Journal of Membrane Science 302 (2007) 20–26

Nanofibrous membrane of wool keratose/silk fibroin blend

for heavy metal ion adsorption
Chang Seok Ki a , Eun Hee Gang a , In Chul Um b , Young Hwan Park a,∗
aDepartment of Biosystems and Biomaterials Science and Engineering, Seoul National University,
Seoul 151-921, Republic of Korea
b Department of Natural Fiber Sciences, Kyungpook National University, Deagu 702-701, Republic of Korea

Received 3 April 2007; received in revised form 1 June 2007; accepted 8 June 2007
Available online 14 June 2007

Abstract
In this study, SF and WK/SF blend nanofibrous membranes were prepared by electrospinning and their performances were evaluated as a heavy
metal ion adsorbent. The electrospun nanofibrous membranes were comprised of randomly oriented ultra-fine fibers of 200–400 nm diameters. As
a result of pressure–flux test, the membranes exhibited similar flux to ordinary microfiltration membrane. Metal ion adsorption test was performed
with Cu2+ as a model heavy metal ion in a stock solution. Due to large surface area and high porosity of electrospun nanofibrous structure, the
nanofibrous membranes exhibited high metal ion capacities compared with common fibrous filters (wool sliver and filter paper). Especially, the
WK/SF blend nanofibrous membrane had an exceptional performance for the adsorption of metal ions. Furthermore, its adsorption capacity has
been maintained after several recycling process (desorption and re-adsorption) which is very advantageous as an affinity membrane. The electrospun
WK/SF nanofibrous membrane is very suitable for removing and recovering heavy metal ions in water.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Electrospinning; Silk fibroin; Wool keratose; Membrane; Heavy metal ion

1. Introduction [12] and an application to an affinity membrane was also reported

Since mid-1990s, many research groups have investigated
electrospinning techniques and electrospun materials. With a
wide variety of materials, organic and/or inorganic materials,
electrospinning techniques have been dramatically developed
[1–6]. Nowadays, the main focus of electrospinning study moves
to the applications of the electrospun materials in various fields,
such as biomedical, electronic, textile and filtration devices
[7–15]. Filter application of the electrospun material is con-
sidered as a special interest for industrialization. Recently,
water permeable nanofibrous membrane is discussed widely.
A permeability of the electrospun nanofibrous membrane was
investigated with varying parameters of the membrane struc-
ture such as fiber diameter, porosity and thickness [11]. It was
reported by Gopal et al. that an electrospun PVDF nanofibrous
membrane could be used for separation of microscale particles
∗ Corresponding author. Tel.: +82 2 880 4622; fax: +82 2 873 2285.
E-mail address: nfchempf@snu.ac.kr (Y.H. Park).
for electrospun cellulose nanofiber having large surface area to
volume ratio [13].
Removal of heavy metal ions existed in water is always
an issue because they cause a serious biological danger. An
exclusion of the heavy metal ion is commonly carried out by
an adsorption based on an ionic interaction between positively
charged metal ions and negatively charged matrices. Based
on this fundamental principle, various techniques and materi-
als have been developed for ion exchange, precipitation and
cementation. An ion exchanger of membrane form, especially, is
effective for continuous water treatment because it can remove
a large amount of metal ions per unit time. Therefore, a large
surface area and many ionizable sites of the matrix are essen-
tial for removing heavy metal ions. From this point of view, an
electrospun protein membrane comprised of ultra-fine fibers is
an excellent candidate due to a large amount of polar groups of
protein.
Wool protein, known as keratin, is composed of various
amino acids, especially containing many hydrophilic amino
acids which have high affinity for ionic species such as metal

0376-7388/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.memsci.2007.06.003
 C.S. Ki et al. / Journal of Membrane Science 302 (2007) 20–26
Fig. 1. Oxidation of wool keratin by performic acid.
ions. However, wool keratin is not soluble in general solvents
and is hardly regenerated due to disulfide cross-links. In order to
dissolve wool keratin, the cystine cross-links should be cloven
by oxidation or reduction and then several fractions are iso-
lated depending on cystine content. The oxidized wool protein
is commonly called by wool keratose (WK). Especially, the cys-
teic acid residues formed by oxidation can yield a capability to
bind positively charged metal ions (Fig. 1) [16–18]. Silk fibroin
(SF) is a major protein comprising silk fiber. SF is a highly
crystalline material with a ␤-sheet conformation and exhibits
a great biocompatibility. Moreover, SF can be easily fabricated
into nanofibrous form by electrospinning. Many research groups
have attempted to apply the SF nanofiber as tissue engineer-
ing scaffold [19–21]. Most of all, both the WK and SF have an
advantage as eco-friendly materials which can be easily obtained
from natural resources and recycled in environment by decom-
position.
In this study, we investigated the characteristics and per-
formance of an electrospun membrane prepared with WK and
SF protein for metal ion adsorption. The nanofibrous SF and
WK/SF blend membrane were fabricated by typical electro-
spinning technique. Especially, the WK was blended with SF for
improving the electrospinnability of pure WK dope and mechan-
ical property. The metal ion adsorption test was performed using
Cu2+ as a model heavy metal ion and a recycling test (des-
orption and re-adsorption) was also carried out for a practical
use of membrane. Special interest is concerned with the nanofi-
brous WK/SF blend membrane for removing heavy metal ions in
water.
2. Experimental
2.1. Materials
The scoured Merino wool fiber was oxidized by immersing
in performic acid of 1:30 liquor ratio at 0 ◦ C for 24 h. The per-
formic acid was prepared by mixing 98% formic acid (Kanto,
Japan) and 30% hydrogen peroxide (DC Chemical, Korea) at
room temperature for 1–2 h. Then, the soluble fraction of oxi-
dized wool fiber was filtered and dried at ambient temperature
for obtaining the WK. On the other hand, SF was prepared as
following procedure. Bombyx mori cocoons were boiled with an
aqueous solution of marseillus soap 0.3 wt.% and sodium car-
bonate 0.2 wt.% at 100 ◦ C for an hour to remove sericin. And
they were rinsed thoroughly in warm distilled water and dried.
Then the degummed SF fibers were dissolved in a ternary sol-
vent system of CaCl2 /H2 O/EtOH (mole ratio = 1/8/2) solution
at 85 ◦ C for 15 min and dialyzed in order to remove salts using
cellulose tube (MWCO: 12,000–14,000) against distilled water
at room temperature for 3 days. Finally the aqueous SF solution
was freeze-dried.
21
2.2. Membrane fabrication
The electrospun nanofibrous membrane was fabricated for
metal ion adsorbent. For the electrospinning, the pure SF and
WK/SF (50/50 mixture ratio) blend dopes were prepared by dis-
solving in 98% formic acid at room temperature for 4 h and the
concentration of the SF and WK/SF dopes were 12% and 15%,
respectively. The solutions were filtered to remove impurities.
Then, each dope solution was placed in a 10 ml syringe with
stainless steel syringe needle (22 G) as an electrode which con-
nected to power supply (Chungpa EMT High Voltage Supply,
Korea). The syringe was loaded in a syringe pump (KD Scien-
tific, USA) to control a flow rate accurately with spinning rate.
Rolling stainless drum was used as a collector for obtaining sheet
type nanofiber assemblies and grounded. The flow rate of dope
solution was controlled to maintain a constant size of droplet
at the tip of the syringe needle. The electrospinning condition
was performed at room temperature and 60% (RH) humidity.
Electric potential and distance to collector were fixed at 12 kV
and 10 cm, respectively.
After the electrospinning process, the electrospun SF and
WK/SF blend nanofiber mat were immersed in methanol for
an hour and dried completely under a tension for preventing
shrinkage at ambient temperature for 2 weeks. Subsequently,
the methanol-treated nanofiber mats were kept in a chamber
saturated with formaldehyde vapor for 24 h for cross-linking
formation and dried in a vacuum oven for a day.
2.3. Membrane characterization
Morphological structure of the membrane was observed
using scanning electron microscope (SEM) (JSM-5410LV,
JEOL, Japan) after gold coating and the mean diameter of the
nanofiber was measured by an image processing software. A spe-
cific surface area of the membrane was obtained by the following
equation:
A
S= (1)
V
where A is total surface area of the nanofiber and V is the vol-
ume. Assuming that the nanofiber is a cylinder, A and V can be
calculated from measured mass and density of the membrane by
Eqs. (2) and (3), respectively:
4m
A = πDL = (2)
ρD
 2
m D
V = =π L (3)
ρ 2
where m is the mass of the membrane and π is the density of
the nanofiber assuming the nanofiber is rigid without pore or
void. D is a mean diameter and L is total length of the nanofiber.
The fiber density, ρ, was obtained by measuring the mass of the
membrane in the air and in liquid (n-hexane) using the following
equation:
m1
ρ= ρh , (4)
m1 − m 2
22 C.S. Ki et al. / Journal of Membrane Science 302 (2007) 20–26

where m1 and m2 are the masses of the membrane in the air and Desorption and re-adsorption were performed in order to ver-
in n-hexane, respectively. And ρh is a density of n-hexane at ify the recycling efficiency of the membrane for the practical use.
measured temperature. For desorption, 1 mM ethylenediaminetetraacetic acid (EDTA)
The porosity (ε) of the membrane can be calculated by mea- solution of 30 ml was flowed through the membrane. Distilled
suring the volume of the membrane and the nanofiber volume water, subsequently, was flowed to wash the membrane for an
by the following equation: hour. To determine the amount of desorbed Cu2+ , the EDTA
solution was sampled and the concentration of Cu2+ was mea-
Vm − V V V
ε= =1− =1− (5) sured. Re-adsorption process was carried out as the same method
Vm Vm Am t of the first adsorption. The concentration of Cu2+ in the stock
where Vm is total volume of the membrane which can be obtained solution was measured and the Cu2+ adsorbing capacity (q) was
by measuring the area (Am ) and thickness (t) of membrane. The determined by the following equation:
thickness was measured by using a dead-weight dial micrometer. q0 − q1
In order to measure the permeability of the membrane, cir- q (μg/mg) = (6)
m
cular shape of 76 mm diameter was stamped out from the
electrospun nanofiber mat and placed in Amicon® cell filled with where q0 and q1 are the amount of Cu2+ ions in the stock solution
350 ml distilled water. The pressure was gradually increased by before and after the adsorption, respectively, and m is the mass
using nitrogen gas and corresponding water flux was measured. of the membrane. The recycling efficiency was expressed by the
following equation:
2.4. Amino acid composition q0 − q2
recycling efficiency (%) = × 100 (7)
q0 − q 1
Each protein of 40 mg was added into a vessel filled with
where q2 is the amount of re-adsorbed Cu2+ ions. The concen-
6N hydrochloric acid and nitrogen gas was purged. Then, the
tration of Cu2+ in the solution was measured by Inductively
protein was hydrolyzed at 150 ◦ C for an hour. By Pico-Tag
Coupled Plasma Emission Spectrometer (ICPS-1000IV, Shi-
method, PTC-amino acid derivatives were prepared using the
madzu, Japan) at 327 nm.
hydrolyzed solution of 50 ␮l. Quantitative amino acid analysis
was performed using high-performance liquid chromatography
3. Results and discussion
and each amino acid contents were calculated from detected
peak area at 254 nm.
3.1. Membrane properties
2.5. Cu2+ adsorption and desorption test SF and WK/SF blend were used for the electrospun mem-
brane because electrospinnability of only WK was very poor
For Cu2+ adsorption, the nanofibrous membrane, cut into cir- although WK has been expected as an excellent material for
cular shape of 26 mm diameter, was placed in filter-holder and metal ion adsorption due to the large amount of polar side
supported by stainless mesh. The diameter of each membrane residues compared with SF. It has been reported that the WK,
was measured and listed in Table 1. The Cu2+ solution was soluble fraction of oxidized wool keratin by performic acid,
prepared by dissolving copper nitrate (II) (Cu(NO3 )2 ) in dis- has only a few thousands Dalton molecular weight which
tilled water and pH value of the solution was adjusted to 7 by gives a poor electrospinnability [22]. On the other hand, the
adding ammonia water. As pH value increased, gel-like copper SF, regenerated by CaCl2 /H2 O/EtOH ternary solvent, has a
hydroxide formed and precipitated. The precipitate was removed molecular weight of a few ten thousands Dalton [23] and
by filtering and the final concentration was 3.49 ␮g/ml. Using exhibits an excellent electrospinnability. The molecular weight
a peristaltic pump (323E/D, Watson-Marlow Bredel, UK), the is a very important parameter for electrospinning because it
Cu2+ solution of 30 ml was forced to circulate through the filter- closely relates to the dope viscosity. Therefore, when the WK
holder. Then, the stock solution passed through the filter-holder was blended with the SF, the electrospinnability was markedly
and flowed into the stock vessel. The solution was stirred dur- improved and uniform nanofiber mat could be obtained. How-
ing the process. The flow rate was 13 ml/min (50 rpm) and the ever, the metal ion adsorption capacity varies with WK/SF blend
circulation time was 30 min. ratio because of different amino acid composition.
The morphological structures of the electrospun nanofibrous
Table 1 membrane of SF and WK/SF blend are shown in Fig. 2. The
Specific surface area and porosity of SF and WK/SF blend nanofibrous
membrane
membrane is composed of nanofibers which are randomly
deposited in a mat and have ca. 300 nm sizes (fiber diameters).
Membranes Fiber diameter Thickness Specific surface Porosity Fig. 2(b) and (d) shows the fabricated nanofibrous membrane
(␮m) (␮m) area (×107 m−1 ) (%)
of SF and WK/SF blend, respectively which were treated by
SF 0.27 ± 0.04 152 1.481 73 methanol for crystallization as well as treated by formaldehyde
WK/SF blend 0.28 ± 0.06 126 1.429 63 vapor for cross-linking. The methanol treatment causes swelling
Wool sliver 13.76 ± 4.26 300 0.029 83
Filter paper 15.83 ± 4.65 118 0.025 52
by increasing the fiber diameter compared with the structure
of the as-spun SF and WK/SF nanofiber (Fig. 2(a) and (c)).
 C.S. Ki et al. / Journal of Membrane Science 302 (2007) 20–26 23

Fig. 2. SEM micrographs of electrospun membrane: (a) SF; (b) methanol and formaldehyde vapor treated SF; (c) WK/SF blend; (d) methanol and formaldehyde
vapor treated WK/SF blend (magnification: 10,000×).

However, cross-linking does not change the mean diameter of

nanofiber.
The dimensions of SF and WK/SF nanofibrous membrane
are listed in Table 1. The mean diameter of nanofiber was
0.27–0.28 ␮m and the thicknesses of membranes were 152 ␮m
and 126 ␮m, respectively. Also, the specific surface area and
porosity were calculated and listed in Table 1. The surface
area per unit volume, specific surface area, of nanofiber mem-
brane was approximately 1.4 × 107 m−1 and the porosity was
in the range of 63–73%. Since the electrospun nanofibrous
membrane has very large surface area, this characteristic prop-
erty is very effective for the adsorption of metal ions and
becomes an advantage for affinity membrane which separates
specific molecules by adsorption based on physical or chem-
ical affinity. The porosity is also an important parameter for
the membrane. Although the nanofibrous membrane has a com-
pact structure, it is an open pore structure and therefore, highly Fig. 3. Pressure–flux profiles for electrospun SF and WK/SF blend nanofibrous
porous structure (60–80% porosity) helps easy permeation of membrane.
liquid.
The permeability of the nanofibrous membranes was evalu- 3.2. Cu2+ adsorbability
ated by examining the pressure–flux profiles, as shown in Fig. 3.
The membranes exhibited a very high water flux. When 0.5 Cu2+ was chosen as a model heavy metal ion because it is
bar pressure was engaged, the flux was about 0.8 t/h m2 . The tinted with light blue color in aqueous solution which is easily
flux steadily increased with pressure and the flux reached up observed by the naked eye or spectrometer. The concentration
to 3 t/h m2 at 4 bar. The water fluxes of the SF and WK/SF of Cu2+ in the stock solution was 3.49 ␮g/mg at pH 7 and the
membranes were almost same because the structural parameters adsorption time was determined by measuring the amount of
(e.g. mean diameter, porosity, thickness) were similar. Such a Cu2+ with time progress. The relative Cu2+ concentration was
pressure–flux value is corresponding to a permeability of micro- then determined by measuring the absorbance of the solution at
filtration (MF) membrane and this result is well agreed with 600 nm using spectrophotometer. As a result, it was found that
previous report about electrospun membrane [12]. the absorbance dropped down and reached an equilibrium state
24 C.S. Ki et al. / Journal of Membrane Science 302 (2007) 20–26

Table 2 Table 3
Cu2+ adsorption capacity of membranes at pH 7 Amino acid composition of silk fibroin and wool keratose
Membranes Capacity (␮g/mg) Amino acid Silk fibroin Wool keratose

SF 1.65 Cysteic acid – 10.49

WK/SF blend 2.88* Aspartic acid 1.96 8.00
Wool sliver 0.71 Glutamic acid 1.67 11.77
Filter paper 0.23 Serine 11.72 10.43
Glycine 37.37 11.42
* Indicates significant difference from Cu2+ adsorption capacity of SF (p < 0.1,
Arginine 0.41 7.40
n = 3). Threonine – 6.54
Alanine 33.72 5.36
after 10 min. Hence, Cu2+ adsorption running time was fixed at Proline 0.16 6.26
Tyrosine 7.10 1.23
30 min. Valine 2.68 6.18
Cu2+ adsorption capacity of SF and WK/SF blend nanofi- Isoleusine 0.91 2.79
brous membrane were calculated and compared with other Leusine 0.89 7.18
filtering materials in Table 2. Wool sliver material was prepared Phenylalanine 1.12 2.24
by pressing in filter-holder compactly while commercial filter Lysine 0.17 2.72
Histidine 0.06 –
paper (No. 4, Advantec, Japan) was shaped into a circle of 26 mm Methionine 0.04 –
diameter and used as another control sample for the comparison.
Their dimension, specific surface area and porosity were listed Each value was calculated as mol% of total amino acids.
in Table 1.
The nanofibrous membrane showed much higher Cu2+ adsorption on WK in the stock solution. Therefore, the WK/SF
adsorption capacity than wool sliver and filter paper due to blend nanofibrous membrane exhibited higher adsorption capac-
extremely larger specific surface area (ca. 50 times) related to ity (2.88 ␮g/mg) than SF membrane (1.65 ␮g/mg) with similar
fiber diameter. When wool sliver membrane is compared with specific surface area and porosity. This is mainly attributed to
filter paper (mostly composed of cellulose material), it can be higher ion adsorbability of WK.
easily found that wool protein has an excellent performance as
a metal ion adsorbent. This is mainly due to the difference in 3.3. Desorption and re-adsorption behavior
chemical structure and composition, especially ionizable groups
which are the binding sites of positive metal ions. When an ion exchanger is used, desorption and re-adsorption
The difference in Cu2+ adsorption capacity between SF and of metal ions are very important for practical use because recy-
WK/SF blend can be also explained by different chemical com- cling of membrane is economically meaningful. Furthermore,
position. When SF and WK protein are compared, a different desorption of once adsorbed metal ions to an adsorbent material
adsorption capacity can rise due to different amino acid compo- gives a great advantage in industry because expensive metals can
sition. There are more non-polar amino acids in SF than WK; be recovered in waste water. Therefore, perfect desorption and
alanine, glycine and serine occupy more than 80% of total amino maintaining the adsorbability in several recycling process are
acid content of SF. These amino acids hardly play a role of bind- essential. For the desorption of metal ions bound to a substrate,
ing site. On the other hand, the WK contains more hydrophilic negatively charged agents are needed. When the ionic power
amino acids which have polar side residues; aspartic acid, glu- of the desorbing agent is greater than that of the substrate, the
tamic acid, arginine and cystine compose more than 35% of total metal ions can be detached. EDTA is very efficient as a metal ion
amino acid content. Table 3 shows the amino acid composition desorbing agent, which can easily and strongly bind positively
of SF and WK. Especially, the cystine content which can be charged metal ion. Therefore, the desorption process was per-
oxidized to cysteic acid (–SO3 H) was about 10%. And the side formed using 1 mM EDTA solution. The amounts of desorbed
and/or terminal groups, which can form binding formation with and re-adsorbed Cu2+ were determined with their efficiencies in
metal ion, are supposed to be mainly carboxylic acid and cys- Table 4. After the desorption proceeded for 30 min, the amount
teic acid side residues (Fig. 4). This results in much more Cu2+ of the desorbed Cu2+ from SF and WK/SF nanofibrous mem-
branes were 1.68 ␮g/mg and 3.13 ␮g/mg, respectively. These
values exceeded the amount of originally adsorbed Cu2+ . It
might be due to measuring error or a tiny amount of Cu2+
derived from other sources. Nevertheless, it was confirmed that
the adsorbed Cu2+ could be completely desorbed by dilute EDTA
solution.
The re-adsorption step was performed with the same stock
solution. After the first recycling process, the SF nanofibrous
membrane re-adsorbed 1.01 ␮g/mg copper (II) ions and the recy-
cling efficiency was 61%, as shown in Table 4. On the other
Fig. 4. Binding formation between Cu2+ ions and carboxylic acid and cysteic hand, the WK/SF nanofibrous membrane exhibited higher recy-
acid residue in WK/SF molecular chains. cling efficiency of 97%. The relatively low recycling efficiency
 C.S. Ki et al. / Journal of Membrane Science 302 (2007) 20–26
Table 4
Efficiency of Cu2+ desorption and re-adsorption in the first recycling process
Membranes Amount of desorbed Desorption
Cu2+ (␮g/mg) efficiency (%)
SF 1.68 (1.65)a 102
WK/SF blend 3.13 (2.88)a 109
a The value is the Cu2+ adsorption capacity.
of SF might be due to the loss of some electrically active
sites during the desorption process with EDTA although the
morphological structure of the membrane was little deformed
(Fig. 5). The fibrous structure of WK/SF nanofibrous mem-
brane was somewhat distorted and swelled. This might be an
effect of hydrophilic nature of WK. It, however, maintained its
own adsorption capacity after the desorption. It is because the
WK has much more metal ion binding sites than SF. Therefore,
the adsorption capacity of WK/SF nanofibrous membrane did
not decrease although some binding sites lost their activity. In
order to evaluate the adsorption capacity change and durability
of the WK/SF blend nanofibrous membrane in continuous recy-
cling, the desorption and re-adsorption process was repeated six
times. Fig. 6 shows the change of the Cu2+ adsorption capac-
ity of WK/SF blend nanofibrous membrane during repeating
recycling. As a result, the adsorption capacity was steadily main-
tained above 90% during the whole process and the membrane
structure was not deformed at all (figure not shown). Therefore,
the WK/SF blend nanofibrous membrane is very suitable mate-
Fig. 5. SEM micrographs of electrospun (a) SF and (b) WK/SF blend nanofi-
brous membrane after the first re-adsorption of Cu2+ (magnification: 10,000×).
25
Amount of First recycling
re-adsorbed Cu2+ efficiency (%)
(␮g/mg)
1.01 61
2.79 97
Fig. 6. Recycling efficiency of electrospun WK/SF blend nanofibrous mem-
brane.
rial for removal or recovery of metal ions in water and has an
economically great benefit for practical use. But the difference
in re-adsorption efficiency between SF and WK/SF membrane
is not clearly understood and more detail studies should be made
in the future.
4. Conclusion
Electrospun nanofibrous membrane, which has large surface
area per unit volume or mass, can be very suitable for removing
specific materials by adsorption based on chemical or physical
affinity. It was confirmed that the SF and WK/SF nanofibrous
membranes exhibited an excellent performance as a heavy metal
ions adsorbent. SF and WK protein have numerous polar groups
which can bind positively charged metal ions. Especially, the
WK is preferred for removing metal ions because it has much
more amino acids having polar side residues than SF. However,
the use of only WK for preparation of nanofibrous membrane
can be hardly performed due to its low electrospinnability as well
as structural instability in water. By blending the WK with SF,
the electrospinnability and structural stability of WK/SF blend
membrane was markedly improved and the WK/SF nanofibrous
membrane exhibited higher Cu2+ adsorption capacity than SF
nanofibrous membrane. Furthermore, the adsorption capacity
of WK/SF nanofibrous membrane was maintained after several
recycling process of adsorption and desorption. Consequently,
this membrane can be a promising material for removing and
recovering heavy metals ion in water.
26 C.S. Ki et al. / Journal of Membrane Science 302 (2007) 20–26
Acknowledgement
The authors of this paper thank the Korea Science and
Engineering Foundation (KOSEF) for sponsoring this research
through the SRC/ERC Program of MOST/KOSEF (R11-2005-
065).
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