Journal of Membrane Science 369 (2011) 499–505
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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 b c
Department of Chemistry, South Dakota School of Mines and Technology, 501 East Saint Joseph Street, Rapid City, SD 57701, USA Biomedical Engineering Program, University of South Dakota, 4800 North Career Avenue, Sioux Falls, SD 57107, USA 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 ∼450 nm were prepared by the technique of electrospinning; amidoxime nano-fibrous membranes were then prepared through treatment of PAN nano-fibrous membranes in hydroxylamine (NH2 OH) aqueous solution. The –C N groups on the surface of PAN nanofibers reacted with NH2 OH molecules and led to the formation of –C(NH2 ) N–OH 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. Morphologies, structures, and antimicrobial efficacies (against Staphylococcus aureus and Escherichia coli) of the membranes of electrospun PAN (ESPAN) nanofibers, ESPAN surface functionalized with amidoxime groups (ASFPAN), ASFPAN coordinated with silver ions (ASFPAN–Ag+ ), and ASFPAN attached with silver nanoparticles (ASFPAN–AgNP) were investigated. The study revealed that, with treatment of ESPAN membranes in 1 M NH2 OH aqueous solution for 5 min, the resulting ASFPAN membranes became antimicrobial without distinguishable morphological variations; further treatment of ASFPAN membranes in 0.1 M AgNO3 aqueous solution for 1 h and the subsequent treatment in 0.01 M KBr aqueous solution for 2 h followed by photo-decomposition made the respective membranes of ASFPAN–Ag+ and ASFPAN–AgNP 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 filtration applications. This study demonstrated a convenient and cost-effective approach 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 mechanical properties, and chemical resistivity [1,2]. Recently, there have been numerous research efforts dedicated to electrospun nano-fibrous membranes for the filtration application [3–7]. The nano-materials processing technique of electrospinning provides a straightforward approach to produce fibers with diameters ranging from tens to hundreds of nanometers [8–10]. 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:
[email protected] (Y. Sun),
[email protected] (H. Fong). 0376-7388/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2010.12.032
The filters of nano-fibrous membranes with antimicrobial functionality have attracted growing attentions due to the concerns about qualities of purified water and/or filtered air as well as the processing costs [5,6,13–15]. 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 surfaces 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 reported methods 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
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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 antimicrobial 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 diameters of ∼450 nm were prepared by the technique of electrospinning; amidoxime nano-fibrous membranes were then prepared through the treatment of PAN nano-fibrous membranes in hydroxylamine (NH2 OH) aqueous solution; the −C N groups on the surface of PAN nanofibers reacted with NH2 OH molecules and led to the formation of −C(NH2 ) N–OH 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. Morphologies, structures, and antimicrobial efficacies (against Staphylococcus aureus and Escherichia coli) of the membranes of as-electrospun PAN (ESPAN) nanofibers, ESPAN surface functionalized with amidoxime groups (ASFPAN), ASFPAN coordinated with silver ions (ASFPAN–Ag+ ), and ASFPAN attached with silver nanoparticles (ASFPAN–AgNP) were investigated. The results indicated that the nano-fibrous membranes of ASFPAN–Ag+ and ASFPAN–AgNP possessed potent antimicrobial functionality, while the ASFPAN membranes were intrinsically antimicrobial and their antimicrobial efficacy increased with prolonging the reaction time with NH2 OH. 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 (NH2 OH), silver nitrate (AgNO3 ), and phosphate buffered saline (PBS) were purchased from the Sigma–Aldrich Chemical Co. (St. Louis, MO) and used without further purification. E. coli (ATCC 15597, Gramnegative bacteria) and S. aureus (ATCC 6538, Gram-positive bacteria) were obtained from the 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 60 ◦ C. Subsequently, the solution was filled in a 30 mL 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 voltage of 25 kV 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 electrospun 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 functionalization. 2.3. Surface functionalization The surface functionalization was carried out by immersion of electrospun PAN nano-fibrous membranes (ESPAN with the dimension being 2 in. × 2 in.) in 1 M NH2 OH aqueous solution at 70 ◦ C for 5, 10, and 20 min. The surface functionalized membranes with amidoxime groups (ASFPAN) were then immersed in 0.1 M AgNO3 aqueous solution at ∼25 ◦ C for 30 min, 1 h, and 16 h to allow the amidoxime groups to coordinate with silver ions. The membranes coordinated with silver ions (ASFPAN–Ag+ ) 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 membranes attached with silver nanoparticles (ASFPAN–AgNP). 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 6 h before characterizations and antimicrobial tests. 2.4. Characterization A Zeiss Supra 40VP field-emission SEM was employed to examine 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 mapping 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 Tensor27 FT-IR spectrometer equipped with a liquid nitrogen cooled mercury–cadmium–telluride (MCT) detector. 2.5. Antimicrobial assessment Antimicrobial assessments were carried out by following a modified AATCC (American Association of Textile Chemists and Colorists) 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, ASFPAN–Ag+ , and ASFPAN–AgNP samples. In the antibacterial tests, both microbial species were grown in broth solutions (Luria–Bertani broth for E. coli, and tryptic soy broth for S. aureus) for 24 h at 37 ◦ C. The bacteria were harvested by centrifuge, washed with phosphate buffered saline (PBS), and then re-suspended in PBS to the density of 107 colony forming units per milliliter (CFU/mL). 100 L of the freshly prepared bacterial suspensions were placed onto the surfaces of two layers of the samples (2.0 ± 0.1 cm2 ). 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 100 L of each diluent were placed onto agar plates (Luria–Bertani agar for E. coli, and tryptic soy agar for S. aureus). Colony forming units on the agar plates were 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 water through different nano-fibrous membranes was determined with an AKTA Purifier (GE Healthcare)
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Fig. 1. Representative SEM images of (1) ESPAN (A); (2) ASFPAN: ESPAN in 1 M NH2 OH aqueous solution at 70 ◦ C for 5 min (ASFPAN-1, B), 10 min (ASFPAN-2, C), and 20 min (ASFPAN-3, D); (3) ASFPAN–Ag+ : ASFPAN-1 in 0.1 M AgNO3 aqueous solution for 30 min (ASFPAN–Ag+ -1, E), 1 h (ASFPAN–Ag+ -2, F), and 16 h (ASFPAN–Ag+ -3, G); ASFPAN-2 in 0.1 M AgNO3 aqueous solution for 16 h (ASFPAN–Ag+ -4, H); ASFPAN-3 in 0.1 M AgNO3 aqueous solution for 16 h (ASFPAN–Ag+ -5, I); (4) ASFPAN–AgNP: ASFPAN–Ag+ -1 and ASFPAN–Ag+ -5 in 0.01 M KBr aqueous solution for 2 h followed by photo-decomposition of AgBr (ASFPAN–AgNP-1 (J) and ASFPAN–AgNP-2 (K), respectively).
by online measurement of pressure. A small scale “coin” membrane 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 (∼0.25 mm) of each nano-fibrous membrane was sandwiched between two micro-porous supports and inserted into the holder. The pressure 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 hysteresis 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 membranes were fluffy and composed of PAN nanofibers with diameters of ∼450 nm (Fig. 1A). After reaction with NH2 OH in water for up to 20 min, the resulting ASFPAN membranes retained the overall morphology while became densely packed. The ASFPAN membranes that reacted with NH2 OH for 5 and 10 min did not show distinguishable variations of fiber size (Fig. 1B and C); whereas those reacted with NH2 OH for 20 min had the average fiber diameter of ∼600 nm (Fig. 1D), representing ∼30% increase in comparison with the original ESPAN nanofibers. It is noteworthy 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 silver nanoparticle formation did not result in appreciable variations of fiber diameters (Fig. 1E–K). Scattered nanoparticles with sizes from 20 to 100 nm were observed on the surface of nanofibers that
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1384
824
Absorbance (a.u.)
I
II
C B
2243
3453
1656
927
Absorbance (a.u.)
502
F E
A 4000
3500
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D 4000
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-1
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Wavenumber (cm )
Absorbance (a.u.)
J I
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3450
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Absorbance (a.u.)
IV
1656 1384 920
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H G 4000
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III
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Fig. 2. FT-IR spectra of (I) ESPAN (A); ESPAN in 0.1 M AgNO3 aqueous solution for 30 min (B); sample “B” washed thoroughly in distilled water (C); spectra were normalized based on the C N band at 2243 cm−1 ; (II) ASFPAN: ESPAN in 70 ◦ C NH2 OH aqueous solution for 5 min (ASFPAN-1, D), 10 min (ASFPAN-2, E), and 20 min (ASFPAN-3, F); (III) ASFPAN–Ag+ : ASFPAN-1 in 0.1 M AgNO3 aqueous solution for 30 min (ASFPAN–Ag+ -1, G), 1 h (ASFPAN–Ag+ -2, H), and 16 h (ASFPAN–Ag+ -3, I); ASFPAN-2 in 0.1 M AgNO3 aqueous solution for 16 h (ASFPAN–Ag+ -4, J); ASFPAN-3 in 0.1 M AgNO3 aqueous solution for 16 h (ASFPAN–Ag+ -5, K); (IV) ASFPAN–AgNP: ASFPAN–Ag+ -1 and ASFPAN–Ag+ -5 in 0.01 M KBr aqueous solution for 2 h followed by photo-decomposition of AgBr (ASFPAN–AgNP-1 (J) and ASFPAN–AgNP-2 (K), respectively). All of the spectra in (II), (III), and (IV) were normalized based on the –CH2 – band centered at 1452 cm−1 , since –CH2 – was not involved in coordination.
were treated in 1 M NH2 OH for 5 min followed by the treatment in 0.1 M AgNO3 for 30 min and the subsequent AgBr formation and photo-decomposition (Fig. 1J). Prolonging the reaction times with NH2 OH (20 min) and AgNO3 (16 h) resulted in more and larger silver nanoparticles on the surface of nanofibers (40–200 nm, Fig. 1K). 3.2. Structure Prior to studying the reaction between ESPAN and NH2 OH as well as the coordination between ASFPAN and silver ions, the adsorption of AgNO3 on ESPAN nano-fibrous membrane was examined by FT-IR. A piece of ESPAN membrane (2 in. × 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 of ∼1383 cm−1 as well as a weak and narrow band at the wavenumber of ∼824 cm−1 ; 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, suggesting that the adsorbed AgNO3 was completely removed despite
the high surface area of ESPAN nano-fibrous membrane. The results indicated that the simple adsorption of AgNO3 on ESPAN membranes 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 characteristic peak of PAN at 2243 cm−1 (assigned to C N) and the characteristic peaks of amidoxime at 3100–3700 cm−1 (broad, assigned to both N–H and O–H), 1656 cm−1 (assigned to C N), and 917–927 cm−1 (assigned to N–O). 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 N–H/O–H and N–O 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 2243 cm−1 of the sample which reacted with NH2 OH 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 60–90 min [20] and 24 h [23] using hydroxylamine hydrochloride. This study revealed that a large amount of amidoxime functional groups could
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Fig. 3. SEM images (A and B) and elemental mapping images of silver (A and B ) for ASFPAN–Ag+ nanofibers: ASFPAN–Ag+ -1 (A and A ); ASFPAN–Ag+ -5 (B and B ). 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 reaction time (such as 5 min) by using NH2 OH aqueous solution. Immersion of ASFPAN in 0.1 M AgNO3 aqueous solution resulted in the complex of ASFPAN–Ag+ . After thorough rinse in distilled water, all of the ASFPAN–Ag+ samples in Fig. 2(III) still had a strong absorption at 1384 cm−1 (assigned to NO3 − ) in their FT-IR spectra, while the N–O vibration was substantially weaker as compared to that in the spectra of ASFPAN. In particular, the comparison between the FT-IR spectra of Fig. 2(III) and Fig. 2(I)C indicated the formation of coordination bonds between amidoxime groups and silver 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 in Scheme 1, coordination bonds could be formed between silver ions and amidoxime groups. Therefore, silver ions were bound onto the surface of ASFPAN nanofibers; 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 (ASFPAN–AgNP) through formation of AgBr followed by photo-decomposition. The strong NO3 − absorption was no longer present in the FT-IR spectra of ASFPAN–AgNP. The characteristic peaks of ASFPAN–AgNP were observed at 3450, 1656, and 923 cm−1 , similar to the corresponding spectra of D and F in Fig. 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 ASFPAN–Ag+ nanofibers, the elemental mapping of silver was acquired from two samples: (1) ASFPAN–Ag+ -1 from 5 min reaction with NH2 OH followed by 30 min immersion in 0.1 M AgNO3 , and (2) ASFPAN–Ag+ -5 from 20 min reaction with NH2 OH followed by 16 h immersion in 0.1 M AgNO3 . Fig. 3A and B showed the respective SEM images of the representative nanofibers of (1) and (2); while Fig. 3A’ and 3B’ showed the silver mapping images of the corresponding
Ag
HO
H O N
NH2 C n
N AgNO3 aq
NH2 NO 3
C n
Scheme 1. The formation of coordination bonds between a silver ion and an amidoxime group.
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Table 1 Antimicrobial efficacies of ESPAN, ASFPAN, ASFPAN–Ag+ and ASFPAN–AgNP against S. aureus and E. 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) ASFPAN-1 ASFPAN-2 ASFPAN-3 ASFPAN–Ag+ -1 ASFPAN–Ag+ -2 ASFPAN–Ag+ -3 ASFPAN–Ag+ -4 ASFPAN–Ag+ -5 ASFPAN–AgNP-1 ASFPAN–AgNP-2
0 0 0 7 1 7 7 7 7 1 7
0 2 3 7 3 7 7 7 7 3 7
0 0 0 7 1 7 7 7 7 1 7
0 2 3 7 3 7 7 7 7 3 7
(1) The concentration of both bacteria was 107 CFU/mL; and (2) the log reduction 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. It was evident that silver existed/distributed evenly on the surface of nanofibers; whereas longer reaction time with NH2 OH led to more amidoxime groups on the fiber surface and thus higher amount of silver ions. This further supported that the coordination interaction existed between silver ions and amidoxime functional groups. 3.3. Antimicrobial effects Antimicrobial efficacies of ESPAN, ASFPAN, ASFPAN–Ag+ , and ASFPAN–AgNP against S. aureus and E. coli were listed in Table 1. As expected, the ESPAN membranes did not possess any antimicrobial functionality against either microorganism within the testing period up to 1 h. Thus under the application conditions, microorganism species can readily contaminate ESPAN membranes, causing serious microorganism buildups. The ASFPAN membranes, however, demonstrated reasonably good antimicrobial activity: ASFPAN-1, which was from the shortest reaction time with NH2 OH (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 NH2 OH increased to 10 min, the antimicrobial efficacy of ASFPAN2 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 NH2 OH to 20 min resulted in a substantial improvement of antimicrobial efficacy to 7-log reduction for both microorganisms (total kill) after 30 min contact. The antimicrobial activity 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 essential 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 replication and growth. During the filtration of water, metal ions 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 NH2 OH (5 min and 10 min) were significantly improved upon binding with silver ions. Silver ions have
been known as a potent antimicrobial agent with low mammal toxicity 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 electron donor groups in biological molecules containing N, S, and/or O [27]. After coordination with silver ions onto the nanofibers, all of ASFPAN–Ag+ samples except ASFPAN–Ag+ -1, which was prepared by immersing ASFPAN-1 in 0.1 M AgNO3 aqueous solution for merely 30 min, demonstrated a total kill of both microorganisms with 30 min contact. ASFPAN–Ag+ -1 only provided a 1-log reduction for both microorganisms after 30 min contact and a 3log 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 silver ions were further converted into 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 ASFPAN–AgNP samples provided similar antimicrobial efficacies as their parent ASFPAN–Ag+ samples. The above results suggested that the incorporation of silver 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 combination 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 ASFPAN–AgNP or ASFPAN–Ag+ sample, it showed very similar antimicrobial potency against the Gram-negative E. coli and 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 Gramnegative cell wall also contains an outer membrane composed by phospholipids and lipopolysaccharides, which face to the external environment. These added protections make the Gram-negative cell wall much less permeable to most antimicrobial agents than the Gram-positive cell wall. Thus, Gram-negative bacteria are usually more difficult to kill than Gram-positive bacteria. In the nanofibrous membranes of ASFPAN–AgNP or ASFPAN–Ag+ , 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 permeability was measured in this study by using the flow rate of water per unit area of membrane per unit pressure drop across the membrane. The measured value of water permeability for the ESPAN
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nano-fibrous membranes was ∼1.7 × 10−7 m/(Pa s); after surface functionalization in NH2 OH, the values of water permeability for the ASFPAN membranes with the reaction times being 5, 10, and 20 min were slightly reduced into ∼1.4 × 10−7 , ∼8.5 × 10−8 , and ∼8.5 × 10−8 m/(Pa s), 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 ) N–OH groups generated through the NH2 OH treatment were much more hydrophilic than the original –C N groups. As evidenced by SEM images (Fig. 1A–D), swollen fibers with larger diameters as well as more densely packed nano-fibrous membranes were observed for the NH2 OH treated membranes, both of which decreased pore sizes among the nanofibers and thus reduced the water permeability. Nonetheless, the further coordination with silver ions and the subsequent formation of silver nanoparticles did not significantly vary the water permeability of the membranes: all of the ASFPAN–Ag+ and ASFPAN–AgNP samples had the values of water permeability between 6 × 10−8 and 2 × 10−7 m/(Pa s). It is noteworthy that the water permeability of the nano-fibrous membranes developed in this study was similar to that of conventional micro-filtration membranes, 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 throughput 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 (ASFPAN) were prepared by electrospinning followed by amidoxime reaction with NH2 OH at 70 ◦ C. 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 5 min in 1 M NH2 OH aqueous solution, the ESPAN nano-fibrous membrane became antimicrobial without significant variation of morphology. Although this membrane had relatively slow antimicrobial 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 AgNO3 aqueous solution for 1 h and the subsequent treatment in 0.01 M KBr aqueous solution for 2 h followed by photo-decomposition made the respective membranes of ASFPAN–Ag+ and ASFPAN–AgNP highly antimicrobial, which were capable of killing the tested microorganisms of S. aureus and E. coli in 30 min. The combination of amidoxime groups with silver ions/nanoparticles into one system was proposed as an effective strategy to achieve dual and/or synergetic effects on anticandidal efficacy. The water permeability test indicated that the prepared nano-fibrous membranes possessed adequate fluid transport properties for typical membrane applications. This study demonstrated a convenient and cost-effective approach 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 Foundation (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).
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References [1] N. Scharnagl, H. Buschatz, Polyacrylonitrile (PAN) membranes for ultra- and microfiltration, Desalination 139 (2001) 191–198. [2] S. Yang, Z. Liu, Preparation and characterization of polyacrylonitrile ultrafiltration membranes, J. Membr. Sci. 222 (2003) 87–98. [3] P. Gibson, H. Schreuder-Gibson, D. Rivin, Transport properties of porous membranes based on electrospun nanofibers, Colloids Surf. A 187–188 (2001) 469–481. [4] Z.M. Huang, Y.Z. Zhang, M. Kotaki, S. Ramakrishna, A review on polymer nanofibers by electropinning and their applications in nanocomposites, Compos. Sci. Technol. 63 (2003) 2223–2253. [5] R.S. Barhate, S. Ramakrishna, Nanofibrous filtering media: filtration problems and solutions from tiny materials, J. Membr. Sci. 296 (2007) 1–8. [6] K. Yoon, B.S. Hsiao, B. Chu, Functional nanofibers for environmental applications, J. Mater. Chem. 18 (2008) 5326–5334. [7] L. Zhang, T.J. Menkhaus, H. Fong, Fabrication and bioseparation studies of adsorptive membranes/felts made from electrospun cellulose acetate nanofibers, J. Membr. Sci. 319 (2008) 176–184. [8] Y. Dzenis, Spinning continuous fibers for nanotechnology, Science 304 (2004) 1917–1919. [9] A. Greiner, J.H. Wendorff, Electrospinning: a fascinating method for the preparation of ultrathin fibres, Angew. Chem. Int. Ed. 46 (2007) 5670–5703. [10] H. Fong, Electrospun polymer, ceramic, carbon/graphite nanofibers and their applications, in: H.S. Nalwa (Ed.), Polymeric Nanostructures and Their Applications, vol. 2: Applications, American Scientific Publishers, Los Angeles, 2005, pp. 451–474. [11] R.S. Barhate, C.K. Loong, S. Ramkrishna, Preparation and characterization of nanofibrous filtering media, J. Membr. Sci. 283 (2006) 209–218. [12] K.M. Yun, C.J. Hogan Jr., Y. Matsubayashi, M. Kawabe, F. Iskandar, K. Okuyama, Nanoparticle filtration by electrospun polymer fibers, Chem. Eng. Sci. 62 (2007) 4751–4759. [13] E.H. Jeong, J. Yang, J.H. Youk, Preparation of polyurethane cationomer nanofiber mats for use in antimicrobial nanofilter applications, Mater. Lett. 61 (2007) 3991–3994. [14] S.J. Kim, Y.S. Nam, D.M. Rhee, H.S. Park, W.H. Park, Preparation and characterization of antimicrobial polycarbonate nanofiberous membrane, Eur. Polym. J. 43 (2007) 3146–3152. [15] C. Yao, X. Li, K.G. Neoh, Z. Shi, E.T. Kang, Surface modification and antibacterial activity of electrospun polyurethane fibrous membranes with quaternary ammonium moieties, J. Membr. Sci. 320 (2008) 259–267. [16] X. Ren, A. Akdag, C. Zhu, L. Kou, S.D. Worley, T.S. Huang, Electrospun polyacrylonitrile nanofibrous biomaterials, J. Biomed. Mater. Res. Part A 91 (2009) 385–390. [17] N.L. Lala, R. Ramaseshan, B. Li, S. Sundarrajan, R.S. Barhate, Y.J. Liu, S. Ramkrishna, Fabrication of nanofibers with antimicrobial functionality used as filters: protection against bacterial contaminants, Biotechnol. Bioeng. 97 (2007) 1357–1365. [18] W. Lin, Y. Lu, H. Zeng, Studies of the preparation, structure, and properties of an acrylic chelating fiber containing amidoxime groups, J. Appl. Polym. Sci. 47 (1993) 45–52. [19] J. Okamoto, T. Sugo, A. Katakai, H. Omichi, Amidoxime-group-containing adsorbents for metal ions synthesized by radiation-induced grafting, J. Appl. Polym. Sci. 30 (1985) 2967–2977. [20] K. Saeed, S. Haider, T.J. Oh, S.Y. Park, Preparation of amidoxime-modified polyacrylonitrile (PAN-oxime) nanofibers and their applications to metal ions adsorption, J. Membr. Sci. 322 (2008) 400–405. [21] V.K. Sharma, R.A. Yngard, Y. Lin, Silver nanoparticles: green synthesis and their antimicrobial activities, Adv. Colloid Interface Sci. 145 (2009) 83–96. [22] J. Coates, Interpretation of infrared spectra—a practical approach, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, John Wiley & Sons Ltd., Chichester, 2000, pp. 10815–10837. [23] L. Chen, L. Bromberg, H. Schreuder-Gibson, J. Walker, T.A. Hatton, G.C. Rutledge, Chemical protection fabrics via surface oximation of electrospun polyacrylonitrile fiber mats, J. Mater. Chem. 19 (2009) 2432–2438. [24] D.L. Verrasest, J.A. Peters, H.C. Kuzee, H.W.C. Raaijmakers, H.V. Bekkum, Modification of inulin with amidoxime groups and coordiantin with copper (II) ions, Carbohydr. Polym. 37 (1998) 209–214. [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. 65–90. [26] R. Bhattacharya, P. Mukherjee, Biological properties of naked metal nanoparticles, Adv. Drug Deliv. Rev. 60 (2008) 1289–1306. [27] R. Kumar, H. Munstedt, Silver ion release from antimicrobial polyamide/silver composites, Biomaterials 26 (2005) 2081–2088. [28] E. Banin, K.M. Brady, E.P. Greenberg, Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm, Appl. Environ. Microbiol. 72 (2006) 2064–2069. [29] R.J.W. Lambert, G.W. Hanlon, S.P. Denyer, The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa, J. Appl. Microbiol. 96 (2004) 244–253.
