Healable Antifouling Films Composed of Partially Hydrolyzed Poly(2

Apr 11, 2017 - Antifouling polymeric films can prevent undesirable adhesion of bacteria but are prone to accidental scratches, leading to a loss of th...
2 downloads 18 Views 5MB Size
Download PDF
Research Article www.acsami.org

Healable Antifouling Films Composed of Partially Hydrolyzed Poly(2ethyl-2-oxazoline) and Poly(acrylic acid) Yixuan Li, Tiezheng Pan, Benhua Ma, Junqiu Liu, and Junqi Sun* State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, People’s Republic of China S Supporting Information *

ABSTRACT: Antifouling polymeric films can prevent undesirable adhesion of bacteria but are prone to accidental scratches, leading to a loss of their antifouling functions. To solve this problem, we report the fabrication of healable antifouling polymeric films by layer-by-layer assembly of partially hydrolyzed poly(2-ethyl-2-oxazoline) (PEtOx-EI-7%) and poly(acrylic acid) (PAA) based on hydrogen-bonding interaction as the driving force. The thermally cross-linked (PAA/PEtOx-EI-7%)*100 films show strong resistance to adhesion of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria due to the high surface and bulk concentration of the antifouling polymer PEtOx-EI-7%. Meanwhile, the dynamic nature of the hydrogenbonding interactions and the high mobility of the polymers in the presence of water enable repeated healing of cuts of several tens of micrometers wide in cross-linked (PAA/PEtOx-EI-7%)*100 films to fully restore their antifouling function. KEYWORDS: antifouling, layer-by-layer assembly, materials science, self-healing, thin films

■

these films after long-term usage can expose the underlying substrates on which bacteria will readily adhere. The adhered bacteria produce various metabolic products including corrosive ammonia and organic acids during their proliferation process, which can further erode the already damaged antifouling films.30 The damaged antifouling films will eventually exfoliate from the underlying substrates and completely lose their antifouling functions. Therefore, it is critical to fabricate antifouling films capable of healing scratches and cuts to extend their service life and enhance their reliability. Previous studies have shown that the healing capacity of polymeric films on solid substrates can be enhanced by increasing their thickness.31−33 This is because the thick polymeric films can provide sufficient polymer supplements to the damaged region to repair severe damages such as deep and wide cuts. However, PEG- or zwitterionic polymer-based antifouling films fabricated by surface-initiated polymerization or covalent coupling methods on solid substrates are usually a

INTRODUCTION Biofouling, especially caused by the undesirable adhesion of bacteria on solid surfaces, poses severe problems across a wide range of applications such as medical devices,1−3 the food industry,4 water purification systems5−7 and ship hulls.8 Once permanently attached to these surfaces, the bacteria proliferate and colonize.1−11 This can deteriorate the surface properties and eventually lead to material failure. Antifouling films can inhibit bacterial colonization by effectively preventing the initial adhesion of bacteria on these surfaces.1,5,7−10,12−21 Poly(ethylene glycol) (PEG) and zwitterionic polymers have been extensively employed for the fabrication of antifouling films through surface immobilization methods such as surfaceinitiated polymerization,1,9,20,22 self-assembly,23 and covalent coupling.15,17,24,25 The strong polymer−water interactions originating from hydrogen bonding or ionic solvation produce a hydration layer on PEG and zwitterionic polymer surfaces.12,13,26−29 The PEG and zwitterionic polymers with a hydration layer can repel the adhesion of bacteria and act as an antifouling film. However, most antifouling films are soft and susceptible to mechanical damage in practical usage. Accidental scratches made on antifouling films or partial exfoliation of © XXXX American Chemical Society

Received: February 27, 2017 Accepted: April 11, 2017 Published: April 11, 2017 A

DOI: 10.1021/acsami.7b02872 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 1. (a) Synthetic route of the partially and fully hydrolyzed PEtOx. (b) 1H NMR spectrum of PEtOx-EI-7%. (c) Hydrolysis degree of hydrolyzed PEtOx as a function of hydrolysis time.

various functional films on solid substrates.31−34,46−53 Herein, we demonstrate that healable antifouling films capable of healing deep and wide cuts can be fabricated by the LbL assembly of partially hydrolyzed PEtOx (PEtOx-EI) and PAA based on hydrogen-bonding interactions as the driving force. The stability of the hydrogen-bonded LbL-assembled films is improved by thermally cross-linking the secondary amine groups in PEtOx-EI and the carboxylic acid groups in PAA. A high concentration of partially hydrolyzed PEtOx on the film surface gives the LbL-assembled PAA/PEtOx-EI films the ability to inhibit the adhesion of both Gram-negative and Gram-positive bacteria. Moreover, the PAA/PEtOx-EI film can heal scratches and cuts several tens of micrometers wide after being immersed in water to restore the original antifouling function of the film in the damaged regions, thanks to the high concentration of PEtOx-EI in the bulk of the antifouling films.

few tens of nanometers thick.1,9,20,22,25 These films are too thin to heal severe mechanical damage several tens of micrometers wide. Grafting PEG and zwitterions onto polymers capable of forming thick films provides an effective way to fabricate micrometer-thick antifouling films.34,35 However, the limited grafting density of PEG or zwitterions on these polymers usually leads to antifouling films with low surface density of antifouling moieties, which largely decreases the antifouling performance of the resultant films. Mésini and co-workers demonstrated that antifouling films composed of phosphorylcholine (PC)-modified poly(acrylic acid) (PAA) are less efficient in fouling resistance.36 Ethylene oxide chains are introduced as spacers between PC and the backbone of PAA to further improve the antifouling performance of the antifouling films. Our previous work shows that the antifouling ability of the alternately deposited PEGylated branched poly(ethylenimine) (bPEI-PEG)/hyaluronic acid (HA) composite films originates from the synergic effect of the incorporated PEG on the film surface and the extremely soft nature of the films.34 This is because soft polymeric films can also resist adhesion of cells and bacteria.37 Moreover, PEG-containing antifouling films are prone to losing their antifouling properties after long-term exposure to oxidative environments because of the oxidative degradation of the polyether structure of PEG.38 Therefore, it is critically important to fabricate antifouling polymeric films that can heal wide and deep cuts and show effective antifouling performance as well as satisfactory stability for long-term applications. Poly(2-ethyl-2-oxazoline) (PEtOx) and its derivatives have attracted wide attention because of their excellent antifouling capacities.39−44 The amide groups of the PEtOx and its derivatives can form hydrogen bonds with water, generating a physical barrier to hinder the fouling process.39,40,44,45 Originating from their peptide-like structures, PEtOx and its derivatives exhibit comparable antifouling ability to PEG and zwitterionic polymers, but are more stable than PEG in oxidative environments.45 Due to the presence of the amide group, PEtOx and its derivatives can be alternately deposited with partner polymers containing hydrogen-bonding donors to fabricate composite films. The layer-by-layer (LbL) assembly technique involving alternate deposition of species with complementary interactions such as electrostatic and hydrogen-bonding interactions has been widely used to fabricate

■

EXPERIMENTAL SECTION

Materials. PEtOx (Mw ∼ 500,000), PAA (35 wt %, Mw ∼ 100,000), poly(allylamine hydrochloride) (PAH) (Mw ∼ 120,000−200,000), bPEI (Mw ∼ 750,000), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Lucifer yellow cadaverine (LYC) was obtained from Biotium. HCl (37%) was purchased from Beijing Chemical Reagents Co. Glutaraldehyde was obtained from Alfa Aesar. All chemical reagents were used without further purification if not specified. The concentration of aqueous polyelectrolyte solutions used for film fabrication was 2 mg mL−1. The pH values of the dipping solutions and water were adjusted with either 1 M HCl or 1 M NaOH. Synthesis and Characterization of Hydrolyzed PEtOx. According to the methods described in previous literature,54,55 the hydrolysis of PEtOx was carried out as follows. First, 10 g of PEtOx was dissolved in 52 mL of deionized water at room temperature. Then 82.5 mL of HCl (37%) was added to the above polymer solution, leading to an aqueous HCl solution with a concentration of 24%. The mixture was then further stirred at 100 °C for various reaction times of 0.5, 1, 1.5, 2, and 6 h. For the PEtOx that was hydrolyzed for 0.5, 1, 1.5, and 2 h, the resulting solutions were cooled at room temperature and then dialyzed in a dialysis bag with a molecular weight cutoff of 8,000−14,000 Da against deionized water for 5 days to remove the propanoic acid and HCl. Finally, the white powders of partially hydrolyzed products were obtained by lyophilization. The PEtOx hydrolyzed for 6 h was directly precipitated from the reaction solution. Then the precipitate was filtrated off and washed with deionized water. Finally, white powders were obtained by lyophilization. The hydrolysis degree of the hydrolyzed PEtOx was determined by 1H NMR B

DOI: 10.1021/acsami.7b02872 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 2. (a) Thickness of the as-prepared (PAA/PEtOx-EI-x%)*n films as a function of the number of film deposition cycles. (b) Thickness of the (PAA/PEtOx-EI-x%)*100 films as a function of hydrolysis degree of PEtOx. (c) CLSM image of a (PAA/PEtOx-EI-7%)*100 film with a top layer of PAA-LYC. (d) Time-dependent stability curves of the (PAA/PEtOx-EI-7%)*100 films before (■) and after (●) thermal cross-linking. Diffusion of the PAA-LYC into the (PAA/PEtOx-EI-7%)*100 films was characterized by an Olympus Fluoview FV1000 confocal laser scanning microscope. Fourier transform infrared (FTIR) spectra were obtained on a Bruker VERTEX 80 V FTIR spectrometer. Optical micrographs were recorded on an Olympus BX51 optical microscope. The mechanical properties of the films were measured on an Agilent Nano Indenter G200 using an XP-style actuator and the continuous stiffness measurement (CSM) method. Young’s modulus was tested with a Berkovich diamond tip in air with an ∼30% relative humidity (RH) at ∼27 °C by the “G-Series CSM Standard Hardness, Modulus and Tip Cal” method. The following three-stage load-time sequence was used for the nanoindentation measurements: (1) loading at a constant strain rate of 0.05 s−1 until the indenter reaches a depth of 2000 nm into the surface; (2) holding at the maximum load for 10 s; (3) unloading 90% of the maximum load with a constant unloading rate, then holding for 75 s at 10% of maximum load for thermal drift correction and unloading completely. The storage modulus of the films in water was tested with a flat-ended cylindrical tip made of diamond with a diameter of 108.5 μm using the “G-Series XP CSM flat punch complex modulus” method. Bacterial Adhesion Test. Escherichia coli BL21 (DE3) and Bacillus subtilis strains were used as model Gram-negative and Gram-positive bacteria to evaluate the antifouling properties of the LbL-assembled films. After resuscitation and purification, 100 μL of the bacterial strains was inoculated in 10 mL of Luria−Bertani (LB) medium (10 mg/mL of tryptone, 5 mg/mL of yeast extract, and 10 mg/mL of NaCl) for 12 h at 37 °C with a shaking speed at 200 rpm. The resulting culture was inoculated into a second culture and diluted to achieve an optical density of 0.5 at 600 nm. The LbL-assembled films deposited on the substrates were incubated in a test tube with 3 mL of the bacterial suspension at 37 °C for 6 h. The films were then removed and gently washed three times with sterile PBS buffer. The adhered bacteria on the films were observed by SEM after fixing with 2.5% glutaraldehyde for 4 h at 4 °C.

spectroscopy. Partially hydrolyzed PEtOx is named as PEtOx-EI-x%, where x represents the hydrolysis degree and is determined to be 7, 13, 22, and 50 (Figure S1 and Figure 1b). Fully hydrolyzed PEtOx is linear poly(ethylene imine), which is denoted LPEI (Figure S1). The 1H NMR spectra of hydrolyzed products were as follows: 1H NMR (500 MHz, D2O) PEtOx-EI-7%, δ 3.56−3.40 (m, 4H, −NCOCH2CH2−), 3.21−3.09 (m, 4H, −CH 2 CH 2 NH−), 2.33−2.17 (m, 2H, −COCH2−), 0.96−0.90 (m, 3H, −CH3); PEtOx-EI-13%, δ 3.61− 3.42 (m, 4H, −N COC H 2 CH 2 −) , 3.2 1−3.18 (m, 4H, −CH2CH2NH−), 2.33−2.17 (m, 2H, −COCH2−), 0.96−0.90 (m, 3H, −CH3); PEtOx-EI-22%, δ 3.58−3.41 (m, 4H, −NCOCH2CH2−), 3.18−3.15 (m, 4H, −CH 2 CH 2 NH−), 2.33−2.17 (m, 2H, −COCH2−), 0.96−0.90 (m, 3H, −CH3); PEtOx-EI-50%, δ 3.51− 3.34 (m, 4H, −N COC H 2 CH 2 −) , 2.6 9−2.57 (m, 4H, −CH2CH2NH−), 2.33−2.23 (m, 2H, −COCH2−), 0.98−0.95 (m, 3H, −CH3); LPEI, δ 3.10 (m, 4H, −CH2CH2NH−). Fabrication of LbL-Assembled (PAA/PEtOx-EI-x%)*n Films. Taking (PAA/PEtOx-EI-7%)*n films, for instance, the fabrication of LbL-assembled PAA/PEtOx-EI-x% films was conducted automatically by a programmable dipping machine (Dipping Robot DR-3, Riegler & Kirstein GmbH). First, freshly cleaned substrates (silicon and glass) were immersed in an aqueous bPEI solution for 15 min to obtain a positively charged surface. The bPEI-modified substrates were immersed into an aqueous PAA solution (pH 3.5, 2 mg mL−1) for 15 min to deposit a layer of PAA, followed by rinsing in three water baths (pH 3.5) for 1 min each before the next layer deposition. Next, the substrates were immersed into an aqueous PEtOx-EI-7% solution (pH 3.5, 2 mg mL−1) for 15 min to obtain a layer of PEtOx-EI-7%, followed by the same water rinsing procedure. The adsorption and rinsing steps were repeated until the desired number of deposition cycles was achieved. No drying step was conducted in the LbL deposition procedure until it was in the last PEtOx-EI-7% layer. The (PAA/PEtOx-EI-7%)*100 films were thermally cross-linked at 130 °C for 30 min in a vacuum oven. Measurements. 1H NMR spectra were obtained on a Bruker AVANCEIII 500 MHz NMR spectrometer. Film thickness was recorded on a Dektak 150 surface profiler using a 0.7 μm stylus tip with a 3 mg stylus force. SEM images were obtained using a Hitachi SU8020 scanning electron microscope under vacuum. All samples were coated with a thin layer of gold (2−3 nm) before SEM imaging.

■

RESULTS AND DISCUSSION Synthesis and Characterization of Hydrolyzed PEtOx. PEtOx was hydrolyzed in an aqueous HCl solution (24%) at 100 °C to gradually convert amide groups of PEtOx into C

DOI: 10.1021/acsami.7b02872 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

increasing PEtOx hydrolysis degree. As the hydrolysis degree of PEtOx increases, the amount of amide groups in the corresponding PEtOx-EI-x% polymers decreases, and the hydrogen-bonding interactions between PEtOx-EI-x% and PAA in (PAA/PEtOx-EI-x%)*100 films gradually become weaker. The electrostatic interactions in (PAA/PEtOx-EI-x %)*100 films, with x being 13, 22, and 50, do not have an obvious increase compared with (PAA/PEtOx-EI-7%)*100 films because their strength is limited by the low amount of carboxylate groups in PAA. Meanwhile, the partially hydrolyzed PEtOx-EI-x% (x = 13, 22, 50) with a higher hydrolysis degree has an increased solubility in water and adopts a more extended configuration in the dipping solution. This also enables the fabrication of (PAA/PEtOx-EI-x%)*100 films with a thinner thickness. The secondary amine groups of fully hydrolyzed PEtOx (also named LPEI) in aqueous solution at pH 3.5 are nearly all positively charged. Therefore, hydrogen-bonding and electrostatic interactions between PAA and LPEI are extremely weak, resulting in LbL-assembled (PAA/LPEI)*100 with a minimum thickness compared to other films (Figure 2b). The decrease of hydrogen-bonding interactions and the more extended configuration of PEtOx-EI-x% in solution explain the decreased film thickness of the (PAA/PEtOx-EI-x%)*100 films with increasing PEtOx hydrolysis degree. The PAA conjugated with luminescent LYC (denoted PAALYC) was deposited as the outermost layer of (PAA/PEtOx-EI7%)*100 film to investigate the rapid LbL assembly process of the (PAA/PEtOx-EI-7%)*n films. The diffusion of PAA-LYC within the (PAA/PEtOx-EI-7%)*100 film was investigated by confocal laser scanning microscopy (CLSM). The crosssectional CLSM image in Figure 2c indicates that the PAALYC can diffuse throughout the entire film, indicating that the “in-diffusion” of PAA leads to the deposition of an excessive amount of PAA when the film is immersed in an aqueous PAA dipping solution. When the film is transferred into an aqueous PEtOx-EI-7% solution, the PAA can diffuse out to complex a large amount of PEtOx-EI-7% and deposit a thick PEtOx-EI-7% layer. The diffusion depth of PAA within the (PAA/PEtOx-EI7%)*n films increases with increasing number of film deposition cycles and gradually becomes constant after 10 deposition cycles. The “in-and-out diffusion” of PAA explains the exponential and rapid deposition of (PAA/PEtOx-EI7%)*n films.57 The (PAA/PEtOx-EI-7%)*100 films were examined as healable antifouling films because the (PAA/ PEtOx-EI-7%)*100 films have the highest amount of unhydrolyzed PEtOx and the highest film thickness when the number of film deposition cycles is the same. Therefore, the (PAA/PEtOx-EI-7%)*100 films can provide sufficient material to enhance damage-healing capacity. Meanwhile, the secondary amine group in PEtOx-EI-7% can be cross-linked with carboxylic acid groups of PAA to improve the stability of the (PAA/PEtOx-EI-7%)*100 films. Enhanced Stability of Thermally Cross-Linked (PAA/ PEtOx-EI-7%)*100 Films. To ensure the application of antifouling films in complicated biological environments, the stability of the as-prepared (PAA/PEtOx-EI-7%)*100 films was examined by immersing the films in normal saline (0.9% NaCl, pH 7.4) at 37 °C. The changes of the film thickness as a function of immersion time were recorded by a surface profilometer. As shown in Figure 2d, the thickness of the asprepared (PAA/PEtOx-EI-7%)*100 films rapidly decreases within 4 days due to the dissociation of hydrogen bonds between PAA and PEtOx-EI-7% in a salty solution. After 14

secondary amine groups (Figure 1a). The hydrolyzed PEtOx is denoted PEtOx-EI-x%, where x represents the hydrolysis degree of the resulting polymers and was determined by 1H NMR spectroscopy (Figure 1b and Figure S1). According to the 1H NMR spectrum in Figure 1b, 7 mol % of amide groups in PEtOx are converted into secondary amine groups after being hydrolyzed for 0.5 h. As the reaction proceeds, the hydrolysis degree of PEtOx-EI-x% quickly increases as the reaction time increases. There is 100% hydrolysis after 6 h of reaction (Figure 1c). LbL Assembly of Polyelectrolyte Films. The PEtOx and the hydrolyzed products with different hydrolysis degrees are LbL assembled with PAA to fabricate PAA/PEtOx-EI-x% composite films with PEtOx-EI-x% being the outermost layer. Taking PEtOx-EI-7%, for instance, the LbL-assembled (PAA/ PEtOx-EI-7%)*n (n represents the number of film deposition cycles; an integral number denotes that PEtOx-EI-7% is the outermost layer) film was fabricated by alternately dipping bPEI-coated substrate in aqueous solutions of PAA (2 mg mL−1, pH 3.5) and PEtOx-EI-7% (2 mg mL−1, pH 3.5) with intermediate water washing (pH 3.5) to remove physically adsorbed polyelectrolytes. FTIR spectra of the PEtOx-EI-7% casting film, PAA casting film, and as-prepared (PAA/PEtOxEI-7%)*10 film were measured to investigate the driving force for the formation of the LbL-assembled PAA/PEtOx-EI-x% films (see the Supporting Information, Figure S2). The characteristic peak at 1720 cm−1 in the PAA film is attributed to the −CO stretching vibration of the carboxylic acid groups in PAA, whereas the corresponding peak in the (PAA/ PEtOx-EI-7%)*10 film shifts to 1729 cm−1. The FTIR spectrum of the PEtOx-EI-7% film shows a characteristic peak of the amide groups at 1645 cm−1. The peak of the amide groups in the (PAA/PEtOx-EI-7%)*10 film appears as a red shift from 1645 to 1615 cm−1. The obvious peak shifts of the carbonyl and amide groups in the FTIR spectra indicate that the hydrogen-bonding interactions between amide groups of PEtOx-EI-7% and carboxylic acid groups of PAA are the primary driving force for the formation of the PAA/PEtOx-EI7% films. The thicknesses of the (PAA/PEtOx-EI-x%)*n films (x is 0, 7, 13, 22, 50, or 100) were determined with a surface profilometer. Then the thickness of these films was plotted as a function of the number of film deposition cycles (Figure 2a). The (PAA/PEtOx-EI-x%)*n films (x is 7, 13, or 22) exhibit a typically exponential deposition behavior in the initial 10 deposition cycles (see the Supporting Information, Figure S3). Afterward, the thickness increases rapidly in a nearly linear manner (Figure 2a). However, the thickness of the (PAA/ PEtOx-EI-x%)*n films, with x being 0, 50, and 100, increases in a slow but almost linear way with increasing numbers of film deposition cycles. Moreover, the thickness of the (PAA/PEtOxEI-x%)*n films with the same deposition cycles is greatly dependent on the hydrolysis degree of PEtOx-EI-x%. Figure 2b indicates the thickness of the (PAA/PEtOx-EI-x%)*100 films using PEtOx of different hydrolysis degrees. Of these, the (PAA/PEtOx-EI-7%)*100 film has a maximum thickness of 26.5 ± 0.2 μm. PAA contains ∼5% negatively charged carboxylate groups in pH 3.5 aqueuos solution.56 Carboxylate groups of PAA can have electrostatic interactions with protonated secondary amine groups of PEtOx-EI-7%. By replacing hydrogen-bonding interactions with stronger electrostatic interactions, (PAA/PEtOx-EI-7%)*100 films have a thicker thickness than (PEtOx/PAA)*100 films. The thickness of the (PAA/PEtOx-EI-x%)*100 films decreases with further D

DOI: 10.1021/acsami.7b02872 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces days of immersion, ∼90% of the original film is disintegrated, indicating that the hydrogen-bonded (PAA/PEtOx-EI7%)*100 films are unstable and require chemical cross-linking to enhance their stability. The (PAA/PEtOx-EI-7%)*100 films were thermally cross-linked at 130 °C for 30 min in a vacuum oven to induce partial amidation of the secondary amine groups and carboxylic acid groups in PEtOx-EI-7% and PAA. The formation of the amide groups was confirmed by an absorbance peak at ∼1645 cm−1 in the FTIR spectrum of the thermally cross-linked (PAA/PEtOx-EI-7%)*10 film (see the Supporting Information, Figure S4). For simplicity, the cross-linked (PAA/ PEtOx-EI-7%)*n films are denoted CL-(PAA/PEtOx-EI-7%)*n films. The thickness of the CL-(PAA/PEtOx-EI-7%)*100 films decreases only ∼10% after being immersed in normal saline for 14 days, showing much enhanced stability after thermal crosslinking (Figure 2d). Antifouling Performance of CL-(PAA/PEtOx-EI7%)*100 Films. The CL-(PAA/PEtOx-EI-7%)*100 films were incubated with Gram-negative and Gram-positive bacteria, E. coli and B. subtilis, respectively, to evaluate their antifouling properties. The bacterial adhesion behavior of the films was characterized by SEM. After incubation with E. coli and B. subtilis at 37 °C for 6 h, very few E. coli and B. subtilis were attached to the surface of the CL-(PAA/PEtOx-EI-7%)*100 films. This confirms that these films have excellent antifouling properties toward Gram-negative and Gram-positive bacteria (Figure 3a(i) and b(i)). The remarkable antifouling performance of the CL-(PAA/PEtOx-EI-7%)*100 films originates from the high surface concentration of the antifouling PEtOx

polymers.39−45 To show the importance of the surface PEtOx in prohibiting bacterial attachment, a four-cycle PAA/PAH film was deposited on the surface of the CL-(PAA/PEtOx-EI7%)*100 film with PAH serving as the outermost layer (donated CL-(PAA/PEtOx-EI-7%)*100/(PAA/PAH)*4 film). The adhesion behavior of the CL-(PAA/PEtOx-EI-7%)*100/ (PAA/PAH)*4 film was investigated. In contrast to the CL(PAA/PEtOx-EI-7%)*100 films, E. coli and B. subtilis clearly attach to the CL-(PAA/PEtOx-EI-7%)*100/(PAA/PAH)*4 films after incubation with E. coli and B. subtilis for 6 h (Figure 3a(ii) and b(ii)). The amount of attached E. coli and B. subtilis on CL-(PAA/ PEtOx-EI-7%)*100 and CL-(PAA/PEtOx-EI-7%)*100/(PAA/ PAH)*4 films was measured via the corresponding SEM images with the attached bacteria. As shown in Figure 3c, the densities of E. coli and B. subtilis on CL-(PAA/PEtOx-EI7%)*100/(PAA/PAH)*4 films are (2.0 ± 0.6) × 104 and (1.0 ± 0.3) × 104 cells/mm2, respectively. The density of E. coli on the CL-(PAA/PEtOx-EI-7%)*100 film is dramatically reduced by 98% in comparison with the CL-(PAA/PEtOx-EI-7%)*100/ (PAA/PAH)*4 film, and the density of the attached B. subtilis on the CL-(PAA/PEtOx-EI-7%)*100 film is