Article

pubs.acs.org/Biomac

Green Synthesis of Silk Fibroin-Silver Nanoparticle Composites with

Effective Antibacterial and Biofilm-Disrupting Properties
Xiang Fei,† Minghui Jia,‡ Xin Du,§ Yuhong Yang,∥ Ren Zhang,∥ Zhengzhong Shao,† Xia Zhao,*,‡
and Xin Chen*,†
†
State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced
Materials, Fudan University, Shanghai, 200433, People’s Republic of China
‡
Department of Otorhinolaryngology−Head and Neck Surgery and §Department of Laboratory Medicine, Huashan Hospital, Fudan
University, Shanghai, 200040, People’s Republic of China
∥
Research Centre for Analysis and Measurement, Fudan University, Shanghai 200433, People’s Republic of China
*
S Supporting Information

ABSTRACT: Natural polymer Bombyx mori silk fibroin is used as a biotemplate to produce silver nanoparticles in situ under
light (both incandescent light and sunlight) at room temperature. Silk fibroin provides multiple functions in the whole reaction
system, serving as the reducing agent of silver, and the dispersing and stabilizing agent of the resulted silver nanoparticles. As the
reaction needs not any other chemicals and only uses light as power source, the synthetic route of silver nanoparticles reported
here is rather environment-friendly and energy-saving. The silk fibroin-silver nanoparticle composite prepared by this method can
be stably stored in a usual environment (room temperature, exposure to light, and so forth) for at least one month. Such a silk
fibroin-silver nanoparticle composite shows an effective antibacterial activity against the methicillin-resistant Staphylcoccus aureus
(S. aureus) and subsequently inhibits the biofilm formation caused by the same bacterium. Moreover, a maturely formed biofilm
created by methicillin-resistant S. aureus can be destroyed by the silk fibroin-silver nanoparticle composite, which meets the
demand of clinical application. Therefore, the silk fibroin-silver nanoparticle composite prepared by this clean and facile method
is expected to be an effective and economical antimicrobial material in biomedical fields.

■ INTRODUCTION
There has been increasing interest in the development of clean
synthetic procedures for nanoproducts targeted for biomedical
applications.1 Utilization of nontoxic chemicals, environ-
mentally benign solvents, and renewable materials are some
of the key issues that merit important consideration in a green
synthetic strategy. In nature, biological systems have developed
biomineralization approaches in the combination of the biology
and nanotechnology, offering unparalleled opportunities for
excellent physicochemical properties in natural biomaterials2
and providing inspiration for material scientists to design
advanced nanomaterial.3−5 As an important component in
biomineralization, various biological systems, such as viruses,6
proteins,7 and peptides7−11 broaden the possible application
fields of the biomineralization in electronics and nano-
biotechnology. They often act as a biotemplate during the
preparation of novel biomaterials and nanodevices in the form
of inorganic−organic hybrid composites.7,10,12−14
Silk fibroin derived from Bombyx mori silkworm silk is a
commonly used biomacromolecule with unique sequence-
specific self-assembly behavior and substrate recognition
properties.15 Silk fibroin has been used as a biomedical material
for long a time because of its good biocompatibility,
controllable biodegradability, and easy fabrication into different
forms, such as fibers, films, gels, and three-dimensional
scaffolds.16,17 As other proteins, silk fibroin is also a good
candidate for a soluble template for biomineralization. In our
previous works, we have proved that silk fibroin plays an
Received: September 22, 2013
Revised: October 20, 2013
Published: October 30, 2013

© 2013 American Chemical Society 4483 dx.doi.org/10.1021/bm4014149 | Biomacromolecules 2013, 14, 4483−4488
Biomacromolecules
important role in the biomineralization process for inducing
and regulating the morphologies and lattice structures of
different inorganic nanostructures.18−22
Nowadays, the development of antibiotic-resistant bacteria
has created a myriad of new challenges within the healthcare
field. In terms of public health, the methicillin-resistant
Staphylcoccus aureus (MRSA) accounts for a considerable part
of the reported cases of S. aureus infections all over the world.
People estimate 20 000 deaths occurred in the United States
annually because of MRSA infections.23 On the other hand,
planktonic bacteria in the nosocomial environment or during
the manufacturing processes of medical supplies can form
colonies on their surfaces to create biofilms, leading to the
wide-spreading of infectious diseases by contacting those
contaminated surfaces. Bacterial biofilms consist of bacteria
and self-secreted extracellular polymeric substances (EPS),24
which are extremely resistant to conventional antibiotics
because of acquired resistance, limited diffusion, and
inactivation of antibiotics in EPS.23,25
Silver nanoparticles (AgNPs) are well-known for their
antibacterial property to a broad spectrum of bacteria and
have been used increasingly in detergents, plastics, food storage
containers, antiseptic sprays, catheters, bandages, and textiles,
and so forth.26 Nevertheless, practical applications of AgNPs
are often hampered by their easy oxidization weakness, which
may cause the loss of the antibacterial activity.27 To overcome
this problem, different organic27−29 and inorganic tem-
plates30−36 have been employed to stabilize AgNPs through
the formation of nanocomposites. However, those methods
always need complex and tedious procedures, and face the
problems of high cost and poor biocompatibility.
In this article, we introduce an easy and environmentally
friendly route to in situ preparation of AgNPs using silk fibroin
as a biotemplate at room temperature under light exposure.
Then, we report the antibacterial and biofilm-disrupting
properties of the resulted silk fibroin-AgNPs composite.
■
EXPERIMENTAL SECTION
Preparation of Regenerated Silk Fibroin (RSF) Solution. The
raw B. mori cocoon silk consists of fibroin fibers that are bound
together by sericin, a hydrophilic gumlike coating protein. The
degumming (removing the sericin) and dissolving process of B. mori
silk followed the established procedures.37 In brief, the silk fibers were
degummed twice with 0.5 wt % NaHCO3 aqueous solution at 100 °C
for 30 min, washed with distilled water, and allowed to air-dry at room
temperature. The degummed B. mori silk fibers were dissolved in 9.3
mol/L LiBr aqueous solution at 60 °C. The silk fibroin-LiBr solution
was dialyzed against deionized water for 72 h at room temperature
with a semipermeable membrane (MEMBRA-CEL, 12 000−14 000
MWCO) to remove the salt. The dialyzed silk fibroin solution was
centrifuged at 6000 r/min for about 5 min and the supernatant was
collected and then stored at 4 °C for further use. The final
concentration of RSF solution was about 4 wt % and was diluted to
1 wt % by adding deionized water.
Preparation of RSF-AgNPs Composite Solution. Five to eighty
milligrams of AgNO3 powders were added into 5 mL of 1 wt % RSF
solution to form a transparent RSF-AgNO3 mixture solution. The final
AgNO3 concentration in the mixture was 1−16 mg/mL. Then, the
RSF-AgNO 3 solution was exposed under the light with an
incandescent bulb (40 W, from Philips) and incubated at room
temperature for 24 h to produce RSF-AgNPs composite solution.
Afterward, RSF-AgNPs composite solution was stored at 4 °C for
further use.
Preparation of Bacterial Suspension. The bacterial strain used
in this study was Sa006, a clinical isolate of MRSA obtained from the
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clinical laboratory of Huashan Hospital affiliated to Fudan University,
which has the ability to form biofilm. A single isolated colony of the
target bacteria was inoculated in 4 mL of Muller−Hinton broth
(MHB) and was cultured to a midlog phase at 37 °C with shaking
(220 r/min). Then, the bacteria liquid in midlog phase was
resuspended in the physiological saline to an optical density (OD)
of 0.5 at 600 nm, which corresponds to the concentration of 1.5 × 108
colony forming units per milliliters (CFU/mL).
Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC) Determination. The anti-
microbial activity of RSF-AgNPs composite was evaluated by
determination of MIC and MBC against Sa006 in vitro. MIC and
MBC testing were performed according to the Clinical and Laboratory
Standards Institute (CLSI, 2011) guidelines for macro-dilution in
broth.38 The bacterial suspension was resuspended in MHB to 2 × 105
CFU/mL and dispensed into nine tubes (1 mL per tube), then equal
volume of the 2-fold serially diluted concentrations of RSF-AgNPs
composite solution were added into the tubes. The final concentration
of AgNPs in these tubes was from 0 to 153.6 mg/L, using pure MHB
medium as a control. MIC was defined as the lowest concentration
that yielded no visible growth of bacteria after 24 h incubation at 37
°C. MBC was determined by plating 100 μL suspension collected from
the MIC test tubes without visible bacteria growth onto the Muller−
Hinton (MH) agar plates and then incubated at 37 °C for another 24
h. The lowest concentration for monoclonal number in the plate was
five or less and defined as the MBC.38 All the tests were performed at
least in triplicate.
Antibacterial Activity of RSF-AgNPs Composite against
Biofilm. In order to test the antibacterial activity of RSF-AgNPs
composite against the maturely formed biofilm, the bacteria
suspension was diluted in tryptic soy broth supplemented with 0.5%
glucose (TSBg) to approximately 1 × 105 CFU/mL. Then, 1 mL of
such bacteria suspension was transferred to a cell culture dish (NEST
Biotechnology Co., Ltd.) to incubate for 24 h at 37 °C without
shaking. After the biofilms were formed in the dishes, the previous
TSBg media were changed by the fresh TSBg containing different
amount of RSF-AgNPs composite and continued to incubate for
another 10 h. The concentration of AgNPs was set as MIC, MBC, 2 ×
MBC, and 5 × MBC, respectively. After 10 h incubation, the dishes
were sequentially rinsed with physiological saline three times to
remove the planktonic bacteria. The viability and structure of the
biofilms were detected by means of confocal laser scanning microscopy
(CLSM) and scanning electron microscopy (SEM).
Characterizations. Transmission electron microscopy (TEM) was
performed with a Hitachi H-600 transmission electron microscope
(Japan) at 75 kV. High-resolution transmission electron microscopy
(HRTEM), selective area electron diffraction (SAED) and energy
dispersive X-ray spectroscopy (EDX) were performed on a JEM-
2010F transmission electron microscope (JEOL, Japan) at an
accelerating voltage of 200 kV. UV−vis spectra were measured by a
Hitachi U-2910 spectrophotometer. For SEM detection, the biofilm
samples were placed in 2.5 wt % glutaraldehyde solution for 24 h, and
then dehydrated in a series of ethanol aqueous solutions with the
increased concentration until the specimens were placed in absolute
ethanol. Afterward, the specimens were critical-point dried in carbon
dioxide, mounted on the scanning electron microscope stubs, sputter
coated with gold palladium, and then examined with a Hitachi SU8010
scanning electron microscope (Japan) at an accelerating voltage of 20
kV. For CLSM detection, the biofilm samples were immersed in 1 mL
of physiological saline in which contained 1.5 μL aliquots of
component A (Syto 9) and component B (propidium iodide) from
the BacLight LIVE/DEAD kit (Invitrogen, Molecular Probes) to stain
in darkness at room temperature for 15 min, Then, they were analyzed
with a Leica TCS SP5 confocal scanning laser microscope (Leica
Microsystems, Germany) at certain wavelengths (for Syto 9 at 515−
530 nm, and for propidium iodide, >600 nm).
dx.doi.org/10.1021/bm4014149 | Biomacromolecules 2013, 14, 4483−4488
Biomacromolecules
Figure 1. (a) Possible mechanism of the reduction of Ag+ ion by Tyr
residues in silk fibroin chain. (b) Fluorescence spectra of RSF/AgNO3
reaction systems with different AgNO3 concentrations (from 0 to 16
mg/mL) after 24 h at room temperature ([RSF] = 1 wt %).
■ RESULTS AND DISCUSSION
In Situ Prepared RSF-AgNPs Composite Solution. B.
mori silk fibroin contains 18 amino acid residues, among which
the tyrosine (Tyr) has strong electron donating property. On
the basis of the previous studies on the synthesis of noble metal
(Ag and Au) nanoparticles with either single Tyr molecule or
polypeptides and proteins containing Try residues in both our
laboratory39 and other research groups,40−43 we believe that silk
fibroin is also capable of reducing Ag+ to Ag by the redox-active
Figure 2. UV−vis spectra (a) and TEM (b−d) images of the synthesized RSF-AgNPs composites ([RSF] = 1 wt %, [AgNO3] = 4 mg/mL).
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nature of Try residues (Figure 1a). In the current study, we set
the RSF concentration to 1 wt % in the reaction system, so we
need to determine the suitable concentration of AgNO3 to be
reduced. Fluorescence analysis was chosen as an effective
method to do this because the loss of fluorescence from Tyr
residues reflected the oxidation degree of the phenolic groups
in them. The more Ag+ ions were reduced, the more Tyr
residues were oxidized, and the less fluorescence intensity was
observed. Figure 1b indicates that the fluorescence intensity
does not change when the AgNO3 concentration is larger than
8 mg/mL. This means almost all of the Tyr residues in silk
fibroin were used (oxidized) at [AgNO3] = 8 mg/mL, so we
decided to set [AgNO3] = 4 mg/mL in our reaction system to
make sure that all the Ag+ ions can be reduced to Ag.
The initial RSF-AgNO3 solution was colorless, but after the
exposure to the light, the color gradually turned to yellow (see
inset in Figure 3b). Such a change was attributed to the size-
and shape-dependent surface plasmon resonance (SPR) of
AgNPs44 formed in the solution in the visible region and was
characterized by UV−vis spectroscopy (Figure 2a). The main
SPR band was at 440 nm, but a small shoulder can be found at
347 nm, suggesting the possible existence of different size and
morphology of AgNPs.45 TEM images (Figure 2b,c) show the
particle size distribution of the AgNPs. The average size of
AgNPs was 12.0 ± 2.1 nm, which was obtained by measuring
100 nanoparticles. HRTEM image of an individual nanoparticle
indicates the d-spacing of the crystallographic plane is 0.23 nm,
which agrees well with the distance of (111) lattice plane of Ag
(Figure 2d).46 The electron diffraction patterns (inset in Figure
2d) prove the single crystal nature of the synthesized AgNPs.
The local elemental composition of the product was confirmed
as Ag element by EDX microanalysis at a single nanocrystal
level (Supporting Information Figure S1). The explanation
about how polypeptides like silk fibroin reduce Ag+ to Ag and
subsequently form AgNPs can be found in the literature.47,48
Figure 3a is the time-resolved UV−vis spectra recorded from
beginning of the reaction to 32 h. The main absorption at 440
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Biomacromolecules
Figure 3. (a) Time-resolved UV−vis spectra of RSF-AgNO3 reaction
solution. (b) The change of the absorption at 440 nm at different
reaction time, and the inset is the corresponding digital photos of the
reaction system. (c) Comparison of UV−vis spectra of RSF-AgNPs
composite solutions: black line, fresh synthesized; red line, stored in
the dark for 30 day; green line, stored in the light for 30 days (the inset
is the corresponding digital photos).
Figure 4. Antibacterial activities of RSF-AgNPs composites. (a) MIC
test shows the MHB liquid medium turbidity assays of bacteria
suspensions after incubation with RSF-AgNPs composite for 24 h at
37 °C. (b) MBC test shows the colonies of the bacteria suspensions
taken from the MIC test tubes and incubated for another 24 h on the
MH agar plate.
nm was plotted as a function of time in Figure 3b and the
corresponding optical photos were shown as an inset. It shows
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a rapid growth period from 0 to 20 h and then levels off.
Therefore, we set the reaction time as 24 h in the present study
to make sure the formation of AgNPs is complete. We should
point out that AgNPs can be also formed if the RSF-AgNO3
solution was exposed under sunlight.49 However, it is not
controllable to use sunlight, so we chose an incandescent lamp
for this fundamental research. For real applications, it would be
no problem to use sunlight and would probably need a long
time to allow the reaction to be completed.
Individual AgNPs are easily oxidized and/or aggregated in air
that may affect their practical uses,50,51 so the stability of AgNPs
has been considered as a critical key for the real application. In
our case, RSF-AgNPs composite formed a stable yellow
colloidal solution and almost had no change for at least one
month. Figure 3c shows the UV−vis spectra as well as the
appearance of RSF-AgNPs composite solutions, which scarcely
change after 30 days storage either in the dark or in the light.
During the AgNPs formation process, silk fibroin chains
connect to the surface of nanoparticles.47,48 Silk fibroin is a
protein, a natural nonionic surfactant, so it surely enhances the
stability of AgNPs in the aqueous dispersions.52 Therefore, silk
fibroin also acts as a dispersing and stabilizing agent of the
synthesized AgNPs like soy protein39 and bovine serum
albumin.53
Antibacterial Property of RSF-AgNPs Composite. In
this article, we evaluated the antibacterial activity of our RSF-
AgNPs composite with a clinical isolated MRSA. We cultured
the bacteria in MHB liquid media containing different amount
of RSF-AgNPs composite for 24 h, and the result was shown in
Figure 4a. It can be found that the bacteria suspension became
turbid if there was no RSF-AgNPs composite in the mixture,
suggesting the bacteria proliferated rapidly in MHB medium.
With the increase in concentration of RSF-AgNPs composite in
MHB medium, the bacteria suspension was still turbid until the
AgNPs concentration in the mixture reached 19.2 mg/L. As the
MIC is defined as the lowest concentration of an antimicrobial
material that can inhibit bacteria growth, so the MIC of AgNPs
in our RSF-AgNPs composite solution is 19.2 mg/L.
Subsequently, we plated the bacteria suspensions in which
the AgNPs concentration was larger than MIC onto the MH
agar plates and incubated at 37 °C for another 24 h. The result
showed that when the AgNPs concentration increased to 76.8
mg/L, the monoclonal number of the bacteria growing on the
MH agar plate was less than 5 (Figure 4b). Therefore, the MBC
of AgNPs in our RSF-AgNPs composite was identified as 76.8
mg/L.
As our RSF-AgNPs composite exhibited quite effective
antimicrobial activity to MRSA, as shown above, in the next
step we tested its effect on the inhibition of the biofilm
formation. The result showed that the biofilms were formed if
the concentration of RSF-AgNPs was no more than a quarter of
MIC (Supporting Information Figure S2a−c). When the
concentration increased to half of MIC, although the bacteria
still grew, the biofilm was failed to form (Supporting
Information Figure S2d). Of course, it was understandable
that MRSA bacteria were killed or their growth was inhibited
when the concentration of RSF-AgNPs reached MIC
(Supporting Information Figure S2e).
In comparison with the inhibition effect on the biofilm
formation by the bacteria, the ability to destroy of RSF-AgNPs
composite toward the maturely formed biofilm created by
MRSA is more promising in real clinical application. Here, we
used SEM and CLSM to examine the structures of the biofilms
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Biomacromolecules
Figure 5. SEM (small magnification, a1−d1; large magnification, a2−d2) and CSLM images (a3−d3, with the same magnification of a1−d1) of
maturely formed MASA biofilm after contact with RSF-AgNPs composite with different concentration at 37 °C for 24 h (a1−a3, MIC; b1−b3,
MBC; c1−c3, 2 × MBC; d1−d3, 5 × MBC). In the CSLM image, the live MASA emit green light, while the dead MASA emit red light.
and the corresponding bacterial activities after these biofilms
were contacted with a different concentration of RSF-AgNPs
composite. The MRSA created a thick and dense biofilm under
the experimental condition (Supporting Information Figure
S3). In the meantime, the proportion of the live MRSA in the
biofilm was very high. When the biofilm contacted the RSF-
AgNPs composite with the concentration of MIC (Figure 5a1−
a3), the structure of the biofilm and the proportion of the live
bacteria in the biofilm seemed to have little difference from the
control. With the increase of AgNP’s concentration to MBC,
the structure of the biofilm became loose, showing an island-
like distribution (Figure 5b1−b3). Although there were still
quite a lot of biofilm structures remaining, the number of dead
bacteria significantly increased, indicating the strong bacter-
icidal ability of RSF-AgNPs composite at MBC. When the
concentration of RSF-AgNPs composite further increased to 2
× MBC (Figure5c1-c3), the biofilm started to break, so most of
the bacteria were washed away by the physiological saline
before the SEM and CLSM observation. Meanwhile, it can be
seen that the bacteria that remained in the cracked biofilm were
almost dead. If we continuously increased the concentration of
RSF-AgNPs composite to 5 × MBC, the biofilm was totally
destroyed (Figure 5d1−d3). Therefore, it clearly indicated that
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our RSF-AgNPs composite had an obvious effect to destroy the
structure of a maturely formed biofilm and kill the
corresponding bacteria.
■
CONCLUSIONS
In this article, silk fibroin was used in situ to synthesize AgNPs
by adding AgNO3 powders into RSF solution and exposing
under 40 W incandescent light at room temperature. The
reaction was found to happen under the sunlight as well. In the
whole synthetic process, silk fibroin acted as the reducing,
dispersing, and stabilizing agent, so no other chemical reagent
was needed. Tyr residues in the silk fibroin backbone were
thought to be responsible for the reduction of Ag+ into AgNPs
in situ. The prepared RSF-AgNPs composite exhibited an
effective antibacterial activity against MRSA and showed strong
ability to destroy the maturely formed biofilm created by the
same bacterium. Therefore, it is a rather green route to produce
AgNPs compared to the reported method, and the product has
the great potential as an antibacterial and biofilm-disrupting
agent in real clinic applications.
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■
ASSOCIATED CONTENT
*
S Supporting Information
EDX spectrum of the synthesized RSF-AgNPs composite,
photos of biofilm formation phenomenon at different RSF-
AgNPs composite concentration, and SEM and CSLM images
of the maturely formed MASA biofilm. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: (X.C.) chenx@fudan.edu.cn. Fax: +86 21 5163 0300.
Tel: +86 21 6564 2866.
*E-mail: (X.Z.) zhaoxiaabc2@126.com. Fax: +86 21 6564 7072.
Tel: +86 21 5288 7073.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. X.F. and M.J. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work is supported by the National High Technology
Research and Development Program of China (863 Program)
(No. 2012AA030309) and the National Natural Science
Foundation of China (No. 21034003, 21274028, and
81271094). We thank Dr. Jinrong Yao and Dr. Lei Huang for
their valuable suggestions and discussions.
■
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