From Homogeneous to Heterogeneous: A Simple Approach to Prepare Polymer Brush Modified Surfaces for Anti-Adhesion of Bacteria

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From Homogeneous to Heterogeneous: A Simple Approach to Prepare Polymer Brush Modified Surfaces for Anti-Adhesion of Bacteria Xiao-Hong Zhanga,1, Hai-Xia Wua,b,1, Lin Huanga, Chuan-Jun Liua, a b

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Key Laboratory of Biomedical Polymers of Ministry of Education, College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, PR China College of Chemistry and Chemical Engineering, Luoyang Normal University, Luoyang, 471022, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Polymer brush P(N-hydroxyethylacrylamide) Surface modification Anti-adhesion of bacteria

In this paper, the hydrophilic p(N-hydroxyethylacrylamide) (PHEAA) brush was grafted from silicon wafer, followed by the decoration of C3F7 groups with low surface energy to the side chains of PHEAA brush. The antiadhesion performance of the modified surfaces was evaluated using Actinomyces naeslundii and Streptococcus mutans. For 4 h incubation, the silicon wafer with PHEAA brush (Si-PHEAA) was able to resist the adhesion of bacteria, while the anti-adhesion of the PHEAA-C3F7 decorated silicon wafer (Si-PHEAA-C3F7) had been improved. When the incubation time was extended to 24 h, the Si-PHEAA-C3F7 still exhibited excellent adhesionrepellent property against bacteria. And among the Si-PHEAA-C3F7 surfaces with 2.6%, 9% and 13% grafting degrees of C3F7, there was no obvious difference in anti-adhesion performance. The results indicated that the method of polymerization prior to decoration is easy to operate, moreover the prepared Si-PHEAA-C3F7 possessed the long-term and high-efficiency anti-adhesion of bacteria ability.

1. Introduction Bacterial infection is a key issue for the application of biomedical materials, especially for medical implants [1–3]. The adherent bacteria and subsequent formation of a biofilm on the material's surface can cause pathogenic infections [4–7]. The attachment of bacteria on the surface starts from the adhesion of bio-macromolecules, such as the proteins from bacterial membrane. After the free bacteria successfully adhere onto the surface, the bacteria would secrete extra-cellular polymeric substance (EPS) [8], including functional proteins, signal molecules, cell factors for proliferation to form a three dimensional network, and finally form the biofilm. Once the formation of the biofilm, it would protect bacteria from the antibiotics drugs and increase drug resistance [9–11]. So how to prevent the adhesion of nonspecific proteins and bacteria is a great challenge for the application of biomedical implant. Great efforts have been made to prevent the adhesion of bacteria. Surface modification via chemical or physical process is widely adopted to realize the regulation of the adhesion process [12,13]. With the development of living polymerization, the polymer brush has become a reliable method for functionalizing the surface due to its robust mechanical stability, controllable brush thickness, and further modification potential [14–17]. The most widely used polymer brush for preventing the adhesion of

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protein or bacteria is the hydrophilic polymer, such as poly(ethylene glycol) (PEG) [18,19]. It can inhibit the adsorption because of the hydration layer created via hydrogen bonds, which constructs a steric barrier to adsorption. However, PEG may lose their adsorption resistance property because of being oxidized in the presence of oxidant and transition metal ions [20,21]. As alternatives of PEG, other hydrophilic polymer such as poly(vinylpyrrolidone) (PVP) [22], poly (glycerol) [23–25], polypeptide [26], polysaccharide [27], and zwitterionic polymers [28–30] were also developed for preventing the adhesion of protein and bacteria. Among them, the zwitterionic polymers, such as poly(carboxybetaine methacrylate) (PSBMA) [31], and poly (sulphobetaine methacrylate) (PCBMA) [32], exhibit super-low fouling performance resulting from the formation a hydration layer via electrostatic interactions. It is proved that the surface with lower surface energy possesses excellent resistance to adhesion. In this case, the adhesion interaction between the protein or bacteria and the surface is weak, the protein or bacteria could be easily removed from the surface. The typical materials with low surface energy include poly(dimethylsiloxane) (PDMS) [33,34], and fluorinated polymers [35,36]. Zuilhof et al. [37] prepared a series of fluoropolymer brushes via surface initiated ATRP, which displayed excellent antifouling performance against protein due to their relative durability and fouling-release feature.

Corresponding author. E-mail address: [email protected] (C.-J. Liu). X-H Zhang and H-X Wu contributed equally to this work.

https://doi.org/10.1016/j.colcom.2018.02.002 Received 30 December 2017; Received in revised form 24 January 2018; Accepted 12 February 2018 2215-0382/ © 2018 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Zhang, X.-H., Colloid and Interface Science Communications (2018), https://doi.org/10.1016/j.colcom.2018.02.002

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2.2. Preparation of Fluoride Grafted Polymer Brush

However, both in terms of the diversity of proteins and the intrinsic amphiphilic nature of most microorganism, the homogeneous polymer brushes decorated surfaces with sole hydrophilic or hydrophobic properties might be insufficient in practical applications [38]. Thus, it is significant to design the heterogeneous surface with the alternation of hydrophilic or hydrophobic groups to restrict the protein or bacteria adsorption because of the weak thermodynamically interactions. For instance, Zhou et al. [39] prepared a series of random binary polymers brushes (POEGMA-PNCA-F15) grafted surfaces via surface initiated atomic transfer radical polymerization (SI-ATRP) from (polydopamine) PDA based mix-catechol initiator, and the polymer brushes modified surfaces exhibited the good anti-adhesion performance and fouling release behavior. Ober et al. [40] developed a set of amphiphilic block copolymers mixed films in poly(dimethylsiloxane) matrix using oligo (ethylene glycol) and fluoroalkyl side chains, and showed us the fine chemical tuning of the block copolymer composition could improve the performance of the surface. In our previous work [41], we synthesized polyacrylamide-ran-perfluorinated methacrylate (PMA-ran-PFMA) brushes modified surfaces to reduce proteins adsorption. The results illustrated that compared with homogeneous PMA and PFMA brush, the amphiphilic PMA-ran-PFMA brush modified surfaces with the 1:3 and 3:1 ratios of PMA/PFMA possessed the best resistance to BSA and Fg, responsively. But the preparation of amphiphilic block or random copolymer brush needs complex synthesis route. Also, it is difficult to controlling the ratio of two components in copolymers just by adjusting the feed ratio of the monomers. For the sake of building long-term and high-efficiency amphiphilic polymer brush modified surface in a simple way, we incorporated the fluorinated segments into hydrophilic polymer brush through postmodification. We prepared p(N-hydroxyethylacrylamide) (PHEAA) brush from silicon wafers by surface initiated atom transfer radical polymerization (SI-ATRP). The PHEAA is hydrophilic and presents excellent performance for preventing the adhesion of bacteria [42]. The PHEAA brush was further decorated with fluorinated alkane (C3F7COCl) via the side chain of PHEAA brush. The resistant for adhesion behavior of the prepared surface was evaluated with the determination of the amount of adsorbed Fg and bacteria.

2.2.1. Amination of Silicon Wafer Silicon wafer was ultrasonically cleaned with acetone and DIW, sequentially. Then the cleaned silicon wafer was immersed into piranha solution and heated to 100 °C for 2 h. Afterward, the surface was rinsed with DIW four times and dried with argon stream to obtain hydroxylated surface (Si-OH). Si-OH surface was immersed into anhydrous toluene concluding 2.5%(v/v) APTES, the system was sealed and placed at 30 °C for 24 h. Then, the surface was rinsed with toluene, ethanol, and DIW sequentially. The substrate was dried with argon stream to obtain aminated surface (Si-NH2). 2.2.2. Preparation of Initiator-Immobilized Surface Anhydrous DCM including 5%(v/v) TEA, as well as the fresh prepared Si-NH2 was sealed in a tube and immersed into the ice bath for 30 min. After the temperature decreased to 0–4 °C, BiBB with a total 5%(v/v) concentration was added dropwise into the solution, and then the reaction was performed at 0 °C for 2 h and kept at room temperature overnight. The substrate was washed with DCM, ethanol, DIW and dried with argon stream to obtain initiator-immobilized surface (Si-initiator). 2.2.3. Polymerization of PHEAA Brush From Initiator-Grafted Silicon Wafer PHEAA brush was prepared through SI-ATRP. Si-initiator, HEAA monomer (1 g, 8.69 mmol), Me6TREN (40 mg, 0.174 mmol), EBIB (25 μL, 0.172 mmol) and 1:1 ethanol/water (4 mL) were successively added into a polymerization tube. Then the mixture was deoxygenated by four freeze-thaw cycles, and during the second cycle, CuBr (25 mg, 0.174 mmol) was added to the tube under the protection of argon. The polymerization was kept with stirring in the oil bath at 29 °C for 72 h and then stopped by exposure to air. The PHEAA grafted silicon wafer was ultrasonically washed four times with deionized water and then dried in an argon stream to obtain the silicon wafer with a PHEAA brush (Si-PHEAA). 2.2.4. Modification of PHEAA Brush With C3F7COCl PHEAA brush modified silicon substrate was put into a tube, then 7.5 mL anhydrous DCM and 105 μL anhydrous TEA were added to the tube sequentially. The tube was placed in an ice bath. After the temperature decrease to 0–4 °C, heptafluorobutyryl chloride (C3F7COCl) with different concentrations in anhydrous DCM was slowly added. The system was sealed and placed at room temperature for 24 h. After reaction, the silicon wafer was taken out and washed with DCM, ethanol, deionized water, respectively, and dried to obtain a series of polymer brush with amphiphilic side chains modified silicon wafers (Si-PHEAAC3F7).

2. Experiment Section 2.1. Materials Silicon wafers [n-doped, (100)-oriented, 0.56 mm thick, 100 mm diameter with one side polished] were purchased from Wacker Chem tronics (Germany) and cut into 1 cm × 1 cm sized samples. 3Aminopropoyltriethoxyesilane(APTES, 97%), 2-bromoisobutyryl bromide (BiBB, 99%), and ethyl-α-bromobutyrate (EBiB, 99%) were obtained from Aladdin Chemistry Co. Ltd. N-Hydroxyethylacrylamide (HEAA, 98%) and Heptafluorobutyryl chloride (98%) were supplied by TCI. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) were purchased from Alfa Aesar. All reagents above were used as received. Acetic acid, triethylamine (TEA), dichloromethane (DCM), N,N-dimethylformamide (DMF), toluene, and copper bromide (CuBr) were obtained from Shanghai Chemical Reagent Co. Ltd. The water in solvent was removed by standard method. CuBr was stirred in acetic acid at 80 °C overnight and washed with ethanol and acetone several times before use. Deionized water (DIW) with 18.2 MΩ.cm resistivity used in all experiments was prepared through a Millipore (Bedford, MA) Milli-Q filtration system. Argon gas was of high-purity grade. Human plasma fibrinogen (Fg) was obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Phosphate buffered saline (PBS, pH, 7.2–7.4; ion strength, 0.01 M) and 24-well culture plate were provided by Becton Dickinson. Live/Dead Bacterial Viability Kit (KGA502) was purchased from KeyGEN Biological science and technology Co. Ltd.

2.2.5. Preparation of C3F7 Decorated Silicon Wafer Fresh prepared Si-NH2 was put into a tube, then 7.5 mL anhydrous DCM and 105 μL anhydrous TEA were added successively. The tube was immersed in ice bath, after the temperature decreased to 0–4 °C, 90 μL heptafluorobutyryl chloride in anhydrous DCM was slowly added. The tube was sealed and placed at room temperature for 24 h. After reaction, the silicon wafer was washed and dried to obtain C3F7 decorated surface (Si-C3F7). 2.3. Characterization of Fluoride Grafted Polymer Brush Surface 2.3.1. Contact Angle Measurement Water static contact angles (WCA) were measured on a OCA20 contact angle goniometer (Data Physics Instruments GmbH, Germany) using the sessile drop method at 25 °C. The volume of water applied on sample surfaces was 2 μL and the value of WCA was obtained from three different positions of each sample. 2

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investigate the anti-adhesion of bacteria performance of the as-modified surfaces. Each kind of bacteria was cultured in a medium containing 15 mg/mL tryptone, 5 mg/mL soytone, and 15 mg/mL NaCl under anaerobic condition at 37 °C for 24 h. After examining the morphology, the single colonies scraped from tablets were cultured by continuously passaging until the third generation in the medium of BHI (brain heart infusion, 10 mL) under anaerobic condition at 37 °C for 16 h. When the OD value of Streptococcus mutans reached 0.30 and that of Actinomyces naeslundii reached 0.50, the bacteria were used in the next attachment assay. After irradiated with ultraviolet for 1 h, the prepared silicon wafers were placed in a 24-well plate, and then 10 μL of bacteria suspension and 1 mL of BHI medium containing 1% sugar were successively added to each well. Bacteria were incubated with the samples in an incubator for 4 or 24 h at 37 °C, and then, the bacterial solution was removed. The silicon wafers were washed with phosphate buffered saline (PBS, pH = 7.4) three times for SEM and fluorescent microscope (FM) observation. Prior to being observed using SEM (FEI QUANTA 200), the samples with bacteria were placed in glutaraldehyde (2.5 wt%) solution for 2 h, followed by dehydration with graded ethanol solutions of 50%, 60%, 70%, 80%, 90%, and 100% and then dried. The SEM images were taken on the surface after sputtering Au. The fluorescent microscope (FM, Leica Dm4000B Germany) was also utilized to evaluate the amount of adhered bacteria on the silicon wafers. Bacteria attached on sample surfaces were subsequently stained with 1 mL of a Live/Dead Bacterial Viability Kit for 15 min in a dark room at ambient temperature based on the principle in previous literature. The sample surfaces with stained bacteria were observed by a FM with 50× magnification, and the images were taken at λex = 488 nm/λem = 520 nm for detection with a Live/Dead Bacterial Viability Kit, which were analyzed by Image Plus software.

2.3.2. X-Ray Photoelectron Spectroscopy The chemical compositions of modified surfaces were recorded on (KRATOS XSAM800) using Mg K (1253.6 eV) as radiation source. The take-off angle of the photoelectron was set at 90° and the binding energies were calibrated by using the containment carbon (C 1s = 284.6 eV). 2.3.3. Atomic Force Microscope The topography of modified surface was characterized by Atomic Force Microscope (AFM) (multimode 8 microscope, Bruker, USA). The surface morphologies were acquired in Scanasyst mode in air using silicone tip cantilevers with a nominal spring constant of 0.4 N m−1, frequency of 70 kHz. The roughness of surface was evaluated with the value of Ra (mean surface roughness), which was determined from 2.0 μm × 2.0 μm area of the surface. The thickness of polymer brushes was acquired by the step-height analysis method. Typically, the sample surfaces were scratched by a sharp blade, and then the AFM probe was carefully loaded and scanned along the edge of the scratch. 2.4. Protein Adsorption Fg was used as the model protein to evaluate the protein-adsorption resistance of modified surface, and the adsorption tests were performed with BCA assay reagent [43]. Typically, the bare and modified silicon wafers with 1 cm × 1 cm area was immersed in PBS solution containing 1 mL 1 mg/mL Fg. The adsorption was incubated in shaker at 37 °C for 24 h. After the adsorption, the samples were taken out and rinsed with PBS three times to remove the non-adsorbed protein. And then, the remaining protein adsorbed on the surfaces was detached in 1 mL Reporter Lysis 1 × Buffer by ultrasonic washing for 30 min at room temperature. The micro BCA protein-analysis kit (no 23235, Pierce, Rockford, IL), based on the bicinchoninic acid (BCA) method was used to determine the concentration of the protein in Reporter Lysis 1 × Buffer by measuring the absorbance at 562 nm by UV–Vis measurement with a Shimadzu UV-200 spectrophotometer. The amount of adsorbed protein in each area was calculated based on the concentration of protein and predetermined calibration curve obtained by measuring the absorbance of 0–2 mg/mL protein solution in Reporter Lysis 1 × Buffer. The reported data is the average of three parallel groups.

3. Results and Discussion 3.1. Characterization of Fluoride Grafted Amphiphilic Polymer Brush The serial fabrication steps of the modified surfaces were present in Scheme 1. The changes of surface wettability were monitored by contact angle goniometer. As shown in Fig. S1 (Supporting information), the water contact angle (WCA) of Si-OH was 16° due to the generation of hydroxyl groups on surface, and it increased to 43° after the amination with APTES. After the immobilization of initiator, the WCA increased to 68°, indicating that the initiator was successfully

2.5. Bacterial Attachment Streptococcus mutans and Actinomyces naeslundii were adopted to

Scheme 1. Schematic representation of the Si wafer grafted with a PHEAA brush and decorated with C3F7.

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S1, Supporting Information), the C]O around 288 eV was mainly attributed to the amide carbon O]CeN in PHEAA. And the ratio of CeO (N, Br)/C]O is 29.0/15.2, which is almost equal to the value of 2/1 in the molecular formula of HEAA. These results confirmed the successful grafting of PHEAA on the Si-initiator surface. In the XPS spectrum of SiPHEAA-C3F7, we found that a new signal appeared at 690 eV assigned to F1s, and the corresponding picture inserted in blank was the magnified XPS spectrum of F1s. Moreover, the new signal of CF2 (291.5 eV) and CF3 (293.5 eV) from the area-normalized C (1s) XPS in table S1 appeared as a result of grafted fluoride. And the ratio of CF2/CF3 was 11.6/4.9, which was consistent with 2/1 in C3F7. By adjusting the reaction condition, a series of Si-PHEAA-C3F7 with different fluorine contents were synthesized. The specific percentage contents of corresponding elements were shown in Table 1, according to the calculation formula—d = (F/ 7 N) × 100%, where “d” represented the grafting degree of C3F7, “F” and “N” respectively represented the atom percentage (at%) of fluorine and nitrogen obtained from XPS, the grafting degrees of C3F7 were 2.6%, 9% and 13.1%. Based on the data analyzed above, it confirmed that the homogeneous PHEAA brush and heterogeneous PHEE brush with amphiphilic side chains were successfully prepared from silicon wafer. The morphologies of modified surfaces were studied with AFM as shown in Fig. S3 (Supporting Information). The surface average roughness (Ra) of Si-initiator, Si-PHEAA, and Si-PHEAA-C3F7 for a 2 μm × 2 μm scan area was 0.6 nm, 1.7 nm, and 2.9 nm, respectively. And there was no remarkable difference among these modified surface in Ra. The thicknesses of grafted polymer brushes were estimated by step-height analysis method. As revealed in (d) from Fig. S3, the crosssectional analysis showed 14.3 nm height difference between silicon wafer surface and the PHEAA brush, suggesting the thickness of PHEAA brush was 14.3 nm. The thickness of C3F7 decorated PHEAA brush was 19.8 nm obtained in the same method. The increase of thickness may be caused from the more rigid conformation of the PHEAA chain after introducing C3F7 groups to the side chains of PHEAA brush.

Fig. 1. The XPS survey spectra for (a) Si-NH2, (b) Si-initiator, (c) Si-PHEAA and (d) SiPHEAA-C3F7 surfaces.

immobilized onto Si-NH2 surface. Further, when the surface was grafted with hydrophilic PHEAA brush, the WCA decreased to 21°, which was consist with the reported reference [44]. After the introduction of C3F7 groups in the side chains of PHEAA brush, the WCA of Si-PHEAA-C3F7 with different fluorine contents increased to 89°, 101° and 103° responsively. These values were closed to the WCA of 109° from homogeneous C3F7 decorated substrate (Si-C3F7) due to the surface enrichment trend of fluorine atoms. In addition to contact angle measurements, the surface chemistry compositions after each step were analyzed by XPS. The XPS spectra of modified silicon wafers and atomic compositions are shown in Fig. 1 and Table 1. Compared with the spectrum of Si-NH2 surface, there was a new peak at 69 eV in the spectrum of Si-initiator surface, which was attributed to Br3d (0.7%). The picture inserted in blank was the magnified XPS spectrum of Br3d. This result indicated that the initiator was successfully immobilized on silicon wafer surface. After HEAA monomer was polymerized from initiator-immobilized surface, the peak of Br3d disappeared, yet the intensity of N1s signal enhanced obviously. As shown in Table 1, the relative content of N atom in SiPHEAA increased to 9.9 (at%) compared to 2.9 (at%) in Si-NH2. What's more, the atomic ratio of C/O/N in Si-PHEAA was 50.3/21.8/9.9 from XPS spectrum, which was close to the calculated value of 5/2/1 in HEAA monomer, illustrating the prepared PHEAA brush was uniform and had a high coverage rate upon decorated surface. It should be noted that the results from XPS only reflect composition of the top layer, which is determined to the detection limitation of XPS. In the high-resolution C1s spectrum of Si-PHEAA (as shown in Table

3.2. Anti-Protein Adsorption Performance To study the antifouling property of the modified surfaces, Fg firstly was exploited as the model protein to evaluate the resistant-protein performance with micro-BCA protein assay reagent. As far as we know that fibrinogen (Fg) exhibits a strong tendency to adsorb on various surfaces, which may lead to thrombogenesis and biofouling of many biomaterials (e.g., stents, catheters, heart valves, and artificial organs [45]). As shown in Fig. S4, the amount of adsorbed Fg in pristine Si was about 36 μg/cm2. After grafting PHEAA brush from the substrate, the adsorbed Fg decreased to 11 μg/cm2. In addition, the serial PHEAAC3F7 decorated surfaces had the minimum adsorbance below 10 μg/ cm2. These results could be explained as follows. As for Si-PHEAA, the hydrophilic polymer brush could combine water molecules in solution by hydrogen bonds to form the hydration layer, as a steric barrier impeding the interaction between proteins and surface [18]. As for the PHEAA-C3F7 modified surface, except for the partial hydroxyl groups not being decorated by heptafluorobutyryl chloride, there were also a lot of C3F7 groups in structure. It is reported that fluorine-containing materials have a relatively low surface energy, which could release the reversibly adsorbed proteins from surface. Therefore, in the protein adsorption tests, Si-PHEAA-C3F7 displayed the best adsorption resistance performance against Fg.

Table 1 Elemental composition of Si-NH2, Si-initiator, Si-PHEAA, and Si-PHEAA- C3F7 surface determined by XPS analysis. Samples

Si-NH2 Si-initiator Si-PHEAA Si-PHEAA-(C3H7)0.026 Si-PHEAA-(C3H7)0.09 Si-PHEAA-(C3H7)0.13 Si-C3F7

Element (at%) C

O

N

Si

Br

F

22.9 40.3 50.3 62.3 63.2 65.4 60.0

39.8 27.9 21.8 21.3 19.4 17.8 14.8

2.8 2.9 9.9 8.4 7.7 7.0 2.5

34.5 28.2 8.0 6.5 4.8 3.4 14.3

– 0.7 – – – – –

– – – 1.5 4.9 6.4 8.4

3.3. Anti-Adhesion of Bacteria Performance Except for the protein adsorption tests, Actinomyces naeslundii and Streptococcus mutans were further employed to assess the bacterial

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Fig. 2. SEM images of the amount of (a) Actinomyces naeslundii and (b) Streptococcus mutant adhered after 4 h incubation on the pristine Si, Si-PHEAA and Si-PHEAA-C3F7 surfaces.

Fig. 3. SEM images of the amount of (a) Actinomyces naeslundii and (b) Streptococcus mutant adhered after 24 h incubation on the pristine Si, Si-PHEAA and Si-PHEAA-C3F7 surfaces.

incubation, the fluorescence images as shown in Fig. 4 suggested that most of the adsorbed bacteria on modified surfaces was live, and only a small amount of dead bacteria appeared as a result natural apoptosis. And the fluorescence intensity of both Actinomyces naeslundii and Streptococcus mutans attached on pristine Si, Si-PHEAA and Si-PHEAAC3F7 decreased in turn. Quantitative analysis of the fluorescence intensity was revealed in Fig. 6 (a). Compared with the pristine Si, the amount of attached bacteria in Si-PHEAA and Si-PHEAA-C3F7 reduced by 73% and 96.6% for Actinomyces naeslundii, and 77%, 86% for Streptococcus mutant. The results confirmed that PHEAA brush modified surface greatly improved the adhesion-resistant performance against the two kinds bacteria. Moreover, after the introduction of C3F7 groups to the side chains of PHEAA brush, the amphiphilic PHEAA-C3F7 decorated surfaces had more high-efficiency anti-adhesion of bacteria performance than homogeneous PHEAA brush. To further investigate the long-term anti-adhesion of bacteria

attachment on the modified surfaces. The amount of attached bacteria on surfaces was qualitatively and quantitatively analyzed by SEM and fluorescence microscope (FM), respectively. The SEM images as shown in Fig. 2 present the total level of adherent bacteria on these modified surfaces after 4 h incubation. High density bacteria was observed on the pristine Si for both Actinomyces naeslundii and Streptococcus mutans. In contrast, fewer bacteria adhered on both Si-PHEAA and Si-PHEAA-C3F7 surfaces, indicating that these surfaces possessed good anti-adhesion property. Among them, the amount of adherent bacteria on Si- PHEAAC3F7 was least, which suggested the PHEAA-C3F7 decorated surface exhibited the best adhesion resistance performance. In addition to the SEM characterization, the anti-adhesion of bacteria feature also was proved through fluorescence microscope. First, the bacteria adsorbed on surface was stained with Live/Dead Bacterial Viability Kit, and then was observed under fluorescence microscope, where the live bacteria present green and the dead bacteria present red [46]. After 4 h

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Fig. 4. Representative fluorescence microscopy images of pristine Si, Si-PHEAA, and Si-PHEAA-C3F7 surfaces by live/dead bacteria staining after 4 h incubation with (a) Actinomyces naeslundii and (b) Streptococcus mutant.

adhesion of bacteria performance of serial PHEAA-C3F7 modified surfaces were evaluated using SEM and FM. As shown in Figs. S5 and S6, after 24 h incubation with Actinomyces naeslundii and Streptococcus mutans, all of these PHEAA-C3F7 modified surfaces had a better antiadhesion property compared to pristine Si, Si-PHEAA and Si-C3F7, and there was no distinct difference among PHEAA-C3F7 modified surfaces. The special characterizations of Actinomyces naeslundii, as well as Streptococcus mutans by SEM and FM were showed in supporting information. This results indicated that in a wide range of grafting degrees of C3F7, the amphiphilic PHEAA-C3F7 modified surfaces exhibited excellent performance of anti-adhesion property.

property, the incubation time of both Actinomyces naeslundii and Streptococcus mutans was extended to 24 h. The SEM images shown in Fig. 3 displayed that there was still a great deal of bacteria adhered on the pristine Si, while no obvious increase of the two kinds bacteria attached on Si-PHEAA and Si-PHEAA-C3F7 surfaces. The fluorescence images shown in Fig. 5 indicated that the fluorescence intensity of the two kinds bacteria attached on both Si-PHEAA and Si-PHEAA-C3F7 weakened greatly, when compared to the pristine Si. Quantitative analysis of the fluorescence intensity was revealed in Fig. 6 (b). Namely, the bacteria adsorbed in Si-PHEAA and Si-PHEAA-C3F7 decreased by 83%, 95% for Actinomyces naeslundii, and 80%, 87% for Streptococcus mutant. The results illustrated that after 24 incubation, both the PHEAA brush and PHEAA-C3F7 decorated surfaces still had anti-attachment property against the two kinds bacteria. And the PHEAA-C3F7 decorated surface possessed a more high-efficiency and long-term anti-adhesion of bacteria property due to the amphiphilic components in the structure [47–49]. To study the effect of the grafting degrees of C3F7 on antifouling performance, we further investigated the fouling-resistant performance of Si-PHEAA-C3F7 with different grafting degrees of C3F7. The anti-

4. Conclusion In summary, we synthesized a series of Si-PHEAA-C3F7 surfaces in a simple way. The PHEAA polymer brush was firstly synthesized by SIATRP from silicon wafer, and then was modified by low surface energy C3F7 groups to obtain amphiphilic polymer brush decorated surface. We evaluated the anti-protein adsorption property using BCA method, and the anti-adhesion of bacteria performance with SEM and FM. The Si-

Fig. 5. Representative fluorescence microscopy images of pristine Si, Si-PHEAA, and Si-PHEAA-C3F7 surfaces by live/dead bacteria staining after 24 h incubation with (a) Actinomyces naeslundii and (b) Streptococcus mutant.

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Fig. 6. Quantitative analysis of the fluorescence intensity from adherent bacteria with (a) 4 h and (b) 24 h incubation.

PHEAA surface had antifouling performance against protein and bacteria. After the introduction of C3F7 groups to the side chains of PHEAA brush, the anti-adhesion of bacteria performance of Si-PHEAA-C3F7 surface was further improved. In a wide range of grafting degrees of C3F7 from 2.6% to 13% the amphiphilic PHEAA-C3F7 modified surfaces exhibited excellent performance of anti-adhesion property.

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