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Stable and efficient loading of silver nanoparticles in spherical polyelectrolyte brushes and the antibacterial effects
Colloids and Surfaces B: Biointerfaces 127 (2015) 148–154
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Stable and efficient loading of silver nanoparticles in spherical polyelectrolyte brushes and the antibacterial effects Xiaochi Liu a,1 , Yisheng Xu a,b,∗,1 , Xiaohan Wang a , Mingfei Shao c,d,∗∗ , Jun Xu a , Jie Wang a , Li Li a , Rui Zhang a , Xuhong Guo a,e,∗ a
State-Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China Zhejiang Provincial Key Laboratory for Chemical & Biochemical Processing Technology of Farm Products, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, 318 Liuhe Road, Hangzhou 310023, China c Harbin Institute of Technology Shenzhen Graduate School, Shenzhen, Guangdong 518055, China d Shenzhen Key Laboratory of Water Resource Utilization and Environmental Pollution Control, Shenzhen, Guangdong 518055, China e Key Laboratory of Materials-Oriented Chemical Engineering of Xinjiang Uygur Autonomous Region, Shihezi University, Xinjiang 832000, China b
a r t i c l e
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Article history: Received 27 November 2014 Received in revised form 22 January 2015 Accepted 23 January 2015 Available online 31 January 2015 Keywords: Spherical polyelectrolyte brush Poly(N-vinylcarbazole) Silver nanoparticles Antibacterial
a b s t r a c t A more efficient and convenient strategy was demonstrated to immobilize silver nanoparticles (NPs) with a crystalline structure into the spherical polyelectrolyte brushes (SPB) as an antibacterial material. The SPB used for surface coating (Ag immobilized PVK–PAA SPB) consists of a poly(N-vinylcarbazole) (PVK) core and poly(acrylic acid) (PAA) chain layers which are anchored onto the surface of PVK core at one end. Well-dispersed silver nanoparticles (diameter ∼ 3.5 nm) then formed and were electrostatically confined in the brush layer. Ag content is controlled by a repeated loading process. Thin film coatings were then constructed by layer-by-layer depositions of positive charged poly(diallyldimethylammonium chloride) (PDDA) and SPB. The multilayer composites display excellent stability as well as antibacterial performance but not for simple PVK–PAA coated surface. The results show that almost complete bacteria growth including both dispersed bacterial cells and biofilms was inhibited over a period of 24 h. This approach opens a novel strategy for stable and efficient immobilization of Ag NPs in fabrication of antibacterial materials. © 2015 Published by Elsevier B.V.
1. Introduction Antibacterial surface coatings or modifications have attracted increasing interests in biomedical and industrial fields because of their important applications in diminish of microorganisms on surfaces of medical devices or in drinking water [1–3]. Layerby-layer (LbL) assemble technique [4,5] as one of the simplest and most popular methods to construct multilayer films [6], has been widely applied in a variety of areas such as catalysis [7], electronic/optical devices [8–10], and biomimetics [11,12]. Since the composition of each layer can be simply altered through LbL assembly, antibacterial or antifouling properties are able to be
∗ Corresponding authors at: State-Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China. ∗∗ Corresponding author at: Harbin Institute of Technology Shenzhen Graduate School, Shenzhen, Guangdong 518055, China. E-mail addresses: [email protected] (Y. Xu), [email protected] (M. Shao), [email protected] (X. Guo). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2015.01.040 0927-7765/© 2015 Published by Elsevier B.V.
realized by introducing some effective materials with bactericidal functionalities [13–15]. Among various antibacterial agents, silver nanoparticles are highly favorable owning to their excellent toxicity to a broad spectrum of microorganisms with low cytotoxicity to human cells [15–18]. In order to fabricate silver-containing films, the stability and durability of the deposited films are required. Some researchers are able to deposit Ag NPs onto the surface of films or membranes by certain surface modifications showing good antibacterial effect [3,19], but the NPs are likely to be eluted from the surface because of weak van der Waals interactions. The loss of Ag particles undoubtedly reduces the antiseptic efficiency dramatically as well as increases the environment contamination. One strategy to resolve this issue is to immobilize Ag NPs into the polymer matrix coated on surfaces. In situ synthesis of Ag NPs has been successfully implemented to stabilize Ag NPs into multilayer composites where silver ions adsorbed into organic layers were exposed to a reducing reagent and Ag NPs are formed within the layers of the films [20,21]. Moreover, a cap region is also allowed to prevent Ag NPs from eluting away [22]. However, this method needs post-treatments of the
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films or the membranes which brings lots of difficulties to scale up. In addition, the in situ reduction of silver ions within solid films leads to broad size distribution of Ag NPs, which may negatively influence the antibacterial performance. As an alternative, ex situ preparation of Ag NPs immobilized multiplayer composites allows a simple way to construct well-structured thin films in large scale [15], but preparation of precursor solution containing Ag NPs is indispensable prior to the construction of multilayer films. So the dispersibility, the stability, and the sustainability of such solutions should be well controlled to avoid the aggregation of NPs [23]. Herein we demonstrated the introduction of silver immobilized spherical polyelectrolyte brushes (SPB) for the fabrication of antibacterial multilayer films. The well-dispersed Ag NPs inside the SPB provide a solution to the above concerns. SPB is composed of a spherical polymer matrix and a polyelectrolyte shell, which has become one of the most attractive materials [24–27]. SPB has been shown to be an ideal accommodation for the synthesis of well-dispersed inorganic nanoparticles with narrow size distribution which has been widely applied as reaction catalysts [28–35]. Due to the electrostatic repulsion among SPB particles, the stability significantly improves and the inorganic NPs immobilized SPB are stable for months. In addition, since metallic ions are captured by polyelectrolyte chains as counterions, almost all of the generated inorganic NPs are distributed within the brush layer and are barely eluted from the polymer matrix [28]. Therefore, silver immobilized SPB is expected to be a promising precursor solution for the fabrication of metal incorporated layer-by-layer thin films. This study presented a way to prepare silver immobilized PVK–PAA SPB followed by fabrication of antibacterial thin films through LbL assembly. Poly(N-vinylcarbazole) (PVK), as a semiconductive polymer [36–38], displays some activity to inhibit the growth of bacteria with very little damage to human cells [39]. Hence PVK was chosen to serve as the core of SPB followed by covalent grafting of PAA shells onto the surface of PVK core by photo-emulsion polymerization. The stability of PVK particles in aqueous solution was significantly enhanced due to electrostatic repulsions. Furthermore, PAA shells were used for silver ion adsorption through electrostatic attractions. The well-dispersed Ag NPs were then encapsulated in the brush layer of PVK SPB by the reduction of silver nitrate precursors. The antimicrobial coating was then achieved by utilizing multifunctional LBL assembly of the as-prepared Ag immobilized PVK–PAA SPB and PDDA. The silver content is tunable by repeated loading of Ag NPs as well as increasing the number of layers. Such coatings show exceedingly excellent bacteria-killing efficiency and long-lasting antimicrobial performance.
2. Experimental 2.1. Materials N-Vinylcarbazole (VCz, 98%) from Aldrich was purified by recrystallization from methanol. Styrene and acrylic acid (AA) from Lingfeng Chemical Reagent Co. Ltd. were used after distillation under reduced pressure to remove the inhibitor and were stored in the refrigerator. Sodium dodecyl sulfate (SDS, 99%), K2 S2 O8 (KPS, 99%), AgNO3 (99.8%) and NaBH4 (96%) were purchased from Sinopharm Chemical Reagent Co. Ltd (SCRC), and poly(diallyldimethylammonium chloride) solution (PDDA, MW: 100,000–200,000, 20 wt%) from Aladdin Industrial Corporation are used as received. Photoinitiator HMEM was synthesized and characterized as reported previously [40]. Pyridine, 2-hydroxy-4 hydroxyethoxy-2-methylpropiophenone (HMP) and methacryloyl chloride (MC) were purchased from J&K chemical Co. Ltd, Technical
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Choices, Inc. and Ciba Specialty Chemicals Inc., respectively. Water was purified with Milli-Q system (Millipore). 2.2. Synthesis of PVK core 0.1 g N-vinylcarbazole was dissolved in 1 ml toluene to form oil phase since N-vinylcarbazole is crystalline solid at room temperature. Then the oil phase was emulsified in 250 ml 0.4 mg/ml SDS solution with vigorous mechanical agitation (700 rpm) for 1 h. After the addition of 0.001 g initiator KPS, the temperature was adjusted to 70 ◦ C. The polymerization reaction continued for 2.5 h under nitrogen atmosphere. At the end of polymerization, the stirring rate was reduced to 300 rpm, and 1 g acetone solution containing 0.1 g photoinitiator HMEM was added at a rate of 6 s per drop (6 s/d). A thin layer of photoinitiator around the PVK core was formed after 2.5 h. Finally the product was purified through dialysis against DI water for three days. 2.3. Synthesis of PVK–PAA SPB The purified PVK core was charged into a home-made photoreactor. 3 g AA monomers were added into the reactor and mixed with the emulsion under vigorous magnetic stirring. The whole reactor was degassed by repeated evacuation and subsequent addition of nitrogen for three times. Photo-emulsion polymerization was then initiated by a UV lamp (wavelength: 200–600 nm, power: 150 W) at room temperature. After 2.5 h, the obtained PVK–PAA SPB were purified by dialysis against DI water to remove undesired small molecules. 2.4. Preparation of silver immobilized PVK SPB PVK SPB were employed as nanoreactors to immobilized Ag NPs onto its surface by the reduction of Ag+ by NaBH4 . In a typical run, 100 ml PVK SPB was charged into a three-necked bottle, then 0.034 g AgNO3 was added in the solution with a stirring rate of 300 rpm. Ag+ ions were thus confined within brush layers as counterions for 2.5 h. The Ag NPs were prepared by the addition of 5 ml 16 mg/ml NaBH4 solution under nitrogen atmosphere. The reduction reaction continued for 1 h and finally the Ag-NPs immobilized PVK SPB was purified through dialysis against DI water to remove unreacted small molecule residues. 2.5. Repeating immobilization of silver nanoparticles onto Ag immobilized PVK-SPB 100 ml of purified Ag immobilized PVK-SPB (marked as 1#) was charged into a three-necked bottle with the addition of AgNO3 . The concentration of Ag+ was adjusted to 2 mmol/L. After stirring for 2.5 h, the Ag+ immobilized Ag immobilized PVK-SPB was treated with 5 ml 16 mg/ml NaBH4 solution for 1 h. The product was dialyzed marked as 2#. Finally 2# was used to repeat above steps to obtain the triply Ag immobilized PVK-SPB which was marked as 3#. 2.6. Surface coating of Ag immobilized PVK–PAA SPB by LbL assembly The microscope glass slides were immersed into piranha solution (3:1 H2 SO4 :H2 O2 ) at 80 ◦ C for 1 h, and washed with sufficient DI water. Then the sides were dipped into 0.5 wt% PDDA solution for 10 min, rinsed with DI water, dried by air flow, and dipped into 0.5 wt% Ag immobilized PVK–PAA SPB solution for 10 min, rinsing and drying to accomplished one layer coating. The procedure was repeated for ten cycles to achieve ten layers coating.
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Fig. 1. (a) Size and size distribution of PVK core (left) and PVK–PAA SPBs (right). The hydrodynamic diameter significantly increases after grafting of PAA chains. (b) Brush thickness of PVK–PAA SPBs as a function of pH () and concentration of AgNO3 at neutral pH (). To study the effect of pH on sizes, pH was adjusted from 2 to 10. During the pH adjustment, the ionic strength was carefully adjusted by adding NaCl to maintain 10 mM [Cl− ] or [Na+ ]. Both experiments were done in triplicate and the standard deviations are indicated by error bars.
2.7. Characterizations The hydrodynamic diameters of SPBs were measured by dynamic light scattering with an equipment of NICOMP 380 ZLS at a scattering angle of 90◦ . Silver content of silver immobilized SPBs was determined by thermogravimetric analysis with a SDT Q600 Simultaneous DSC–TGA instrument and the samples were measured under nitrogen with a heating rate of 10 ◦ C/min. UV–vis spectra were recorded by a SHIMADZU UV-3250 equipment. Samples for transmission electron microscopy (TEM) were performed by adding one drop of emulsion on a copper grid (300 mesh) and then air dried completely. The TEM images were then collected by a JEOL JME-1400 transmission electron microscope at an acceleration voltage of 100 kV. Atomic force microscopy (AFM) observations were performed with a Multimode Nanoscope IIIa unit (Bruker AXS), and the samples for AFM were prepared by pipetting one drop of SPB suspensions onto freshly cleaved mica surface and then air dried. 2.8. Evaluation of the antibacterial property by fluorescent microscopy Escherichia coli strain K-12 BW25113 obtained from the E. coli Genetic Stock Center (Department of Biology, Yale University) was used in this study as the test strain. After 20 min autoclave at 121 ◦ C, microscopic slides coated with Ag immobilized PVK–PAA and PVK–PAA slides as well as the bare slides in duplicates were vertically immersed in a beaker containing 500 ml sterilized LB broth by a home-made slide holder. The above mentioned biofilm growth apparatus was shaken incubated at 75 rpm and 28 ◦ C for 24 h after inoculation with 5 ml E. coli strain K-12 seed liquid. After cultivation, the test slides were carefully removed, vertically held on a Whiteman filter paper to remove any attached liquid media. Then the samples were air dried for 10 min before fluorescent staining. 5 l of staining solution (10 mg/ml 4 ,6-diamidino-2-phenylindole (DAPI) in water) was added on top of the slides. After 10 min of dark incubation, eight fields were randomly selected and photographed with a fluorescence microscope (Nikon Eclipse E600, Japan) equipped with a 100× objective. The total number of cells was counted respectively according to these images. 3. Results and discussion 3.1. Characteristics of PVK–PAA SPBs PVK core was synthesized through conventional emulsion polymerization. Unlike conventional monomers such as styrene,
N-vinylcarbazole is in solid state at room temperature and it was dissolved in organic solvent to generate an oil phase. The size of obtained PVK particles can vary from 150 to 250 nm depending on the concentration of monomer in toluene [41]. After UV exposure, PAA chains grew from the surface of PVK core through a “grafting from” method. DLS results (Fig. 1a) indicate a dramatic increase of hydrodynamic diameter from 175 to 722 nm implying successful grafting of PAA chains [42]. Similar to PS-PAA SPB reported before, the obtained PVK–PAA SPB is also pH responsive. To avoid the effect of ionic strength on brush thickness, we carefully kept the ionic strength at 10 mM by adding NaCl. Fig. 1b shows the relationship between brush thickness and pH. Dramatic increase in brush thickness was observed from pH 3 to 7 due to the electrostatic repulsion of the negatively charged PAA chains arising from the deprotonation of carboxylic groups. When pH is higher than 7, the brush thickness reaches a plateau suggesting nearly complete deprotonation of carboxylic groups, and the maximum brush thickness can reach up to 350 nm. As known, ionic strength has a significant impact on hydrodynamic diameters of SPB, we then carefully studied the variation of brush thickness in a series of AgNO3 concentrations since AgNO3 is the subsequent precursor needed for silver NP formation. As presented in Fig. 1b, the brush thickness generally shrinks with Ag+ concentration. When CAgNO3 is lower than 0.01 mM, there is no obvious difference in brush thickness. However, an intense shrinkage can be noticed as the CAgNO3 is higher than 0.1 mM, this is because that counterions are confined in brush layer at low salt concentration and the osmotic pressures are different inside and outside the brush layer leading to the extension of PAA chains. While at high salt concentrations, the extra Ag+ screens the electrostatic interactions accounting for the collapse of brush layer. The brush thickness reaches a minimum when CAgNO3 is adjusted to 5 mM where the SPB solution becomes unstable and aggregation took place. To ensure the stability of PVK–PAA SPB with relatively higher content of silver NPs, the final AgNO3 loading concentration of 2 mM was chosen in the experiment for preparation of Ag immobilized SPB. 3.2. Preparation of Ag nanoparticles in PVK–PAA SPB The preparation of silver NPs in PVK–PAA SPB is illustrated in Scheme 1. The PVK–PAA brushes provide a strong negative electric field to capture Ag+ . In other words, it is more like a nanoreactor for Ag+ reduction. Since Ag+ are captured through electrostatic interactions and pH certainly affects the degree of ionization of PAA chains, SPB was exhaustively dialyzed, and the pH of the solution is close
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Scheme 1. Preparation of silver NPs immobilized PVK–PAA SPBs by reducing silver ions adsorbed within the brush layer. Repeating loading process was used to enhance the content of silver NPs.
to neutral, which is favorable for adsorption of silver ions. With the addition of AgNO3 , Ag+ ions are captured within the brush layer as counterions through electrostatic interactions. A dramatic decrease in brush thickness was observed due to the electrostatic screening effect among brushes at high Ag+ concentration. After reduction of Ag+ by NaBH4 , the silver NPs are immobilized inside the brush layer. The brush layer slightly swells probably because of the increased electrostatic repulsion among PAA chains due to the disappearance of Ag+ . In addition, the active sites of PAA chains are released so as to adsorb silver ions again. Compared with original SPB, the brush thickness of Ag immobilized SPB is slightly smaller, which may result from the possible coordination interactions between silver NPs and carboxyl groups in PAA chains. The same behaviors can be observed in repeated loading process [35]. More silver NPs are confined in brush layer when more silver ions are reduced. The UV–vis spectra of PVK–PAA SPB and Ag immobilized PVK–PAA SPB are displayed in Fig. 25 to confirm the immobilization of Ag NPs. The two characteristic peaks of PVK can be observed at 330 and 350 nm in both samples. After reduction by NaBH4 , the white SPB solution turns to dark brown and the spectrum exhibits a broad adsorption at 450 nm corresponding to the characteristic peak of silver NPs. The presence of the new peak at the wavelength of 450 nm implies the formation of Ag NPs inside PVK–PAA SPB. The SPB solution exhibits excellent colloidal stability after months at
Fig. 2. UV–vis spectra of PVK–PAA SPB and Ag immobilized PVK–PAA SPB.
room temperature. Furthermore, no shift of maximum adsorption wavelength was observed indicating the good stability of immobilization of silver NPs inside the PVK–PAA SPB. Fig. 3 presents the HRTEM images of Ag immobilized PVK–PAA SPB repeatedly loaded for different times, which were marked as 1#, 2# and 3# respectively. From the TEM images, it can be seen that small silver nanoparticles have been embedded in the brush layer which are mainly located on the surface of PVK core. While small amount of particles are close to the core surface, this may result from the drying-induced shrinkage during the preparation of TEM samples. Since PAA chains are very long (brush thickness is still about 50 nm at high salt concentration before aggregation, Fig. 1b), it can therefore be concluded that most of the silver nanoparticles are in the brush layer. From the TEM image of sample 2# (Fig. 3b), it can be clearly observed that an increasing number of small silver nanoparticles are homogeneously generated onto the surface of PVK core compared to 1#. The diameter of silver nanoparticles is about 3.5 ± 1.5 nm, and the size distribution is much narrower compared to the Ag NPs prepared in situ within the films [18]. By local up-scaling, the lattice fringes of the crystalline silver particles can be observed (Fig. 3d). No aggregation appears in both 2# SPB solution and corresponding TEM images. This indicates that both PVK–PAA SPB and Ag NPs are still stable after repeated immobilizations. After loading for the third time, the SPB solution became brownish. Fig. 3c demonstrates the morphology of 3#. The PVK core is covered with Ag NPs at high loading density. The Ag NPs are in relative good size distribution with an average diameter of 3.5 nm, but a small amount of Ag NPs are obviously bigger than others (marked by blue circles, see Fig. 3c) with a diameter of about 9 nm. As a result, we suppose that vast majority of silver ions are heterogeneous nucleated in the active sites of PAA brush layer and are subsequently reduced to Ag NPs. Since most silver ions are fixed by PAA chains through electrostatic interactions, the diffusion of silver ions is restricted and the newly generated Ag NPs are relatively small (diameter ∼ 3.5 nm). However, in the process of loading for the third time, the newly formed Ag atoms deposit onto the surface of pre-formed Ag NPs leading to the growth of Ag NPs. From the comparison of TEM pictures of 1#, 2# and 3#, it can be concluded that the content of silver NPs is significantly enhanced through repeated loading method. The enhancement of silver content is also reflected by TGA results (Fig. 4). The mass loss of samples 1#, 2# and 3# was investigated under an air environment. The TGA
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Fig. 3. TEM images of Ag@PVK–PAA SPBs for different loading repeats: (a) loading once, 1#, (b) loading twice, 2#, and (c) loading three times, 3#. Several large Ag NPs are formed by continued growth on nanoparticles from previous loading, marked with blued dotted circles. (d) Partially enlarged figure of (b) and the fully crystalline structure of Ag was shown in red dotted circle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
curve of 1# exhibits a significant weight loss when the temperature reaches 250 ◦ C, which arises from the rapid decomposition of organic polymers. After heating to 600 ◦ C, a total weight loss of 65% is observed. The TGA curves of 2# and 3# display similar trend of weight loss, while the residual weight percentages are different, which are 47.7% and 57.4% respectively. All three samples are very stable. Even after storage in room temperature for three months, no visible aggregation or sediment of silver particles were observed in the solutions. This is because PAA shells of Ag immobilized PVK–PAA SPB are negatively charged, the electrostatic repulsion among particles hinders the approaching of neighboring particles. However, when the repeating loading processes continue for more than three times (4 or 5 times), black sediment of silver particles can be noticed after
Fig. 4. TGA results of Ag immobilized PVK–PAA SPB with different loading times. The samples are 1#, 2# and 3# respectively from bottom to top.
addition of NaBH4 . This means after10 continuous silver loading6, the adsorption capability of PAA shells 11for silver ions7 reaches maximum, and superfluous silver ions remain outside the brush leading to aggregation after reduction.
3.3. Antibacterial performance of Ag immobilized PVK–PAA coating As shown in Fig. 5, bacterial cells grew in two different patterns on bare glass slides as well as PVK–PAA coated slides (control): (1) evenly distributed single cells (Fig. 5a for bare glass and b for PVK–PAA coated substrate), and (2) dense biofilm (Fig. 5c for bare glass and d for PVK–PAA coated substrate). These growth patterns correspond to the different phases of biofilm development [43]. Dispersed single bacterial cells indicate the initial reversible or irreversible attachment, while the dense biofilm mainly corresponds to the architecture development or mature biofilm formation phases. An increase of cell attachment on PVK–PAA coating compared with bare slide was observed which is likely caused by the electrostatic interactions between positively charged PDDA and negatively charged cells. However, the bacterial growth is completely inhibited on Ag immobilized PVK–PAA coated slide as shown in Fig. 125e regardless of the type of cell growth. In comparison one layer coating was also performed on the antibacterial test but the inhibition effect is poor (not shown). The average anchored single cells on different type of slides were quantified and the data were shown in Fig. 5f to better reflect the antibacterial performance of Ag coating by our method. A possible explanation is that the bacterial cells are inactivated rapidly during its initial attachment which hinders the subsequent extracellular polymeric substance (EPS) secretion, irreversible attachment, and biofilm formation.
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Fig. 5. Bacterial cell growth on different substrates including bare (a, c), PVK–PAA coated (b, d), and Ag immobilized PVK–PAA coated (e) glass slides monitored by fluorescent microscopy. In detail, (a) mature biofilm formed on bare glass slide; (b) mature biofilm formed on PVK–PAA coated slide; (c) dispersed bacteria attached on bare glass slide; (d) dispersed bacteria attached on PVK–PAA coated slide; (e) no bacteria attached on Ag immobilized PVK–PAA coated slide with 10 LbL assembly (sample 3# with three times Ag loading on SPB was used as anionic layer). Scale bar represents 10 m; (f) density of cell growth on different substrates. Three regions on the slide were picked on each sample to calculate the average and standard deviations of cells.
4. Conclusions A simple method for preparation and immobilization of silver NPs in spherical polyelectrolyte brushes followed by an LbL assembly on a surface for anti-bacterial purpose was developed. The amount of Ag NPs immobilized inside the SPB brushes can be significantly enhanced by repeated loading process. The resulted coatings after LbL assembly show excellent stability and durability since the Ag NPs are immobilized through coordination forces. The cell growth in both dispersed and biofilm patterns on PVK–PAA coated slide is more significant than that for bare slide which is probably due to the enhanced adsorption of cells caused by the electrostatic interactions between cells and polycations. In the presence of Ag immobilized, the bacterial growth is almost completely inhibited possibly because that the Ag NPs direct the cell disruption during initial cell attachment. Acknowledgements We gratefully acknowledge the support of the National Natural Science Foundation of China (21306049, 51273063 and 21476143), the Fundamental Research Funds for the Central Universities8 (222201314029) and the Open Project of State Key Laboratory of Chemical Engineering (SKL-ChE-14C01) to this work. References [1] K. Lewis, A.M. Klibanov, Surpassing nature: rational design of sterile-surface materials, Trends Biotechnol. 23 (2005) 343–348. [2] D. Saeki, S. Nagao, I. Sawada, Y. Ohmukai, T. Maruyama, H. Matsuyama, Development of antibacterial polyamide reverse osmosis membrane modified with a covalently immobilized enzyme, J. Membr. Sci. 428 (2013) 403–409. [3] B. Moshe, R.Z. Katherine, G. Qi, K. Yan, P.G. Emmanuel, E. Menachem, Surface functionalization of thin-film composite membranes with copper nanoparticles for antimicrobial surface properties, Environ. Sci. Technol. 48 (2014) 384–393. [4] G. Decher, J.D. Hong, J. Schmitt, Buildup of ultrathin multilayer films by a selfassembly process: consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces, Thin Solid Films 210/211 (1992) 831–835.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Stable and efficient loading of silver nanoparticles in spherical polyelectrolyte brushes and the antibacterial effects Xiaochi Liu a,1 , Yisheng Xu a,b,∗,1 , Xiaohan Wang a , Mingfei Shao c,d,∗∗ , Jun Xu a , Jie Wang a , Li Li a , Rui Zhang a , Xuhong Guo a,e,∗ a
State-Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China Zhejiang Provincial Key Laboratory for Chemical & Biochemical Processing Technology of Farm Products, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, 318 Liuhe Road, Hangzhou 310023, China c Harbin Institute of Technology Shenzhen Graduate School, Shenzhen, Guangdong 518055, China d Shenzhen Key Laboratory of Water Resource Utilization and Environmental Pollution Control, Shenzhen, Guangdong 518055, China e Key Laboratory of Materials-Oriented Chemical Engineering of Xinjiang Uygur Autonomous Region, Shihezi University, Xinjiang 832000, China b
a r t i c l e
i n f o
Article history: Received 27 November 2014 Received in revised form 22 January 2015 Accepted 23 January 2015 Available online 31 January 2015 Keywords: Spherical polyelectrolyte brush Poly(N-vinylcarbazole) Silver nanoparticles Antibacterial
a b s t r a c t A more efficient and convenient strategy was demonstrated to immobilize silver nanoparticles (NPs) with a crystalline structure into the spherical polyelectrolyte brushes (SPB) as an antibacterial material. The SPB used for surface coating (Ag immobilized PVK–PAA SPB) consists of a poly(N-vinylcarbazole) (PVK) core and poly(acrylic acid) (PAA) chain layers which are anchored onto the surface of PVK core at one end. Well-dispersed silver nanoparticles (diameter ∼ 3.5 nm) then formed and were electrostatically confined in the brush layer. Ag content is controlled by a repeated loading process. Thin film coatings were then constructed by layer-by-layer depositions of positive charged poly(diallyldimethylammonium chloride) (PDDA) and SPB. The multilayer composites display excellent stability as well as antibacterial performance but not for simple PVK–PAA coated surface. The results show that almost complete bacteria growth including both dispersed bacterial cells and biofilms was inhibited over a period of 24 h. This approach opens a novel strategy for stable and efficient immobilization of Ag NPs in fabrication of antibacterial materials. © 2015 Published by Elsevier B.V.
1. Introduction Antibacterial surface coatings or modifications have attracted increasing interests in biomedical and industrial fields because of their important applications in diminish of microorganisms on surfaces of medical devices or in drinking water [1–3]. Layerby-layer (LbL) assemble technique [4,5] as one of the simplest and most popular methods to construct multilayer films [6], has been widely applied in a variety of areas such as catalysis [7], electronic/optical devices [8–10], and biomimetics [11,12]. Since the composition of each layer can be simply altered through LbL assembly, antibacterial or antifouling properties are able to be
∗ Corresponding authors at: State-Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai 200237, China. ∗∗ Corresponding author at: Harbin Institute of Technology Shenzhen Graduate School, Shenzhen, Guangdong 518055, China. E-mail addresses: [email protected] (Y. Xu), [email protected] (M. Shao), [email protected] (X. Guo). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2015.01.040 0927-7765/© 2015 Published by Elsevier B.V.
realized by introducing some effective materials with bactericidal functionalities [13–15]. Among various antibacterial agents, silver nanoparticles are highly favorable owning to their excellent toxicity to a broad spectrum of microorganisms with low cytotoxicity to human cells [15–18]. In order to fabricate silver-containing films, the stability and durability of the deposited films are required. Some researchers are able to deposit Ag NPs onto the surface of films or membranes by certain surface modifications showing good antibacterial effect [3,19], but the NPs are likely to be eluted from the surface because of weak van der Waals interactions. The loss of Ag particles undoubtedly reduces the antiseptic efficiency dramatically as well as increases the environment contamination. One strategy to resolve this issue is to immobilize Ag NPs into the polymer matrix coated on surfaces. In situ synthesis of Ag NPs has been successfully implemented to stabilize Ag NPs into multilayer composites where silver ions adsorbed into organic layers were exposed to a reducing reagent and Ag NPs are formed within the layers of the films [20,21]. Moreover, a cap region is also allowed to prevent Ag NPs from eluting away [22]. However, this method needs post-treatments of the
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films or the membranes which brings lots of difficulties to scale up. In addition, the in situ reduction of silver ions within solid films leads to broad size distribution of Ag NPs, which may negatively influence the antibacterial performance. As an alternative, ex situ preparation of Ag NPs immobilized multiplayer composites allows a simple way to construct well-structured thin films in large scale [15], but preparation of precursor solution containing Ag NPs is indispensable prior to the construction of multilayer films. So the dispersibility, the stability, and the sustainability of such solutions should be well controlled to avoid the aggregation of NPs [23]. Herein we demonstrated the introduction of silver immobilized spherical polyelectrolyte brushes (SPB) for the fabrication of antibacterial multilayer films. The well-dispersed Ag NPs inside the SPB provide a solution to the above concerns. SPB is composed of a spherical polymer matrix and a polyelectrolyte shell, which has become one of the most attractive materials [24–27]. SPB has been shown to be an ideal accommodation for the synthesis of well-dispersed inorganic nanoparticles with narrow size distribution which has been widely applied as reaction catalysts [28–35]. Due to the electrostatic repulsion among SPB particles, the stability significantly improves and the inorganic NPs immobilized SPB are stable for months. In addition, since metallic ions are captured by polyelectrolyte chains as counterions, almost all of the generated inorganic NPs are distributed within the brush layer and are barely eluted from the polymer matrix [28]. Therefore, silver immobilized SPB is expected to be a promising precursor solution for the fabrication of metal incorporated layer-by-layer thin films. This study presented a way to prepare silver immobilized PVK–PAA SPB followed by fabrication of antibacterial thin films through LbL assembly. Poly(N-vinylcarbazole) (PVK), as a semiconductive polymer [36–38], displays some activity to inhibit the growth of bacteria with very little damage to human cells [39]. Hence PVK was chosen to serve as the core of SPB followed by covalent grafting of PAA shells onto the surface of PVK core by photo-emulsion polymerization. The stability of PVK particles in aqueous solution was significantly enhanced due to electrostatic repulsions. Furthermore, PAA shells were used for silver ion adsorption through electrostatic attractions. The well-dispersed Ag NPs were then encapsulated in the brush layer of PVK SPB by the reduction of silver nitrate precursors. The antimicrobial coating was then achieved by utilizing multifunctional LBL assembly of the as-prepared Ag immobilized PVK–PAA SPB and PDDA. The silver content is tunable by repeated loading of Ag NPs as well as increasing the number of layers. Such coatings show exceedingly excellent bacteria-killing efficiency and long-lasting antimicrobial performance.
2. Experimental 2.1. Materials N-Vinylcarbazole (VCz, 98%) from Aldrich was purified by recrystallization from methanol. Styrene and acrylic acid (AA) from Lingfeng Chemical Reagent Co. Ltd. were used after distillation under reduced pressure to remove the inhibitor and were stored in the refrigerator. Sodium dodecyl sulfate (SDS, 99%), K2 S2 O8 (KPS, 99%), AgNO3 (99.8%) and NaBH4 (96%) were purchased from Sinopharm Chemical Reagent Co. Ltd (SCRC), and poly(diallyldimethylammonium chloride) solution (PDDA, MW: 100,000–200,000, 20 wt%) from Aladdin Industrial Corporation are used as received. Photoinitiator HMEM was synthesized and characterized as reported previously [40]. Pyridine, 2-hydroxy-4 hydroxyethoxy-2-methylpropiophenone (HMP) and methacryloyl chloride (MC) were purchased from J&K chemical Co. Ltd, Technical
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Choices, Inc. and Ciba Specialty Chemicals Inc., respectively. Water was purified with Milli-Q system (Millipore). 2.2. Synthesis of PVK core 0.1 g N-vinylcarbazole was dissolved in 1 ml toluene to form oil phase since N-vinylcarbazole is crystalline solid at room temperature. Then the oil phase was emulsified in 250 ml 0.4 mg/ml SDS solution with vigorous mechanical agitation (700 rpm) for 1 h. After the addition of 0.001 g initiator KPS, the temperature was adjusted to 70 ◦ C. The polymerization reaction continued for 2.5 h under nitrogen atmosphere. At the end of polymerization, the stirring rate was reduced to 300 rpm, and 1 g acetone solution containing 0.1 g photoinitiator HMEM was added at a rate of 6 s per drop (6 s/d). A thin layer of photoinitiator around the PVK core was formed after 2.5 h. Finally the product was purified through dialysis against DI water for three days. 2.3. Synthesis of PVK–PAA SPB The purified PVK core was charged into a home-made photoreactor. 3 g AA monomers were added into the reactor and mixed with the emulsion under vigorous magnetic stirring. The whole reactor was degassed by repeated evacuation and subsequent addition of nitrogen for three times. Photo-emulsion polymerization was then initiated by a UV lamp (wavelength: 200–600 nm, power: 150 W) at room temperature. After 2.5 h, the obtained PVK–PAA SPB were purified by dialysis against DI water to remove undesired small molecules. 2.4. Preparation of silver immobilized PVK SPB PVK SPB were employed as nanoreactors to immobilized Ag NPs onto its surface by the reduction of Ag+ by NaBH4 . In a typical run, 100 ml PVK SPB was charged into a three-necked bottle, then 0.034 g AgNO3 was added in the solution with a stirring rate of 300 rpm. Ag+ ions were thus confined within brush layers as counterions for 2.5 h. The Ag NPs were prepared by the addition of 5 ml 16 mg/ml NaBH4 solution under nitrogen atmosphere. The reduction reaction continued for 1 h and finally the Ag-NPs immobilized PVK SPB was purified through dialysis against DI water to remove unreacted small molecule residues. 2.5. Repeating immobilization of silver nanoparticles onto Ag immobilized PVK-SPB 100 ml of purified Ag immobilized PVK-SPB (marked as 1#) was charged into a three-necked bottle with the addition of AgNO3 . The concentration of Ag+ was adjusted to 2 mmol/L. After stirring for 2.5 h, the Ag+ immobilized Ag immobilized PVK-SPB was treated with 5 ml 16 mg/ml NaBH4 solution for 1 h. The product was dialyzed marked as 2#. Finally 2# was used to repeat above steps to obtain the triply Ag immobilized PVK-SPB which was marked as 3#. 2.6. Surface coating of Ag immobilized PVK–PAA SPB by LbL assembly The microscope glass slides were immersed into piranha solution (3:1 H2 SO4 :H2 O2 ) at 80 ◦ C for 1 h, and washed with sufficient DI water. Then the sides were dipped into 0.5 wt% PDDA solution for 10 min, rinsed with DI water, dried by air flow, and dipped into 0.5 wt% Ag immobilized PVK–PAA SPB solution for 10 min, rinsing and drying to accomplished one layer coating. The procedure was repeated for ten cycles to achieve ten layers coating.
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Fig. 1. (a) Size and size distribution of PVK core (left) and PVK–PAA SPBs (right). The hydrodynamic diameter significantly increases after grafting of PAA chains. (b) Brush thickness of PVK–PAA SPBs as a function of pH () and concentration of AgNO3 at neutral pH (). To study the effect of pH on sizes, pH was adjusted from 2 to 10. During the pH adjustment, the ionic strength was carefully adjusted by adding NaCl to maintain 10 mM [Cl− ] or [Na+ ]. Both experiments were done in triplicate and the standard deviations are indicated by error bars.
2.7. Characterizations The hydrodynamic diameters of SPBs were measured by dynamic light scattering with an equipment of NICOMP 380 ZLS at a scattering angle of 90◦ . Silver content of silver immobilized SPBs was determined by thermogravimetric analysis with a SDT Q600 Simultaneous DSC–TGA instrument and the samples were measured under nitrogen with a heating rate of 10 ◦ C/min. UV–vis spectra were recorded by a SHIMADZU UV-3250 equipment. Samples for transmission electron microscopy (TEM) were performed by adding one drop of emulsion on a copper grid (300 mesh) and then air dried completely. The TEM images were then collected by a JEOL JME-1400 transmission electron microscope at an acceleration voltage of 100 kV. Atomic force microscopy (AFM) observations were performed with a Multimode Nanoscope IIIa unit (Bruker AXS), and the samples for AFM were prepared by pipetting one drop of SPB suspensions onto freshly cleaved mica surface and then air dried. 2.8. Evaluation of the antibacterial property by fluorescent microscopy Escherichia coli strain K-12 BW25113 obtained from the E. coli Genetic Stock Center (Department of Biology, Yale University) was used in this study as the test strain. After 20 min autoclave at 121 ◦ C, microscopic slides coated with Ag immobilized PVK–PAA and PVK–PAA slides as well as the bare slides in duplicates were vertically immersed in a beaker containing 500 ml sterilized LB broth by a home-made slide holder. The above mentioned biofilm growth apparatus was shaken incubated at 75 rpm and 28 ◦ C for 24 h after inoculation with 5 ml E. coli strain K-12 seed liquid. After cultivation, the test slides were carefully removed, vertically held on a Whiteman filter paper to remove any attached liquid media. Then the samples were air dried for 10 min before fluorescent staining. 5 l of staining solution (10 mg/ml 4 ,6-diamidino-2-phenylindole (DAPI) in water) was added on top of the slides. After 10 min of dark incubation, eight fields were randomly selected and photographed with a fluorescence microscope (Nikon Eclipse E600, Japan) equipped with a 100× objective. The total number of cells was counted respectively according to these images. 3. Results and discussion 3.1. Characteristics of PVK–PAA SPBs PVK core was synthesized through conventional emulsion polymerization. Unlike conventional monomers such as styrene,
N-vinylcarbazole is in solid state at room temperature and it was dissolved in organic solvent to generate an oil phase. The size of obtained PVK particles can vary from 150 to 250 nm depending on the concentration of monomer in toluene [41]. After UV exposure, PAA chains grew from the surface of PVK core through a “grafting from” method. DLS results (Fig. 1a) indicate a dramatic increase of hydrodynamic diameter from 175 to 722 nm implying successful grafting of PAA chains [42]. Similar to PS-PAA SPB reported before, the obtained PVK–PAA SPB is also pH responsive. To avoid the effect of ionic strength on brush thickness, we carefully kept the ionic strength at 10 mM by adding NaCl. Fig. 1b shows the relationship between brush thickness and pH. Dramatic increase in brush thickness was observed from pH 3 to 7 due to the electrostatic repulsion of the negatively charged PAA chains arising from the deprotonation of carboxylic groups. When pH is higher than 7, the brush thickness reaches a plateau suggesting nearly complete deprotonation of carboxylic groups, and the maximum brush thickness can reach up to 350 nm. As known, ionic strength has a significant impact on hydrodynamic diameters of SPB, we then carefully studied the variation of brush thickness in a series of AgNO3 concentrations since AgNO3 is the subsequent precursor needed for silver NP formation. As presented in Fig. 1b, the brush thickness generally shrinks with Ag+ concentration. When CAgNO3 is lower than 0.01 mM, there is no obvious difference in brush thickness. However, an intense shrinkage can be noticed as the CAgNO3 is higher than 0.1 mM, this is because that counterions are confined in brush layer at low salt concentration and the osmotic pressures are different inside and outside the brush layer leading to the extension of PAA chains. While at high salt concentrations, the extra Ag+ screens the electrostatic interactions accounting for the collapse of brush layer. The brush thickness reaches a minimum when CAgNO3 is adjusted to 5 mM where the SPB solution becomes unstable and aggregation took place. To ensure the stability of PVK–PAA SPB with relatively higher content of silver NPs, the final AgNO3 loading concentration of 2 mM was chosen in the experiment for preparation of Ag immobilized SPB. 3.2. Preparation of Ag nanoparticles in PVK–PAA SPB The preparation of silver NPs in PVK–PAA SPB is illustrated in Scheme 1. The PVK–PAA brushes provide a strong negative electric field to capture Ag+ . In other words, it is more like a nanoreactor for Ag+ reduction. Since Ag+ are captured through electrostatic interactions and pH certainly affects the degree of ionization of PAA chains, SPB was exhaustively dialyzed, and the pH of the solution is close
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Scheme 1. Preparation of silver NPs immobilized PVK–PAA SPBs by reducing silver ions adsorbed within the brush layer. Repeating loading process was used to enhance the content of silver NPs.
to neutral, which is favorable for adsorption of silver ions. With the addition of AgNO3 , Ag+ ions are captured within the brush layer as counterions through electrostatic interactions. A dramatic decrease in brush thickness was observed due to the electrostatic screening effect among brushes at high Ag+ concentration. After reduction of Ag+ by NaBH4 , the silver NPs are immobilized inside the brush layer. The brush layer slightly swells probably because of the increased electrostatic repulsion among PAA chains due to the disappearance of Ag+ . In addition, the active sites of PAA chains are released so as to adsorb silver ions again. Compared with original SPB, the brush thickness of Ag immobilized SPB is slightly smaller, which may result from the possible coordination interactions between silver NPs and carboxyl groups in PAA chains. The same behaviors can be observed in repeated loading process [35]. More silver NPs are confined in brush layer when more silver ions are reduced. The UV–vis spectra of PVK–PAA SPB and Ag immobilized PVK–PAA SPB are displayed in Fig. 25 to confirm the immobilization of Ag NPs. The two characteristic peaks of PVK can be observed at 330 and 350 nm in both samples. After reduction by NaBH4 , the white SPB solution turns to dark brown and the spectrum exhibits a broad adsorption at 450 nm corresponding to the characteristic peak of silver NPs. The presence of the new peak at the wavelength of 450 nm implies the formation of Ag NPs inside PVK–PAA SPB. The SPB solution exhibits excellent colloidal stability after months at
Fig. 2. UV–vis spectra of PVK–PAA SPB and Ag immobilized PVK–PAA SPB.
room temperature. Furthermore, no shift of maximum adsorption wavelength was observed indicating the good stability of immobilization of silver NPs inside the PVK–PAA SPB. Fig. 3 presents the HRTEM images of Ag immobilized PVK–PAA SPB repeatedly loaded for different times, which were marked as 1#, 2# and 3# respectively. From the TEM images, it can be seen that small silver nanoparticles have been embedded in the brush layer which are mainly located on the surface of PVK core. While small amount of particles are close to the core surface, this may result from the drying-induced shrinkage during the preparation of TEM samples. Since PAA chains are very long (brush thickness is still about 50 nm at high salt concentration before aggregation, Fig. 1b), it can therefore be concluded that most of the silver nanoparticles are in the brush layer. From the TEM image of sample 2# (Fig. 3b), it can be clearly observed that an increasing number of small silver nanoparticles are homogeneously generated onto the surface of PVK core compared to 1#. The diameter of silver nanoparticles is about 3.5 ± 1.5 nm, and the size distribution is much narrower compared to the Ag NPs prepared in situ within the films [18]. By local up-scaling, the lattice fringes of the crystalline silver particles can be observed (Fig. 3d). No aggregation appears in both 2# SPB solution and corresponding TEM images. This indicates that both PVK–PAA SPB and Ag NPs are still stable after repeated immobilizations. After loading for the third time, the SPB solution became brownish. Fig. 3c demonstrates the morphology of 3#. The PVK core is covered with Ag NPs at high loading density. The Ag NPs are in relative good size distribution with an average diameter of 3.5 nm, but a small amount of Ag NPs are obviously bigger than others (marked by blue circles, see Fig. 3c) with a diameter of about 9 nm. As a result, we suppose that vast majority of silver ions are heterogeneous nucleated in the active sites of PAA brush layer and are subsequently reduced to Ag NPs. Since most silver ions are fixed by PAA chains through electrostatic interactions, the diffusion of silver ions is restricted and the newly generated Ag NPs are relatively small (diameter ∼ 3.5 nm). However, in the process of loading for the third time, the newly formed Ag atoms deposit onto the surface of pre-formed Ag NPs leading to the growth of Ag NPs. From the comparison of TEM pictures of 1#, 2# and 3#, it can be concluded that the content of silver NPs is significantly enhanced through repeated loading method. The enhancement of silver content is also reflected by TGA results (Fig. 4). The mass loss of samples 1#, 2# and 3# was investigated under an air environment. The TGA
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Fig. 3. TEM images of Ag@PVK–PAA SPBs for different loading repeats: (a) loading once, 1#, (b) loading twice, 2#, and (c) loading three times, 3#. Several large Ag NPs are formed by continued growth on nanoparticles from previous loading, marked with blued dotted circles. (d) Partially enlarged figure of (b) and the fully crystalline structure of Ag was shown in red dotted circle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
curve of 1# exhibits a significant weight loss when the temperature reaches 250 ◦ C, which arises from the rapid decomposition of organic polymers. After heating to 600 ◦ C, a total weight loss of 65% is observed. The TGA curves of 2# and 3# display similar trend of weight loss, while the residual weight percentages are different, which are 47.7% and 57.4% respectively. All three samples are very stable. Even after storage in room temperature for three months, no visible aggregation or sediment of silver particles were observed in the solutions. This is because PAA shells of Ag immobilized PVK–PAA SPB are negatively charged, the electrostatic repulsion among particles hinders the approaching of neighboring particles. However, when the repeating loading processes continue for more than three times (4 or 5 times), black sediment of silver particles can be noticed after
Fig. 4. TGA results of Ag immobilized PVK–PAA SPB with different loading times. The samples are 1#, 2# and 3# respectively from bottom to top.
addition of NaBH4 . This means after10 continuous silver loading6, the adsorption capability of PAA shells 11for silver ions7 reaches maximum, and superfluous silver ions remain outside the brush leading to aggregation after reduction.
3.3. Antibacterial performance of Ag immobilized PVK–PAA coating As shown in Fig. 5, bacterial cells grew in two different patterns on bare glass slides as well as PVK–PAA coated slides (control): (1) evenly distributed single cells (Fig. 5a for bare glass and b for PVK–PAA coated substrate), and (2) dense biofilm (Fig. 5c for bare glass and d for PVK–PAA coated substrate). These growth patterns correspond to the different phases of biofilm development [43]. Dispersed single bacterial cells indicate the initial reversible or irreversible attachment, while the dense biofilm mainly corresponds to the architecture development or mature biofilm formation phases. An increase of cell attachment on PVK–PAA coating compared with bare slide was observed which is likely caused by the electrostatic interactions between positively charged PDDA and negatively charged cells. However, the bacterial growth is completely inhibited on Ag immobilized PVK–PAA coated slide as shown in Fig. 125e regardless of the type of cell growth. In comparison one layer coating was also performed on the antibacterial test but the inhibition effect is poor (not shown). The average anchored single cells on different type of slides were quantified and the data were shown in Fig. 5f to better reflect the antibacterial performance of Ag coating by our method. A possible explanation is that the bacterial cells are inactivated rapidly during its initial attachment which hinders the subsequent extracellular polymeric substance (EPS) secretion, irreversible attachment, and biofilm formation.
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Fig. 5. Bacterial cell growth on different substrates including bare (a, c), PVK–PAA coated (b, d), and Ag immobilized PVK–PAA coated (e) glass slides monitored by fluorescent microscopy. In detail, (a) mature biofilm formed on bare glass slide; (b) mature biofilm formed on PVK–PAA coated slide; (c) dispersed bacteria attached on bare glass slide; (d) dispersed bacteria attached on PVK–PAA coated slide; (e) no bacteria attached on Ag immobilized PVK–PAA coated slide with 10 LbL assembly (sample 3# with three times Ag loading on SPB was used as anionic layer). Scale bar represents 10 m; (f) density of cell growth on different substrates. Three regions on the slide were picked on each sample to calculate the average and standard deviations of cells.
4. Conclusions A simple method for preparation and immobilization of silver NPs in spherical polyelectrolyte brushes followed by an LbL assembly on a surface for anti-bacterial purpose was developed. The amount of Ag NPs immobilized inside the SPB brushes can be significantly enhanced by repeated loading process. The resulted coatings after LbL assembly show excellent stability and durability since the Ag NPs are immobilized through coordination forces. The cell growth in both dispersed and biofilm patterns on PVK–PAA coated slide is more significant than that for bare slide which is probably due to the enhanced adsorption of cells caused by the electrostatic interactions between cells and polycations. In the presence of Ag immobilized, the bacterial growth is almost completely inhibited possibly because that the Ag NPs direct the cell disruption during initial cell attachment. Acknowledgements We gratefully acknowledge the support of the National Natural Science Foundation of China (21306049, 51273063 and 21476143), the Fundamental Research Funds for the Central Universities8 (222201314029) and the Open Project of State Key Laboratory of Chemical Engineering (SKL-ChE-14C01) to this work. References [1] K. Lewis, A.M. Klibanov, Surpassing nature: rational design of sterile-surface materials, Trends Biotechnol. 23 (2005) 343–348. [2] D. Saeki, S. Nagao, I. Sawada, Y. Ohmukai, T. Maruyama, H. Matsuyama, Development of antibacterial polyamide reverse osmosis membrane modified with a covalently immobilized enzyme, J. Membr. Sci. 428 (2013) 403–409. [3] B. Moshe, R.Z. Katherine, G. Qi, K. Yan, P.G. Emmanuel, E. Menachem, Surface functionalization of thin-film composite membranes with copper nanoparticles for antimicrobial surface properties, Environ. Sci. Technol. 48 (2014) 384–393. [4] G. Decher, J.D. Hong, J. Schmitt, Buildup of ultrathin multilayer films by a selfassembly process: consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces, Thin Solid Films 210/211 (1992) 831–835.
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