Autonomous and Continuous Stimuli-Responsive Polymer Surface for Antibacterial Application through Enzymatic Self-Propagating Reactions

Article abstract of DOI:10.1002/chem.201703067

Stimuli-responsive polymer materials inspired by biological materials have invoked increasing research interest; however, they still suffer from limitations such as finite amplified responses and poor sensitivity of the unstimulated parts. Herein, a new strategy for creating H⁺-responsive polymer surfaces that are capable of transforming specific local fleeting stimuli into global macroscopic changes is described. The introduction of self-propagating reactions into the polymer-surface systems endows them with excellent stimuli-amplifying properties and response of the unstimulated parts. On the basis of this design, a polymer and enzymatic reaction were employed to enable a specific response to a stimulus and then lead to macroscopic changes of the surface. It is further shown that the prepared H⁺-responsive polymer surfaces can be employed for antibacterial application. This work provides a good example of achieving autonomously reconfigurable materials that respond to local fleeting stimuli.

Full text of DOI:10.1002/chem.201703067

A Journal of
Accepted Article
Title: Autonomous and Continuous Stimuli-Responsive Polymer
Surface for Antibacterial Application via Enzymatic Self-
Propagating Reactions
Authors: Xiaogang Qu, Chao Ding, Zhengqing Yan, and Jinsong Ren
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To be cited as: Chem. Eur. J. 10.1002/chem.201703067
Link to VoR: http://dx.doi.org/10.1002/chem.201703067
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Autonomous and Continuous Stimuli-Responsive Polymer
Surface for Antibacterial Application via Enzymatic Self-
Propagating Reactions
Chao Ding,a,b Zhengqing Yan,a,b Jinsong Ren,a and Xiaogang Qu*a
Abstract: Stimuli-responsive polymer materials inspired by
biological materials have invoked increasing research interest;
however still suffer from limitations such as finite amplified
responses and poor sensitivity of the unstimulated parts. Here, we
describe a new strategy for creating H+ responsive polymer surfaces
that are capable of transforming specific local, fleeting stimuli into
global, macroscopic changes. The introduction of self-propagating
reactions into the polymer surface systems endows them with
excellent stimuli amplification properties and calls up response of the
unstimulated parts. Based on this design, a polymer and enzymatic
reaction are empolyed, which enable specific stimuli response and
then lead to macroscopic changes of the surface. We further show
that the prepared H+ responsive polymer surfaces can be empolyed
for antibacterial application. This work provides a good example for
achieving the autonomously reconfigureable materials that respond
to local, fleeting stimuli.
of bio-mimetic applications for such polymer materials, with
the most studied systems being chemical responsive
materials [2a, 2b, 2d, 5]
.
For achieving the desired functionalized surface,
modification procedures have long been of significant
interest. Particularly, numerous methods have been
developed to impart antibacterial properties to inert
surfaces. Two strategies for design of antibacterial surfaces
are often empolyed. One is to coat the surfaces with non-
fouling materials that can effectively prevent the attachment
[6]
of bacteria
. The other is to link bacterial killing
[7]
compounds that can efficiently kill various bacteria
.
However, the functionalized surfaces for antibacterial are
always on their activated state which is a great concern
about the biocompatibility. Accordingly, it is highly desirable
to develop a surface that switches the macroscopic
properties in response to bacteria attacking and turns the
entire surface into the defending forms.
By the inspiration of leaves, we presented a new strategy
for design of stimuli-responsive polymer surface systems
that exhibit the ability to transform local, fleeting stimuli to
global macroscopic properties change of the surface
(Scheme 1). In the design demonstration, we illustrated the
construction of the self-propagating polymer surfaces and
the capability of the responsive surface that switches from
biocompatible to bacterium killing (Scheme 1), thus
providing a new way for amplifying a stimuli-responsive
event into a macroscopic change on the surface. The global
and autonomous properties changes of the polymer
surfaces are attributed to the self-propagating reactions
arose from the polymers that are functionalized with specific
repeating units. These self-propagating reactions endow
the surfaces spontaneous communications with the distant
portions while also change the properties of the surfaces
without requiring additional stimuli. Here we are particularly
interested in the polymer materials that respond to pH
Introduction
The surface of materials determines their interactions with
the surrounding environment,[1] and surface chemistry has
been received great interest, particularly in the design and
study of nature-inspired responsive and adaptive surfaces.[2]
The surfaces of natural leaves react autonomously and
macroscopically to specific stimuli which are controlled by
the biological materials inside the leaves.[3] These biological
materials are able to transform local, usually fleeting stimuli
(like touch, heat, etc) from one part of the leaf to a response
of the whole leaf via self-propagating reactions. The stimuli-
responsive polymer materials that respond to the local
changes via self-propagating reactions (light, chemicals,
etc.), which impart an autonomous, macroscopic response
to the system, have attracted much attention in recent bio-
inspired surface design
switchability like the biological materials enabled a variety
[4]
. The responsiveness and
change of the local surrounding medium, like boronate
[6b, 8]
ester complexes that hydrolysis at acidic condition.
In
the designed polymer surfaces, hydrolysis of prepared
glucose boronate ester polymer (Scheme 1c) with H+ stimuli
releases glucose that can further react with the immobilized
glucose oxidases and continue the self-propagating
reactions. The spreading of the self-propagating reactions
within the surface leads the global macroscopic properties
change. In brief, this work shows: (i) a bio-inspired surface
system which contains self-propagating reactions thus
enabling stimuli spreading, (ii) the surface can be
autonomously in response to local and even fleeting stimuli
to kill bacteria. Our work may offer a new way for achieving
[a]
[b]
C. Ding, Z. Q. Yan, Prof. J. S. Ren, Prof. X. G. Qu*
Laboratory of Chemical Biology and State Key Laboratory of Rare
Earth Resource Utilization, Changchun Institute of Applied
Chemistry,
Chinese Academy of Sciences,
Changchun, Jilin 130022, China
E-mail: xqu@ciac.ac.cn
C. Ding, Z. Q. Yan
University of Chinese Academy of Sciences,
Beijing 100039, China
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201703067
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the autonomously reconfigure materials that respond to
local, fleeting stimuli.
Scheme 1. Schematic illustration of a) the preparation precedures of the polymer surface, b) the nature Venus flytrap that respond to brief touches and c) the leaf-
inspired surface that transform local, fleeting stimuli into global, macroscopic properties changes of the surface. The bacterium invasion releases H+ that initiates
the self-propagating reactions within the surface that amplifies the H2O2 and H+ output, the spreading of H2O2 and H+ leads to the whole properties change of the
surface. The bottom half of c) depicts the specific boronate ester polymer and self-propagating reaction A+B→C+A that provides the global change across the
whole surface.
propagating reactions that change the properties of the
surface as a whole.
Results and Discussion
Scheme 1a depicts the preparation progress of the surface we
designed to simulate nature leaves. With the help of Au
nanoparticles (Figure S1, the average size was 55.8 nm), we
first constructed nanohloes on the glass surface for enzyme
immobilization.[9] The glass surfaces were cleaned with piranha
solution and subsequent immersion in the etching solution. The
nanoholes formed on the surfaces were characterized by AFM
and SEM. As shown in Figure 1a and S2, 10 nm deep
nanoholes formed after etching. We then immobilized GOx onto
the surface by immersing the surface in the GOx solution at 4 oC
for 2 h. Subsequently, the surface was rinsed with water to
remove weakly absorbed GOx. The AFM image in Figure 1b
indicated the nanoholes were filled with GOx. Further results of
XPS and ATR spectra (Figure S3 and S4) exhibited the
successful immobilization of GOx due to the appearance of N 1s
peak at 399.2 eV in Figure S3 and characteristic amide A (3278
cm-1), amide I (1640 cm-1), amide II (1539 cm-1) and amide III
(1248 cm-1) bands of GOx in Figure S4. The activity assay of the
free GOx and immobilized GOx showed that GOx after
immobilization kept most of its activity (Figure S5 and table S1).
The last step was the deposition of the polymer on the surface
Design and construction of the surface that responds to
local and fleeting stimuli via self-propagating reactions
The design for the bio-inspired surface requires a specific
functionalized polymer and an enzymatic reaction, where
the repeating functionalities of the polymer are glucose
boronate esters and the immobilized enzyme is glucose
oxidase (GOx) that leads to a reaction with glucose
(Scheme 1). The acid responsive boronate esters (the
structure in Scheme 1c) on the surface respond to a local
fleeting H+ stimulus by undergoing a hydrolysis reaction that
releases glucose, the released glucose diffuses across the
surface freely and initiates the self-propagating reaction
(the reaction in Scheme 1c) by reacting with GOx to
produce H2O2 and H+. The H+ reacts further with the other
boronate esters of the polymer, which ultimately leads to
release of glucose and then propagates the release of H2O2
and H+ which change global surface properties. Upon
completion of the self-propagating reactions, the former
surface has been transformed to H+ and H2O2 concentrated
surface and could be used to kill bacteria. As the above
illustration, the surface designed can be tuned by the self-
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through the hydrogen bond formation between silanol groups on
Characterization of the surface in response to a local
fleeting stimulus and the change of its macroscopic
properties
the surface with the nitrogen atom on the pyrrolidone group of
[10]
the polymer.
Prior to the deposition, we prepared and
characterized the monomer N-acryloyl-3-aminophenylboronic
acid (Figure S6) and its boronate ester (Figure S7). Free radical
polymerization of the monomers provided the polymers (Figure
S8 and S9). The H+ responsive property of the boronate ester
polymer was characterized by optical transmittance
measurement (Figure 2). As shown in Figure 2, the precipitation
temperature for boronate ester polymer was larger than N-
acryloyl-3-aminophenylboronic acid polymer at basic conditions,
while exhibited no differences at acid conditions. The larger
precipitation temperature at basic conditions could be attributed
to the maintain of boronate ester polymer that contains
hydrophilic glucose, however at acid condition the hydrolysis of
hydrophilic boronate esters to uncharged hydrophobic boronic
acid lead to similar precipitation temperature. Overall, the results
indicated that the prepared boronate ester polymer was stable at
basic condition and hydrolyzed at acid condition. After the
successful preparation and characterization of the acid
responsive polymer, the deposition of the polymer to the
designed surface was conducted. As results, the surface height
raised and the peak at 189 eV corresponded to B 1s presented
due to the polymer deposition, as evident from the AFM and
XPS imaging analysis (Figure 1 and S3).
After successful construction of the responsive surface, we then
estimated the feasibility of the proposed self-propagating
reaction in the surfaces. The fluorescence micrograph was used
to verify the self-propagating response of the surface to the
external H+ stimuli. The pH responsive carboxyfluorescein was
used as a reporter of the pH change on the polymer surface
(Figure S10). As shown in Figure 3, after adding a drop of 0.1 M
HCl solution at the corner of the surface the fluorescence
changed across the surface quickly, as
a
comparison
experiment the surface deposited with only polymer showed a
slower propagation (Figure S11) which indicated the self-
propagating reaction took place along the responsive surface.
Figure 3. The fluorescence micrographes of the prepared surface at different
time after adding a drop of 0.1 M HCl at a corner of the surface were
demonstrated. Scale bar is 25 m.
Global change in antibacterial effect of the surface in
response to a local bacterium stimulus
Since the surface performed successfully the self-propagating
property as designed, we applied the surface to an antibacterial
system. The bacterium E. coli was used as a model for the
investigation of the antibacterial properties of the designed
surface systems. Immersion of each surface in 5.0 mM pH 7.4
PBS solutions to provide a suitable environment for self-
propagating reaction was first carried out. Then a part of the
surface was stained with bacteria E. coli, while the other part
was kept unstained. After 6 h incubation, the bacteria presented
on the unstained part of the surface were characterized by using
Figure 1. AFM images of the surface after a) Au nanoparticles assisted
surface etching, b) enzyme immobilization and c) polymer deposition.
a fluorescence assay as well as standard plate count method [11]
.
The SEM was used to observe bacteria E. coli with different
surface treatment, as shown in Figure 4a and 4b bacteria E. coli
treated with glass and glass modified with polymer only were
both with smooth and intact cell walls, which demonstrated that
these two surfaces showed little toxicity against bacteria E. coli.
However, for the designed surface the bacterial cell walls
Figure 2. Temperature dependent optical transmittance of a) the N-acryloyl-3-
aminophenylboronic acid polymer and b) the prepared H+ responsive boronate
ester polymer at various pH values.
became rough and broken (Figure 4c) since H2O2 can oxidize
[12]
and destroy the bacterial cell membranes.
Propidium (PI)
staining was used to further verify the cell membrane conditions
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and the results demonstrated that neither the glass nor the
polymer surface alone was able to have a significantly affect on
the viability of bacteria E. coli as shown in Figure 4d, 4e, 4g and
4h. However, for the designed surface a significant loss in
viability was exhibited by the substantial increase in the PI
permeable red fluorescent bacteria (Figure 4f and 4i). It is
unlikely that the polymers themselves contributed to the
observed fluorescence, as the results of the glass modified with
polymer only showed low red fluorescence (Figure 4e and 4h).
Further results of the plate count method were consent to the
fluorescence-based assessment, as shown in Figure
5 a
significant decrease in the number of E. coli colonies was
observed for the designed surface (Figure 5c and 5d) compared
to the glass surface (Figure 5a and 5d) and the glass surface
modified with polymer only (Figure 5b and 5d). The occurrence
of dead bacteria on the unstained parts of the surfaces indicated
that the unstained parts exhibited antibacterial properties. The
appearance of antibacterial properties in the unstained parts
could be attributed to the communication via self-propagating
reactions between the stained parts and unstained parts of the
surfaces. Thus, the prepared polymer surface systems, behaved
similarly like nature leaves in their abilities to impart macroscopic
responses to local fleeting stimuli, could be used for antibaterial
application.
Figure 5. The number of E. coli colonies after the treatment with a) glass, b)
glass modified with polymer only, c) glass modified with GOx and polymer
and d) the quantified results of the palte count method (the data were from
three independent experiments).
Conclusions
In summary, a surface with the ability to respond to local and
fleeting stimuli and translate the stimuli globally via self-
propagating reactions was designed and constructed. The self-
propagating reaction initiated by H+ released by bacteria could
lead to the entire properties change of the surface and amplify
the release of H2O2 to endow the surface with the antibacterial
property. The designed surface exhibited significant antibacterial
properties against H+ release E. coli bacteria. This bio-inspired
design may provide new insights into developing the
autonomously reconfigure materials that respond to local,
fleeting stimuli.
Figure 4. SEM and fluorescence microscope images of E. coli treated with
glass (a, d, g), glass modified with polymer only (b, e, h) and glass modified
with GOx and polymer (c, f, i). Scale bars for fluorescence microscope images
are 20 m.
Experimental Section
Materials and methods
All reagents were commercially available and used without further
purification. Acryloyl chloride (96%, contains 200ppm MEHQ as
stabilizer) and N-vinyl-2-pyrrolidone (99%) were purchased from
Aladdin Chemistry. 3-aminophenylboronic acid hydrochloride (98%),
2, 2-azobis (2-methylpropionitrile) (AIBN, 98%), glucose oxidase,
and N-isopropylacrylamide (97%) were obtained from Sigma-
Aldrich. 5(6)-carboxyfluorescein (99%, exhibit pH-dependent
absorption spectra with a pKa~6.5 (pH5~9)) was obtained from
Acros organics. Escherichia coli (E. coli, ATCC 25922) bacterial
strains were obtained from Chuanxiang Biotechnology, Ltd.
(Shanghai, China). Unless specified otherwise, all other reagents
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were of analytical grade. Ultra-pure water (>18.2 MΩ cm-1, Milli-Q,
Millipore) was used for the preparation of all the buffers and for all
experiments.
pyrrolidone (2.5 mmol, 277 mg) and AIBN (0.1 mmol, 16 mg) were
dissolved in 10 mL of 1, 4-dioxane. The reaction was taken place in
a two-necked flask with a cooling condenser and started by heating
to 70 oC under nitrogen protection. After 6 h reaction, a highly
viscous solution was obtained and added dropwise to 80 mL diethyl
ether for precipitation. The precipitate was filtrated and washed with
diethyl ether for three times. The obtained product was dried under
vacuum and characterized by 600 MHz 1H NMR spectrum.
X-ray photoelectron spectra (XPS) were acquired on an
ESCALab220i-XL electron spectrometer from VG scientific with 300
W Al Kα radiations as the X-ray source for excitation. Scanning
electron microscope (SEM) images were recorded with a HITACHI
S-4800 FE-Scanning electron microscope. Attenuated total
reflectance-Fourier transform infrared spectroscopy (ATR-FTIR)
spectra were recorded on a Bruker Vertex 70 FTIR spectrometer.
Ultraviolet-vis spectroscopy (UV-vis) absorbance measurements
were carried out on a JASCO V-550 UV-vis spectrophotometer,
equipped with a Peltier temperature control accessory. All spectra
were recorded in a 1.0 cm path length cell. Atomic force microscopy
(AFM) images were performed using a Nanoscope V multimode
atomic force microscope (Veeco Instruments, Santa Barbara, CA,
USA) in tapping mode. Fluorescence images were captured using
an Olympus BX-51 optical equipped with a CCD camera. All the
photographs were taken by a Canon camera.
The pH reponsive properties of prepared polymers
Polymers were dissolved in 5.0 mM PBS solutions with various pH
values (1.0 mg mL-1). The optical transmittance of the polymer
solutions at different temperatures was measured at 500 nm using
UV-vis spectrophotometer.
The Au-assisted chemical etching processes
The etching of glass surface (2.0×2.0 cm) assisted by Au
nanoparticles was performed following the previous reports [9]. Prior
to the etching, glass substrates were cleaned by ultrasonication in
ethanol and water for 30 min each and followed by freshly prepared
3:1 concentrated H2SO4/30% H2O2 solution for 1h. After washing
with water, an aqueous solution containing HF, H2O2 and Au
nanoparticles was dropped on the surface and etched at 50 oC for
30 min. After etching, the samples were washed with water and
ethanol.
Preparation of N-acryloyl-3-aminophenylboronic acid and N-
acryloyl-3-aminophenylboronic acid glucose ester
The reaction of acryloyl chloride with 3-aminophenylboronic acid
was used to prepare N-acryloyl-3-aminophenylboronic acid
according to the previous report [13]. 3-aminophenylboronic acid
hydrochloride (10 mmol, 1.73g) was dissolved in 25.0 mL 2.0 M
NaOH solution and cooled with ice bath. Acryloyl chloride (10 mmol,
0.8 mL) was added to the solution dropwise with vigorous stirring at
Immobilization of glucose oxidase (GOx) and enzyme activity
evaluation
4
oC. After stirring for one hour with an ice bath, the reaction was
completed at room temperature overnight. 2.0 M HCl was added to
the reaction solution dropwise to adjust pH to 1.0. The precipitate
was separated by filtration and then washed by cold water for three
times. Subsequently, the precipitate was dissolved in 40 mL water
on heating to 60 oC and the insoluble precipitate was filtered off.
The product was finally obtained by crystallization of the solution in
a refrigerator overnight. The product was filtered off, washed with
cold water, and dried under vacuum. The structure of the product
was confirmed by the 600 MHz 1H NMR spectrum.
The etched glass surfaces were rinsed with 5.0 mM pH 5.0 PBS
and then immersed in the GOx solution (160 
g·mL–1 in 5.0 mM pH
5.0 PBS) for 2 h at 4 oC to achieve GOx immobilization [15]. The
glass surfaces were then thoroughly rinsed with water and stored in
a refrigerator 4 oC [16]. After GOx immobilization, the activity of the
free GOx and immobilized GOx were assessed by monitoring the
oxidation rate of glucose. Briefly, 10
L 40.0 mM ABTS in 5.0 mM
pH 5.0 PBS, 10 L 50 L


g·mL–1 HRP in 5.0 mM pH 5.0 PBS, 10
glucose with different concentration (20, 40, 80, 160, 200, 320, 400
and 800 mM in 5.0 mM pH 5.0 PBS), the immobilized GOx and 370
N-acryloyl-3-aminophenylboronic acid glucose ester was
o
L 5.0 mM pH 5.0 PBS were reacted at 37 C and the absorbance
[14]
prepared according to the previous reports
after the successful
at 417 nm was recorded with the UV-vis spectrophotometer. The
activity assays were repeated with series of appropriate
synthesis of N-acryloyl-3-aminophenylboronic acid. The solution of
N-acryloyl-3-aminophenylboronic acid (5 mmol, 955 mg), glucose
(5.25 mmol, 945 mg) and 20 mL of dichloromethane were added to
a dried flask. The solution was stirred for 30 minutes under N2
atmosphere at room temperature and then magnesium sulfate (5
mmol, 600 mg) was added. The solution was further stirred for 4 h
under N2 atmosphere to complete the reaction. Subsequently, the
solution was filtered and washed with dichloromethane. The filtrate
was concentrated with rotary evaporator under reduced pressure
and dried in vacuum to obtain the desired product. The structure of
the product was confirmed by the 600 MHz 1H NMR spectrum.
a
concentrations of glucose to get a series of catalytic rates, followed
by fitting the Michaelis−Menten equation to obtain the constants
Vmax and Km. All the samples are run in triplicate.
Deposition of the polymer onto the surface
The deposition of the polymer was carried out via a typical
procedure of absorption.[10] The surfaces after enzyme
immobilization were immersed into the polymer solution (10.0 mg
mL-1, 5.0 mM pH 5.0 PBS) for 4 h, then the surfaces were
thoroughly rinsed with 5.0 mM pH 5.0 PBS to remove the unbound
polymer.
Copolymerization of prepared N-acryloyl-3-aminophenylboronic
acid or N-acryloyl-3-aminophenylboronic acid glucose ester with N-
vinyl-2-pyrrolidone and N-isopropylacrylamide
Bacterial culture and antibacterial experiments
The polymers were synthesized via the free radical polymerization
[13]
Mono-colony of E. coli on solid Luria-Bertani (LB) agar plate was
transferred to 20 mL of liquid LB culture medium and grown at 37
oC for 12 h under 180 rpm rotation. Then the bacteria were diluted
with broth to 105 cfu·mL–1.[12] The bacteria concentrations were
.
N-acryloyl-3-aminophenylboronic acid (0.5 mmol, 95.5 mg) or N-
acryloyl-3-aminophenylboronic acid glucose ester (0.5 mmol, 167
mg), N-isopropylacrylamide (7.0 mmol, 791 mg), N-vinyl-2-
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determined by measuring the optical density at 600 nm (OD600 nm).
The prepard surfaces were stained with the prepared bacteria
solution (10.0 L) at one corner for 3 h. Then, the unstained part of
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surface was rinsed with 5.0 mM pH 7.4 PBS and the obtained
solution was placed on solid medium by spread plate method and
cultured at 37 oC for 24 h before observing the number of the
bacteria colonies. The number of colony forming units was counted
through visual inspection. Control experiments were performed in
parallel with glass surface and surface modified with only polymer.
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Fluorescence assay for the integrity of bacteria cell membranes
After rinsed with 5.0 mM pH 7.4 PBS for the unstained part of the
surface, the cell death and the extent of bacterial cell invasion were
analyzed by propidium iodide (PI) staining. The solution was
stained with PI (10

g·mL–1) for 10 min in the dark and washed
twice with PBS, then the dead bacteria were visualized with a
fluorescence microscope [11]. The observed areas were randomly
photographed for each sample.
Acknowledgements
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This work was supported by 973 Project (2012CB720602), and
NSFC (21210002, 21431007, 21533008).
Keywords: autonomous and continuous response • stimuli-
responsive polymer • surface • self-propagating reactions • local
fleeting stimuli
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10.1002/chem.201703067
Chemistry - A European Journal
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pH responsive polymer surfaces
that are capable of transforming
specific local, fleeting stimuli into
Chao Ding, Zhengqing Yan, Jinsong
Ren, and Xiaogang Qu*
Page No. – Page No.
global,
changes were designed by the
introduction of self-propagating
reactions into the polymer surface
systems. The prepared H+ responsive
polymer surfaces can be empolyed for
antibacterial application and this
design strategy offers methods for
macroscopic
properties
Autonomous and Continuous Stimuli-
Responsive Polymer Surface for
Antibacterial Application via
Enzymatic Self-Propagating
Reactions
achieving
autonomously
reconfigureable materials.
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