7/27/2019 1-s2.0-S0376738810009865-main

1/7

Journal of Membrane Science 369 (2011) 499505

Contents lists available atScienceDirect

Journal of Membrane Science

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m e m s c i

Antimicrobial nano-fibrous membranes developed from electrospun

polyacrylonitrile nanofibers

Lifeng Zhang a, Jie Luo b, Todd J. Menkhaus c, Hemanthram Varadaraju c, Yuyu Sun b,, Hao Fong a,

a Department of Chemistry, South Dakota School of Mines and Technology, 501 East Saint Joseph Street, Rapid City, SD 57701, USAb Biomedical Engineering Program, University of South Dakota, 4800 North Career Avenue, Sioux Falls, SD 57107, USAc Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, 501 East Saint Joseph Street, Rapid City, SD 57701, USA

a r t i c l e i n f o

Article history:Received 31 August 2010

Received in revised form

10 December 2010

Accepted 13 December 2010

Available online 21 December 2010

Keywords:

Electrospinning

Polyacrylonitrile

Amidoxime

Silver

Antimicrobial

a b s t r a c t

In this study, polyacrylonitrile (PAN) nano-fibrous membranes with fiber diameters of 450nm wereprepared by the technique of electrospinning; amidoxime nano-fibrous membranes were then prepared

through treatment of PAN nano-fibrous membranes in hydroxylamine (NH2OH) aqueous solution. The

C N groups on the surface of PAN nanofibers reacted with NH2OH molecules and led to the formation

of C(NH2) NOH groups, which were used for coordination of Ag+ ions. Subsequently, the coordinated

Ag+ ions were converted into silver nanoparticles (AgNP) with sizes being tens of nanometers. Mor-

phologies, structures, and antimicrobial efficacies (against Staphylococcus aureusandEscherichia coli) of

the membranes of electrospun PAN (ESPAN) nanofibers, ESPAN surface functionalized with amidoxime

groups (ASFPAN), ASFPAN coordinated with silver ions (ASFPANAg+), and ASFPAN attached with sil-

ver nanoparticles (ASFPANAgNP) were investigated. The study revealed that, with treatment of ESPAN

membranes in1 M NH2OH aqueous solution for5 min, theresulting ASFPANmembranes becameantimi-

crobial without distinguishable morphological variations; further treatment of ASFPAN membranes in

0.1MAgNO3 aqueoussolutionfor 1 h andthesubsequenttreatmentin 0.01 M KBraqueoussolution for2 h

followed by photo-decomposition made the respective membranes of ASFPANAg + and ASFPANAgNP

highly antimicrobial, which were capable of killing the tested microorganisms in 30 min. The water

permeability test indicated that these membranes possessed adequate transport properties for filtrationapplications. Thisstudy demonstrated a convenient andcost-effectiveapproach to develop antimicrobial

nano-fibrous membranes that are particularly useful for the filtration of water and/or air.

2010 Elsevier B.V. All rights reserved.

1. Introduction

Polyacrylonitrile (PAN) fibrous membranes have been widely

adopted in filtration due to thermal stability, high mechanicalprop-

erties, and chemical resistivity [1,2]. Recently, there have been

numerous research efforts dedicated to electrospun nano-fibrous

membranes for the filtration application [37]. The nano-materials

processingtechnique of electrospinning provides a straightforward

approach to produce fibers with diameters ranging from tens to

hundreds of nanometers [810]. Electrospun PAN nano-fibrous

membranes have been of particular interests due to extraordinary

properties including small fiber diameters and the concomitant

large specific surface areas, as well as capabilities to control pore

sizes among nanofibers and to incorporate antimicrobial agents at

nanoscale[11,12].

Corresponding author. Tel.: +1 605 367 7776; fax: +1 605 782 3280. Corresponding author. Tel.: +1 605 394 1229; fax: +1 605 394 1232.

E-mail addresses: Yuyu.Sun@usd.edu(Y. Sun),Hao.Fong@sdsmt.edu(H. Fong).

The filters of nano-fibrous membranes with antimicrobial func-

tionality have attracted growing attentions due to the concerns

about qualities of purified water and/or filtered air as well as the

processing costs[5,6,1315]. Water and air filters (particularly

those operating in the dark and damp conditions) are constantly

subject to attacks from environmental microorganisms. The

microorganisms (such as bacteria) that can be readily captured

by the filters grow rapidly, resulting in the formation of biofilms.

Consequently, the buildups of microorganisms on the filter sur-

faces deteriorate the qualities of purified water and/or filtered air;

additionally, they also have the unfavorable effects on the flow of

water and/or air. Moreover, the contaminated filters with biofilms

are difficult to clean; usually, high pressure is required during the

operation. This in turn increases the costs. To our best knowledge,

there have been very few reports on electrospun PAN nano-fibrous

membranes with antimicrobial functionality[16,17];whereas the

reportedmethods are generally to incorporate antimicrobial agents

(such as N-halamine and silver ions/nanoparticles) directly into

spin dopes, thus the molecules/particles of antimicrobial agents

are distributed throughout the nanofibers. This direct-spinning

approach, however, often leads to low antimicrobial efficacy

0376-7388/$ see front matter 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.memsci.2010.12.032

http://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.memsci.2010.12.032http://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.memsci.2010.12.032http://www.sciencedirect.com/science/journal/03767388http://www.elsevier.com/locate/memscimailto:Yuyu.Sun@usd.edumailto:Hao.Fong@sdsmt.eduhttp://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.memsci.2010.12.032http://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.memsci.2010.12.032mailto:Hao.Fong@sdsmt.edumailto:Yuyu.Sun@usd.eduhttp://www.elsevier.com/locate/memscihttp://www.sciencedirect.com/science/journal/03767388http://localhost/var/www/apps/conversion/tmp/scratch_5/dx.doi.org/10.1016/j.memsci.2010.12.032

7/27/2019 1-s2.0-S0376738810009865-main

2/7

500 L. Zhang et al. / Journal of Membrane Science369 (2011) 499505

primarily because the high content of antimicrobial agents can

seriously affect the process of electrospinning and/or deteriorate

the properties of the resulting nanofibers. It was hypothesized that

a potential solution to these problems was to introduce antimicro-

bial functionality onto nanofiber surfaces after the nanofibers were

produced. It is known that the nitrile groups (C N) in PAN can

be chemically converted into amidoxime groups (C(NH2) NOH)

[18];the amidoxime groups can coordinate with a wide range of

metal ions including silver ions[19,20],and the coordinated silver

ions can be reduced into silver nanoparticles. It is noteworthy that

both silver ions and silver nanoparticles are antimicrobial agents

with high antimicrobial efficacy[17,21].

In this study, PAN nano-fibrous membranes with fiber diame-

tersof450nm werepreparedby the techniqueof electrospinning;

amidoxime nano-fibrous membranes were then prepared through

the treatment of PAN nano-fibrous membranes in hydroxylamine

(NH2OH) aqueous solution; the C N groups on the surface of

PAN nanofibers reacted with NH2OH molecules and led to the

formation ofC(NH2) NOH groups, which were used for coor-

dination of Ag+ ions. Subsequently, the coordinated Ag+ ions were

converted into silver nanoparticles (AgNP) with sizes being tens

of nanometers. Morphologies, structures, and antimicrobial effi-

cacies (against Staphylococcus aureus and Escherichia coli) of the

membranes of as-electrospun PAN (ESPAN) nanofibers, ESPAN sur-face functionalized with amidoxime groups (ASFPAN), ASFPAN

coordinated with silver ions (ASFPANAg+), and ASFPAN attached

with silver nanoparticles (ASFPANAgNP) were investigated. The

results indicated that the nano-fibrous membranes of ASFPANAg+

and ASFPANAgNP possessed potent antimicrobial functionality,

while the ASFPAN membranes were intrinsically antimicrobial and

their antimicrobial efficacy increased with prolonging the reac-

tion time with NH2OH. Additionally, the results acquired from

water permeability test indicated that the prepared membranes

possessed adequate transport properties for typical membrane

applications.

2. Experimental

2.1. Materials

The PAN used in this study was the Special Acrylic Fibers (S.A.F.

3K) provided by the Courtaulds, Ltd. (Nottingham, UK). Acetone,

N,N-dimethylformamide (DMF), hydroxylamine (NH2OH), silver

nitrate (AgNO3), and phosphate buffered saline (PBS) were pur-

chased from the SigmaAldrich Chemical Co. (St. Louis, MO) and

used without further purification. E. coli (ATCC 15597, Gram-

negative bacteria) and S. aureus (ATCC 6538, Gram-positive

bacteria) were obtained fromthe American Type Culture Collection

(ATCC, Manassas, VA).

2.2. Electrospinning

The PAN fibers of S.A.F. 3K were first immersed in acetone

overnight to remove the surface oil, they were then dried and

used to prepare a 14 wt.% solution in DMF at 60C. Subsequently,

the solution was filled in a 30mL BD Luer-LokTM tip plastic

syringe having an 18 gauge stainless-steel needle with 90 blunt

end. The electrospinning setup included an ES30P high voltage

power supply, purchased from the Gamma High Voltage Research,

Inc. (Ormond Beach, FL), and a nanofiber collector of electrically

grounded aluminum foil that covered a laboratory-produced roller

with the diameter of 10 in. The collector was placed at 9 in. below

the tip of needle. During electrospinning, a positive high volt-

age of 25kV was applied to the needle; and the solution feed

rate of 1.3 mL/h was maintained using a KDS 200 syringe pump

purchased from the KD Scientific Inc. (Holliston, MA). The elec-

trospun PAN nano-fibrous membranes could be readily peeled

off from the aluminum foil, and the obtained membranes were

stored in a desiccator before the subsequent surface functional-

ization.

2.3. Surface functionalization

The surface functionalization was carried out by immersion of

electrospun PANnano-fibrous membranes(ESPAN with the dimen-

sion being 2 in.2in.) in 1M NH2OH aqueous solution at 70C

for 5, 10, and 20 min. The surface functionalized membranes with

amidoxime groups (ASFPAN) were then immersed in 0.1 M AgNO3aqueous solution at 25 C for 30 min, 1 h, and 16h to allow the

amidoxime groups to coordinate with silver ions. The membranes

coordinated with silver ions (ASFPANAg+) were further treated

in 0.01 M KBr aqueous solution for 2 h, and this was followed by

immersion in methanol and exposure to intensive visible light for

10 min in a Triad 2000 chamber on each side to prepare the mem-

branes attached with silver nanoparticles (ASFPANAgNP). All of

the treated membranes were thoroughly rinsed in distilled water

after each step followed by being dried in an oven at 70 C for 6h

before characterizations and antimicrobial tests.

2.4. Characterization

A Zeiss Supra 40VP field-emission SEM was employed to exam-

ine the morphologies of the prepared nano-fibrous membranes.

Prior to SEM examination, all specimens were sputter-coated

with carbon to avoid charge accumulation. The silver map-

ping on individual nanofibers was acquired from a Hitachi

H-7000 TEM equipped with an H-7110 scanning module and

an IXRF energy-dispersive X-ray spectrometer. FT-IR spectra of

nano-fibrous membranes were obtained from Bruker Tensor-

27 FT-IR spectrometer equipped with a liquid nitrogen cooled

mercurycadmiumtelluride (MCT) detector.

2.5. Antimicrobial assessment

Antimicrobial assessments were carried out by following a

modifiedAATCC (AmericanAssociation of Textile Chemists and Col-

orists) Test Method 100-1999. E. coli and S. aureus were selected

as representative examples of Gram-negative and Gram-positive

bacteria, respectively, to evaluate the antibacterial properties of

ESPAN, ASFPAN, ASFPANAg+, and ASFPANAgNP samples. In the

antibacterial tests, both microbial species were grown in broth

solutions (LuriaBertani broth for E. coli, and tryptic soy broth for

S. aureus) for 24h at 37 C. The bacteria were harvested by cen-

trifuge, washed with phosphate buffered saline (PBS), and then

re-suspended in PBS to the density of 107 colony forming units per

milliliter(CFU/mL). 100L of the freshly prepared bacterialsuspen-

sions were placed onto the surfaces of two layers of the samples(2.00.1cm2). After a certain period of contact time, the sample

layers were transferred into 10 mL of sterilized PBS and vortexed

for 2 min to transfer the adherent bacteria into PBS. The solution

was then diluted serially, and 100L of each diluent were placed

onto agar plates (LuriaBertani agar forE. coli, and tryptic soy agar

for S. aureus). Colonyforming units on theagar plateswere counted

after incubation at 37 C for 24 h. Each test was repeated for three

times, and the lowest log reduction level of the three tests (i.e., the

weakest antimicrobial efficacy observed) was reported.

2.6. Water permeability test

The permeability of waterthrough different nano-fibrous mem-

branes was determined with an AKTA Purifier (GE Healthcare)

7/27/2019 1-s2.0-S0376738810009865-main

3/7

L. Zhang et al. / Journal of Membrane Science369 (2011) 499505 501

Fig. 1. Representative SEM images of (1) ESPAN (A); (2) ASFPAN: ESPAN in 1 M NH2OH aqueous solution at 70C for 5 min (ASFPAN-1, B), 10min (ASFPAN-2, C), and 20 min

(ASFPAN-3, D); (3) ASFPANAg+: ASFPAN-1 in 0.1M AgNO3aqueous solution for 30min (ASFPANAg+-1, E), 1 h (ASFPANAg+-2, F), and 16 h (ASFPANAg+-3, G); ASFPAN-2

in0.1M AgNO3 aqueous solution for 16h (ASFPANAg+-4, H);ASFPAN-3in 0.1M AgNO3 aqueous solutionfor 16h (ASFPANAg

+-5, I); (4) ASFPANAgNP: ASFPANAg+-1and

ASFPANAg+-5 in 0.01 M KBr aqueous solution for 2 h followed by photo-decomposition of AgBr (ASFPANAgNP-1 (J) and ASFPANAgNP-2 (K), respectively).

by online measurement of pressure. A small scale coin mem-

brane adsorption holder from the Pall Corporation (Pensacola, FL,

product number MSTG18H16) was utilized for the tests. The unit

allowed for 1.5 cm2 of effective filtration area, and was sealed

with an O-ring to prevent possible leakage. One layer (0.25mm)

of each nano-fibrous membrane was sandwiched between twomicro-porous supports and inserted into the holder. The pres-

sure drop was then measured for flow rates ramping from 5.0

to 25.0 mL/min, with stable pressure measured at each flow rate

before increasing to the next. After reaching 25.0 mL/min, the flow

rate was reversed and pressure measured to ensure no hystere-

sis was occurring due to irreversible compaction of the fibers. The

pressure drop of the system only, with the membrane holder and

micro-porous supports in place, but with no nanofiber membrane

present, was evaluated at the same flow rates shown above. The

system pressure drop was subtracted from the measured pressure

drop with the membrane in place to calculate permeability of the

membrane at each flow rate. A minimum of 7 flow rates and the

corresponding pressure readings were made for each nano-fibrous

membrane.

3. Results and discussion

3.1. Morphology

ESPAN membraneswere fluffy andcomposedof PANnanofibers

with diameters of

450nm (Fig. 1A). After reaction with NH2OHinwater for up to 20 min, the resulting ASFPAN membranes retained

the overall morphology while became densely packed. The ASF-

PAN membranes that reacted with NH2OH for 5 and 10min did

not show distinguishable variations of fiber size (Fig. 1Band C);

whereas those reacted with NH2OH for 20min had the average

fiber diameter of600 nm (Fig. 1D), representing 30% increase

in comparison with the original ESPAN nanofibers. It is note-

worthy that PAN is hydrophobic while amidoxime is much more

hydrophilic; therefore, the nanofibers will be swollen by water

if a large amount of nitrile groups are converted into amidoxime

groups. The coordination with silver ions and the following sil-

ver nanoparticle formation did not result in appreciable variations

of fiber diameters (Fig. 1EK). Scattered nanoparticles with sizes

from 20 to 100 nm were observed on the surface of nanofibers that

7/27/2019 1-s2.0-S0376738810009865-main

4/7

502 L. Zhang et al. / Journal of Membrane Science369 (2011) 499505

5001000150020002500300035004000

B

C

Wavenumber (cm-1)

1384

A

Absorbance(a.u.)

I 824

5001000150020002500300035004000

Wavenumber (cm-1)

Absorbance(a.u.)

D

E

F

16562243 927II 3453

5001000150020002500300035004000

Wavenumber (cm-1)

13841656

Absorbance(a.u.)

III 920

G

H

I

J

K

5001000150020002500300035004000

Wavenumber (cm-1)

L

M

Absorbance(a.u.)

IV 3450 2243 9231656

Fig. 2. FT-IR spectra of (I) ESPAN (A); ESPAN in 0.1M AgNO3 aqueous solution for 30 min (B); sample B washed thoroughly in distilled water (C); spectra were normalizedbased on the C N band at 2243cm1; (II) ASFPAN: ESPAN in 70 C NH2 OH aqueous solution for 5min (ASFPAN-1, D), 10 min (ASFPAN-2, E), and 20 min (ASFPAN-3, F); (III)

ASFPANAg+: ASFPAN-1 in 0.1 M AgNO3 aqueous solution for 30min (ASFPANAg+-1, G), 1 h (ASFPANAg+-2, H), and 16 h (ASFPANAg+-3, I); ASFPAN-2 in 0.1M AgNO3

aqueous solutionfor 16h (ASFPANAg+-4,J); ASFPAN-3in 0.1M AgNO3aqueoussolution for 16h (ASFPANAg+-5, K); (IV)ASFPANAgNP: ASFPANAg+-1 and ASFPANAg+-5

in 0.01 M KBr aqueous solution for 2 h followed by photo-decomposition of AgBr (ASFPANAgNP-1 (J) and ASFPANAgNP-2 (K), respectively). All of the spectra in (II), (III),

and (IV) were normalized based on the CH2 band centered at 1452cm1, since CH2 was not involved in coordination.

were treated in 1 M NH2OH for 5 min followed by the treatment

in 0.1M AgNO3 for 30min and the subsequent AgBr formation and

photo-decomposition (Fig. 1J). Prolonging the reaction times with

NH2OH (20 min) and AgNO3(16 h) resulted in more and larger sil-

ver nanoparticles on thesurface of nanofibers(40200nm, Fig.1K).

3.2. Structure

Prior to studying the reaction between ESPAN and NH 2OH as

well as the coordination between ASFPAN and silver ions, the

adsorption of AgNO3on ESPANnano-fibrous membrane was exam-

ined by FT-IR. A piece of ESPAN membrane (2in.2 in.) was

immersed in 0.1 M AgNO3 aqueous solution for 30 min and then

rinsed thoroughly with distilled water. As shown in FT-IR spectra

in Fig. 2(I), the sample before water rinse had an intense and broad

band at the wavenumber of1383cm1 aswell asa weakandnar-

row band at the wavenumber of824cm1; these two bands were

attributed to nitrate ions (NO3) in AgNO3[22], and they indicated

a large amount of AgNO3 remained on the surface of nanofibers

after the adsorption. However, the sample after thorough rinse in

distilled water showed no such peaks in its FT-IR spectrum, sug-

gesting that the adsorbed AgNO3was completely removed despite

thehigh surface area of ESPANnano-fibrousmembrane. Theresults

indicated that the simple adsorption of AgNO3 on ESPAN mem-

branes would not retain silver ions on the surface of nanofibers

under the in-use conditions of water/air filtration.

The FT-IR spectra of ASFPAN in Fig. 2(II) showed the char-

acteristic peak of PAN at 2243 cm1 (assigned to C N) and the

characteristic peaks of amidoxime at 31003700 cm1 (broad,

assigned to both NH and OH), 1656cm1

(assigned to C N), and917927 cm1 (assigned to NO). With the increase of reaction

time from 5 to 20 min, the intensities of the characteristic peak

of PAN and the characteristic peaks of amidoxime decreased and

increased, respectively. The maximums of peaks for NH/OH and

NO shifted to lower wavenumbers with increase of reaction time.

This indicated that hydrogen bonds formed among amidoxime

groups and/or between amidoxime groups and water molecules.

The extremely weak peak at 2243cm1 of the sample which

reacted with NH2OH for 20 min suggested that the nitrile groups

were close to be completely converted into amidoxime groups in

the nanofibers under such a condition. Other amidoxime-related

researches adopted the longer reaction time such as 6090 min

[20]and 24 h[23]using hydroxylamine hydrochloride. This study

revealed that a large amountof amidoximefunctional groupscould

7/27/2019 1-s2.0-S0376738810009865-main

5/7

L. Zhang et al. / Journal of Membrane Science369 (2011) 499505 503

Fig. 3. SEM images(A andB) andelementalmappingimagesof silver (A and B) for ASFPANAg+ nanofibers: ASFPANAg+-1(Aand A); ASFPANAg+-5(BandB). The images

of A and B were acquired using the same mapping time.

be generated on the surface of PAN nanofibers in a very short reac-

tion time (such as 5 min) by using NH2OH aqueous solution.

Immersion of ASFPANin 0.1M AgNO3aqueous solution resulted

in the complex of ASFPANAg+. After thorough rinse in distilled

water, all of the ASFPANAg+ samples in Fig. 2(III) still had a strong

absorption at 1384cm1 (assigned to NO3) in their FT-IR spec-

tra, while the NOvibration was substantially weaker as compared

to that in the spectra of ASFPAN. In particular, the comparison

between the FT-IR spectra ofFig. 2(III) andFig. 2(I)C indicated the

formation of coordination bonds between amidoxime groups andsilver ions. Amidoxime is a bidentate ligand because both N and

O atoms can contribute their lone-pair electrons for the formation

of coordination bonds[24,25].As illustrated inScheme 1,coordi-

nation bonds could be formed between silver ions and amidoxime

groups. Therefore, silver ions were bound onto the surface of ASF-

PANnanofibers; accordingly, the counter anions of NO3 were also

attached to the nanofiber surface thus could be detected by FT-IR.

Fig. 2(IV) showed the FT-IR spectra of the ASFPAN membranes

attached with silver nanoparticles (ASFPANAgNP) through for-

mation of AgBr followed by photo-decomposition. The strong

NO3 absorption was no longer present in the FT-IR spectra of

ASFPANAgNP. The characteristic peaks of ASFPANAgNP were

observed at 3450, 1656, and923 cm1, similar to the corresponding

spectra of D and F inFig. 2(II). It was evident that the coordination

between silver ions and amidoxime groups was no longer present,

indicating that most of the coordinated silver ions, if not all, were

converted into silver nanoparticles.

To understand the distribution of silver on ASFPANAg+

nanofibers, the elementalmapping of silver was acquired from two

samples: (1) ASFPANAg+-1 from 5 min reaction with NH2OH fol-

lowed by30 minimmersion in 0.1M AgNO3, and(2) ASFPANAg+-5

from 20 min reaction with NH2OH followed by 16 h immersion in

0.1M AgNO3. Fig. 3A and B showed the respective SEM images

of the representative nanofibers of (1) and (2); while Fig. 3Aand 3B showed the silver mapping images of the corresponding

C

N

Ag

NH2

n

C

N

O

NH2

n

H

AgNO3 aq NO3

OH

Scheme 1. The formation of coordination bonds between a silver ion and an ami-

doxime group.

7/27/2019 1-s2.0-S0376738810009865-main

6/7

504 L. Zhang et al. / Journal of Membrane Science369 (2011) 499505

Table 1

Antimicrobial efficaciesof ESPAN, ASFPAN,ASFPANAg+ and ASFPANAgNP against

S. aureusandE. coli.

Log reduction after different contact time with bacteria

Bacteria S. aureus E. coli

Contact time 30 min 60 min 30 min 60 min

ESPAN (Control) 0 0 0 0

ASFPAN-1 0 2 0 2

ASFPAN-2 0 3 0 3ASFPAN-3 7 7 7 7

ASFPANAg+-1 1 3 1 3

ASFPANAg+-2 7 7 7 7

ASFPANAg+-3 7 7 7 7

ASFPANAg+-4 7 7 7 7

ASFPANAg+-5 7 7 7 7

ASFPANAgNP-1 1 3 1 3

ASFPANAgNP-2 7 7 7 7

(1)The concentration of both bacteriawas 107 CFU/mL; and (2)the logreduction of

0 indicated no kill, while the log reduction of 7 indicated total kill.

nanofibers, respectively. The green areas and their intensities were

corresponding to the distribution and abundance of silver; i.e., the

location of green areas indicated the presence of silver, while the

brightness of green areas represented the abundance of silver. Itwas evident that silver existed/distributed evenly on the surface of

nanofibers; whereas longer reaction time with NH2OH led tomore

amidoxime groups on the fiber surface and thus higher amount of

silverions. This further supported that the coordination interaction

existed between silver ions and amidoxime functional groups.

3.3. Antimicrobial effects

Antimicrobial efficacies of ESPAN, ASFPAN, ASFPANAg+, and

ASFPANAgNP againstS. aureus and E. coli were listed inTable 1.

As expected, the ESPAN membranes did not possess any antimi-

crobial functionality against either microorganism within the

testing period up to 1 h. Thus under the application conditions,

microorganism species can readily contaminate ESPAN mem-branes, causing serious microorganism buildups.

The ASFPAN membranes, however, demonstrated reasonably

good antimicrobial activity: ASFPAN-1, which was from the short-

est reaction time with NH2OH (5 min), showed 2-log reduction for

both microorganisms (0 indicating no kill and 7 indicating

total kill) after 1 h contact; nonetheless, no antimicrobial effect

was observed within 30 min contact. When the reaction time with

NH2OH increased to 10 min, the antimicrobial efficacy of ASFPAN-

2 increased to 3-log reduction for both microorganisms after 1 h

contact. However, the antimicrobial efficacy remained to be 0

within 30 min contact for both microorganisms. Further increase

of reaction time with NH2OH to 20 min resulted in a substantial

improvement of antimicrobial efficacy to 7-log reduction for both

microorganisms (total kill) after 30min contact. The antimicrobialactivity of the ASFPAN membranes is associated with the strong

capacity of amidoxime groups to bind with metal ions (such as

Mg2+ and Ca2+) through coordination. These metal ions are essen-

tial for the stability and replication of the outer layers of bacterial

cell membranes. The coordination between amidoxime groups and

metal ions will compete with bacteria for the metal ions that are

essential for microbial survival, therefore inhibiting cellular repli-

cationand growth. Duringthe filtrationof water,metalions such as

Mg2+ and Ca2+ would be continuously supplied by the stream; thus

the ASFPAN membranes might not be able to effectively prevent

the buildups of microorganisms.

The antimicrobial efficacies of nano-fibrous membranes from

shorter reaction time with NH2OH (5 min and 10min) were sig-

nificantly improved upon binding with silver ions. Silver ions have

been known as a potentantimicrobial agent with lowmammal tox-

icity for thousands of years [26]. Although the detailed mechanism

of antimicrobial effect for silver ions remains controversial, the

previous research results suggested that the antimicrobial activity

might be originated from the strong binding capability to elec-

tron donor groups in biological molecules containing N, S, and/or

O[27].After coordination with silver ions onto the nanofibers, all

of ASFPANAg+ samples except ASFPANAg+-1, which was pre-

pared by immersing ASFPAN-1 in 0.1 M AgNO3

aqueous solution

for merely 30 min, demonstrated a total kill of both microorgan-

isms with 30 min contact. ASFPANAg+-1 only provided a 1-log

reduction for both microorganisms after 30 min contact and a 3-

log reduction after 1 h contact, most likely due to the low amount

of silver ions on this sample.

Since silver ions can be easily denatured by a wide range of

inorganic, organic, and/or biological compounds, leading to the

reduced antimicrobial efficacy in real applications, the coordinated

silverions were further convertedinto silver nanoparticles, a much

more stable form of silver to achieve the longevity of antimicrobial

functionality [21]. The antimicrobial activity of silver nanoparticles

might be originated from their capability to attach on the surface

of cell membranes thus disturbing permeability and respiration

functions of the microbes [21]. It is intriguing to note that all of

ASFPANAgNP samples provided similar antimicrobial efficacies astheir parent ASFPANAg+ samples.

The above results suggested that the incorporation of sil-

ver ions or silver nanoparticles onto ASFPAN membrane might

have dual effects on antimicrobial efficacy: on one hand, silver

ions/nanoparticles are potent antimicrobial agents that can kill

microbial cells [26], on the other hand, the amidoxime groups

on the membranes possess antimicrobial functionality through

competing for metal ions with the cells. Therefore, the combina-

tion of amidoxime functional groups and silver ions/nanoparticles

into one system could provide synergetic effects on anticandidal

efficacy. Indeed, ethylenediaminetetraacetic acid (EDTA), a widely

used chelating agent, has been found to compete with bacteria for

metal ions and disrupt cell membranes, which can substantially

enhance the anticandidal activity of other antimicrobial agents[28,29].It is also noteworthy that, for a specific ASFPANAgNP or

ASFPANAg+ sample, it showed very similar antimicrobial potency

against the Gram-negativeE. coliand the Gram-positive S. aureus.

It has been known that, unlike the wall of Gram-positive cells, the

wall of Gram-negative cells contains a thin peptidoglycan layer

adjacent to the cytoplasmic membrane. In addition, the Gram-

negative cell wall also contains an outer membrane composed by

phospholipids and lipopolysaccharides, which face to the exter-

nal environment. Theseadded protections makethe Gram-negative

cellwall muchless permeableto mostantimicrobial agentsthan the

Gram-positive cell wall. Thus, Gram-negative bacteria are usually

more difficult to kill than Gram-positive bacteria. In the nano-

fibrous membranes of ASFPANAgNP or ASFPANAg+, however,

because the amidoxime groups can damage bacteria cell walls,the differences associated with Gram-negative and Gram-positive

cell walls become less evident. Therefore, the samples showed the

similar antimicrobial activities against both classes of the bacterial

cells.

3.4. Water permeability

To determine whether the prepared materials would actually

possess properties that could make them attractive as membranes,

the fluid transport properties of nano-fibrous membranes were

studied by evaluating the water permeability. The water perme-

ability was measured in this study by using the flow rate of water

per unit area of membrane per unit pressure drop across the mem-

brane. The measured value of water permeability for the ESPAN

7/27/2019 1-s2.0-S0376738810009865-main

7/7

L. Zhang et al. / Journal of Membrane Science369 (2011) 499505 505

nano-fibrous membranes was 1.7107 m/(Pas); after surface

functionalization in NH2OH, the values of water permeability for

the ASFPAN membranes with the reaction times being 5, 10, and

20 min were slightly reduced into 1.4107, 8.5108, and

8.5108 m/(Pas), respectively. The slight reductions of water

permeability were attributed to the increase of hydrophilicity on

the surface of nano-fibrous membranes; i.e., the C(NH2) NOH

groups generated through the NH2OH treatment were much more

hydrophilic than the original C N groups. As evidenced by SEM

images (Fig. 1AD), swollen fibers with larger diameters as well as

more densely packed nano-fibrous membranes were observed for

the NH2OH treated membranes, both of which decreasedpore sizes

among the nanofibers and thus reduced the water permeability.

Nonetheless, the further coordination with silver ions and the sub-

sequent formation of silver nanoparticles did not significantly vary

the water permeability of the membranes: all of the ASFPANAg+

and ASFPANAgNP samples had the values of water permeability

between 6108 and 2107 m/(Pas). It is noteworthy that the

water permeability of the nano-fibrous membranes developed in

thisstudy was similar to thatof conventional micro-filtration mem-

branes, while it was higher than that of the typical nano-filtration

membranes for the applications of sized-based filtration and/or

separation involving the liquid flow (upon communication with

membrane vendors). This suggested that the processing through-put for the nano-fibrous materials developed in this study would

be adequate for typical membrane applications.

4. Conclusion

The surface functionalized PAN nano-fibrous membranes (ASF-

PAN) were prepared by electrospinning followed by amidoxime

reaction with NH2OH at 70C. Silver ions were then bound onto

the surface of nanofibers through coordination with amidoxime

functional groups, and the coordinated silver ions were further

reduced into silver nanoparticles. With a very short treatment time

of 5min in 1M NH2OH aqueous solution, the ESPAN nano-fibrous

membrane became antimicrobial without significant variation of

morphology. Although this membrane had relatively slow antimi-crobial action, it might still be applicable for the filtration of water

and/or air where the buildups of microorganisms occurred in days.

Further treatment of ASFPAN membranes in 0.1 M AgNO3aqueous

solution for 1 h and the subsequent treatment in 0.01 M KBr aque-

ous solution for 2 h followed by photo-decomposition made the

respective membranes of ASFPANAg+ and ASFPANAgNP highly

antimicrobial, which were capable of killing the tested microor-

ganisms of S. aureus and E. coli in 30min. The combination of

amidoxime groups with silver ions/nanoparticles into one system

was proposed as an effective strategy to achieve dual and/or syner-

getic effects on anticandidal efficacy. The water permeability test

indicated that the prepared nano-fibrous membranes possessed

adequate fluid transport properties for typical membrane appli-

cations. This study demonstrated a convenient and cost-effectiveapproach to develop antimicrobial nano-fibrous membranes that

would be particularly suitable for the filtration of water and/or air.

Acknowledgements

This research was supported by the National Science Founda-

tion (NSF) under the grant number of CBET-0827844. The authors

would also acknowledge the joint Biomedical Engineering (BME)

Program between the University of South Dakota (USD) and the

South Dakota School of Mines and Technology (SDSM&T).

References

[1] N. Scharnagl, H. Buschatz, Polyacrylonitrile (PAN) membranes for ultra- andmicrofiltration, Desalination 139 (2001) 191198.

[2] S. Yang, Z. Liu,Preparation and characterization of polyacrylonitrile ultrafiltra-tion membranes, J. Membr. Sci. 222 (2003) 8798.

[3] P. Gibson, H. Schreuder-Gibson, D. Rivin, Transport properties of porous mem-branes based on electrospun nanofibers, Colloids Surf. A 187188 (2001)469481.

[4] Z.M. Huang, Y.Z. Zhang, M. Kotaki, S. Ramakrishna, A review on polymernanofibers by electropinning and their applications in nanocomposites, Com-

pos. Sci. Technol. 63 (2003) 22232253.[5] R.S. Barhate, S. Ramakrishna, Nanofibrous filtering media: filtration problems

and solutions from tiny materials, J. Membr. Sci. 296 (2007) 18.[6] K. Yoon, B.S. Hsiao, B. Chu, Functional nanofibers for environmental applica-

tions, J. Mater. Chem. 18 (2008) 53265334.[7] L. Zhang, T.J. Menkhaus, H. Fong, Fabrication and bioseparation studies

of adsorptive membranes/felts made from electrospun cellulose acetatenanofibers, J. Membr. Sci. 319 (2008) 176184.

[8] Y. Dzenis, Spinning continuous fibers for nanotechnology, Science 304 (2004)19171919.

[9] A. Greiner, J.H. Wendorff,Electrospinning: a fascinating method for the prepa-ration of ultrathin fibres, Angew. Chem. Int. Ed. 46 (2007) 56705703.

[10] H. Fong, Electrospun polymer, ceramic, carbon/graphite nanofibers and theirapplications, in: H.S. Nalwa (Ed.), Polymeric Nanostructures and Their Appli-cations, vol. 2: Applications, American Scientific Publishers, Los Angeles,2005,pp. 451474.

[11] R.S. Barhate, C.K. Loong, S. Ramkrishna, Preparation and characterization ofnanofibrous filtering media, J. Membr. Sci. 283 (2006) 209218.

[12] K.M. Yun, C.J. Hogan Jr., Y. Matsubayashi, M. Kawabe, F. Iskandar, K. Okuyama,Nanoparticlefiltration by electrospun polymerfibers,Chem. Eng.Sci. 62(2007)47514759.

[13] E.H.Jeong,J. Yang, J.H.Youk,Preparationof polyurethanecationomernanofibermats for use in antimicrobial nanofilter applications, Mater. Lett. 61 (2007)39913994.

[14] S.J. Kim, Y.S. Nam, D.M. Rhee, H.S. Park, W.H. Park, Preparation and character-ization of antimicrobial polycarbonate nanofiberous membrane, Eur. Polym. J.43 (2007) 31463152.

[15] C. Yao, X. Li, K.G. Neoh, Z. Shi, E.T. Kang, Surface modification and antibacte-rial activity of electrospun polyurethane fibrous membranes with quaternaryammonium moieties, J. Membr. Sci. 320 (2008) 259267.

[16] X. Ren, A. Akdag, C. Zhu, L. Kou, S.D. Worley, T.S. Huang, Electrospun poly-acrylonitrile nanofibrous biomaterials, J. Biomed. Mater. Res. Part A 91 (2009)385390.

[17] N.L. Lala, R. Ramaseshan, B. Li, S. Sundarrajan, R.S. Barhate, Y.J. Liu, S. Ramkr-ishna, Fabrication of nanofibers with antimicrobial functionality used asfilters:protection againstbacterial contaminants,Biotechnol. Bioeng.97 (2007)13571365.

[18] W. Lin, Y. Lu, H. Zeng, Studies of the preparation, structure, and properties ofan acrylic chelating fiber containing amidoxime groups, J. Appl. Polym. Sci. 47(1993) 4552.

[19] J. Okamoto,T. Sugo, A. Katakai,H. Omichi, Amidoxime-group-containingadsor-bents for metal ions synthesized by radiation-induced grafting, J. Appl. Polym.Sci. 30 (1985) 29672977.

[20] K. Saeed, S. Haider, T.J. Oh, S.Y. Park, Preparation of amidoxime-modifiedpolyacrylonitrile (PAN-oxime) nanofibers and their applications to metal ionsadsorption, J. Membr. Sci. 322 (2008) 400405.

[21] V.K.Sharma,R.A. Yngard, Y. Lin,Silver nanoparticles: green synthesis and theirantimicrobial activities, Adv. Colloid Interface Sci. 145 (2009) 8396.

[22] J. Coates, Interpretation of infrared spectraa practical approach, in: R.A.Meyers (Ed.), Encyclopedia of Analytical Chemistry, John Wiley & Sons Ltd.,Chichester, 2000, pp. 1081510837.

[23] L. Chen, L. Bromberg, H. Schreuder-Gibson,J. Walker,T.A. Hatton,G.C. Rutledge,Chemicalprotection fabrics viasurfaceoximationof electrospun polyacryloni-trile fiber mats, J. Mater. Chem. 19 (2009) 24322438.

[24] D.L. Verrasest, J.A. Peters, H.C. Kuzee, H.W.C. Raaijmakers, H.V. Bekkum, Modi-fication of inulin withamidoxime groups and coordiantin withcopper(II) ions,

Carbohydr. Polym. 37 (1998) 209214.[25] M.V. Dinu, E.S. Dragan, Synthesis and applications of some organic chelating

sorbents, in: E.S. Dragan (Ed.), New Trends in Ionic (Co) Polymers and Hybrids,Nova Science Publishers Inc., New York, 2007, pp. 6590.

[26] R. Bhattacharya, P. Mukherjee, Biologicalproperties of naked metal nanoparti-cles, Adv. Drug Deliv. Rev. 60 (2008) 12891306.

[27] R. Kumar, H. Munstedt, Silver ion release from antimicrobial polyamide/silvercomposites, Biomaterials 26 (2005) 20812088.

[28] E. Banin, K.M. Brady, E.P. Greenberg, Chelator-induced dispersal and killing ofPseudomonas aeruginosacells in a biofilm, Appl. Environ. Microbiol. 72 (2006)20642069.

[29] R.J.W. Lambert, G.W. Hanlon, S.P. Denyer, The synergistic effect ofEDTA/antimicrobial combinations onPseudomonas aeruginosa, J. Appl. Micro-biol. 96 (2004) 244253.