datbt

Fig. 8. SEM images and EDX spectra (shown as the inset) of (a–a1) Ag:Cu

ays and reasonably effective for five days despite using reduced

mounts of expensive Ag metal. We now elucidate the antibac-erial activities of bimetal-grown ACFs/CNFs which are found toe significantly superior to the substrate Ag:Cu-ACF. Fig. 12 showshe number of colonies grown for seven days for ACF/CNF-based

Fig. 9. FTIR spectra of ACF, Ag:Cu (1:1)-ACF and ACF/CNF samples.

-ACF/CNF, (b–b1) Ag:Cu (3:1)-ACF/CNF and (c–c1) Ag:Cu (1:3)-ACF/CNF.

materials. There are four salient observations to be made from the

figure: (1) the Ag:Cu-ACF/CNF based materials were significantlysuperior to the respective ACF substrate-based materials (withoutnanofibers). (2) Ag-ACF/CNF was effective in killing the bacteria. (3)Cu-ACF/CNF inhibited growth for only one day. The colonies began

Fig. 10. Tensile strength of Ag-, Cu- and Ag:Cu (1:1)-ACF/CNF samples.

318 S. Singh et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 311– 319

FE

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ig. 11. Evaluation of antibacterial effects of Ag-, Cu- and Ag:Cu-ACF samples against. coli.

o appear on the following day and grew thereafter. However, lesshan half the number of colonies was observed for Cu-ACF/CNFhen compared to Cu-ACF within the same time period. (4) The

imetal-grown ACF/CNFs were able to either completely inhibit oruppress the growth of E. coli to an insignificant level over sevenays of incubation. As observed, Ag:Cu (1:1)- and Ag:Cu (3:1)-CF/CNF showed strong antibacterial effects against E. coli for sevenays and were nearly as effective as Ag-ACF/CNF. Specifically, noolony of E. coli was found after four days for Ag:Cu (1:1)-ACF/CNFamples and after 6 days for Ag:Cu (3:1)-ACF/CNF samples. Two orhree colonies were observed after seven days in both materials.owever, Ag:Cu (1:3)-ACF/CNF was observed to be less superior to

he other prepared bimetal ACF/CNF, despite also suppressing therowth of E. coli for one day.

The reason for the superior antibacterial activity of Ag:Cu-CF/CNF over that of a single metal Ag-ACF/CNF substrate, despite

he former requiring relatively less Ag, is attributed to the uniqueistribution of the two metals in the materials. The superiorntibacterial performance of Ag:Cu-ACF/CNF than that of Ag:Cu-CF is because of unique asymmetric distributions of two metals

n the multi-scale web of ACF/CNF. Mechanistically, the superiorerformance of ACF/CNF compared to ACF is attributed to thentrapment of bacteria in the multi-scale web of the ACF/CNF

omposite and the exposure of the bacteria to the Cu metals thatre located on the tips of the grown CNFs, providing relatively moreontact time to bacteria. The expensive Ag, which is inherently

ig. 12. Evaluation of antibacterial effect of Ag-, Cu- and Ag:Cu-ACF/CNF against. coli.

Fig. 13. Evaluation of antibacterial effect of Ag-, Cu- and Ag:Cu-ACF/CNF samplesagainst S. aureus.

more effective than the less expensive Cu as antibacterial agents,adhered to the ACF surface. This way, the strong antibacterial Agions produced near the surface in contact with water requiredrelatively less exposure time to be effective against the entrappedbacteria in the fiber-web. However, Cu ions near the top of thefibers in the web are relatively more exposed to the bacteria inthe surrounding medium. The overall effect results not only insuperior antibacterial activity, but the partial replacement of moreexpensive Ag than less expensive Cu also.

Fig. 13 describes the antibacterial effect of the prepared Ag-, Cu-,Ag:Cu-ACF/CNF samples against S. aureus, a gram-positive bacteria.As shown, all materials including Ag:Cu (1:3) are effective, killingthe bacteria. Interestingly, Cu-based materials are as effective asAg-based materials against S. aureus. This is also confirmed fromthe literature where Cu has been shown to have strong antibacterialeffect against gram-positive bacteria [27,28]. From the above data,it can be concluded that Ag:Cu (1:1)-ACF/CNF is equally as effectiveas Ag-ACF/CNF against gram-negative and gram-positive bacteriain water, despite using reduced amounts of Ag.

All tests performed on bimetals-ACF and bimetals-ACF/CNF,controls were prepared using the same protocol of diluting 1 ml ofthe bacterial LB broth solution. The samples of ACF only (withoutmetals) were also tested following the same protocol. Uncount-able number of colonies was found even after 7-days in controlsas well as in ACFs only. The LB broth contained sufficient nutrientsfor bacteria in controls and in ACF samples. After ultrasonciationof Ag:Cu-ACF/CNF or single metal-ACF/CNF, metal nanoparticlesat the tips of CNF may be removed. However, metals will remainadhered to the ACF surface. Therefore, control experiments could beperformed for ACF only, as mentioned earlier. It may also be men-tioned that the antibacterial characteristics of ACF/CNF is likely tobe the same as that of ACF (without metals).

We have also compared the antibacterial activity of the preparedAg:Cu-ACF/CNF to different antibacterial materials described in theliterature. As mentioned earlier, several methods have been devel-oped to produce Ag and Cu Nps in the aqueous solution using strongreducing agents. In some studies, Ag or Cu Nps were prepared insolution and transferred to different supports, such as activatedcarbon, silica, polyethylene, titanium–aluminum composite, TiO2and glass for antibacterial applications [1,8,10–17]. Recently, ACFand ACF/CNF were used as supports for Ag and Cu Nps for simi-lar applications. Table 4 presents the comparative data. As shown

in the table, Ag:Cu (1:1)-ACF/CNF has superior antibacterial effectsthan that of the materials discussed in the literature, with the lattershown to be inhibiting the growth of the gram-negative or gram-positive or both bacteria for three days or less.

S. Singh et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 443 (2014) 311– 319 319

Table 4A comparison of the antibacterial activity of Ag:Cu-ACF/CNF with data from the literature.

Reference Material Bacteria Initial no ofbacteria per ml

Ag loading(w/w %)

Antibacterial activity Duration

Present study Ag:Cu (1:1)- and (3:1)-ACF/CNF E. coli and S. aureus 107–108 9.0 and12.0

100% inhibition 7 days

[6] Silica micro-bead + 0.05 MAgNO3 + NaBH4

E. coli 6 × 106 0.63 100% inhibition 24 h

[8] AgNO3 + organic–inorganic hybrid sols E. coli, S. aureus ∼105 – ∼99% inhibition 24 h[11] Ag and Cu nanoparticles + polyethylene E. coli 105 10.0 100% Inhibition for Ag

and 95% inhibition for Cu24 h

[12] Ag and Cu ions + stainlesssteel + titanium materials

S. aureus 105 1.52 80–100% inhibition 24 h

[15] Hollow silica/sliver (SiO2/Ag) beads E. coli, S. aureus – 9.21 100% inhibition 24 h[16] Silver NPs embedded PS-DVB resin E. coli 105 11.7 100% inhibition 3 h

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[17] AgNO3 + ACF E. coli, S. aureus

[18] Ag-ACF/CNF E. coli, S. aureus

. Conclusions

The hierarchical carbon web of ACFs/CNFs prepared using Agnd Cu bimetal Nps was found to have lethal effects against bothram-negative E. coli and gram-positive S. aureus bacteria in water.he novelty of the prepared material is the asymmetric dispersionf Ag and Cu Nps created in the web, with the former adheredo the ACF surface and the latter attached to the tips of theVD-grown CNFs. Furthermore, the amount of Ag metal in theimetal Ag:Cu ACF/CNF was considerably less (approximately 50%

ess) than that in the single metal Ag-ACF/CNF, yet having similarntibacterial properties. Such bimetal-incorporated carbon micro-anofibers-based materials suppressed bacterial growth in water

or seven days and exhibited superior antibacterial properties thanhe materials discussed in the literature as antibacterial agents.he multi-scale web of the Ag-Cu Nps dispersed in ACF/CNF is aotential material for water purifiers for the purpose of controllingacterial growth in water.

cknowledgements

The authors acknowledge support from DMSRDE (Kanpur, India)n the form of a research grant and from the DST, New Delhi throughts Unit of Excellence on Soft Nanofabrication at IIT Kanpur. Theuthors are also thankful to Kynol Inc. (Tokyo, Japan) for supplyinghe ACFs.

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