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Bioinspired Hierarchical Surface Structures with Tunable Wettability for Regulating Bacteria Adhesion Xiao-Qiu Dou,† Di Zhang,† Chuanliang Feng,*,† and Lei Jiang‡ †
State Key Lab of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, P. R. China and ‡Beijing National Laboratory for Molecular Science (BNLMS), Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China
ABSTRACT To circumvent the influence from varied topographies,
the systematic study of wettability regulated Gram-positive bacteria adhesion is carried out on bioinspired hierarchical structures duplicated from rose petal structures. With the process of tuning the interfacial chemical composition of the self-assembled films from supramolecular gelators, the varied wettable surfaces from superhydrophilicity to superhydrophobicity can be obtained. The investigation of Gram-positive bacteria adhesion on the hierarchical surfaces reveals that Gram-positive bacteria adhesion is crucially mediated by peptidoglycan due to its different interaction mechanisms with wettable surfaces. The study makes it possible to systematically study the influence mechanism of wettability regulated bacteria adhesion and provides a sight to make the bioinspired topographies in order to investigate wettability regulated bioadhesion. KEYWORDS: bacteria adhesion . wettability . supramolecular gelator . hierarchical structures . peptidoglycan
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ettability is a fundamental property of material surfaces and plays an important role on bacteria adhesion.110 The partial reason for the phenomena may ascribe to the interfacial properties of the polysaccharides (e.g., glycoproteins) existing in bacterial shells.1113 In general, moderate wettable surfaces prefer to attract bacteria1422 and superwettable surfaces often inhibit bacteria adhesion due to their specific self-cleaning property.2329 Thus, to control bacteria adhesion on substrates, the desired surface wettability is required, which is usually achieved by modifying surface chemical composition and topographies; typically, the variation of surface topographies is always inevitable. However, the variation may also bring unexpected influence elements on bacterial behaviors at the same time, leading to less dependent on surface wettability for bacteria adhesion.30 This makes it impossible to systematically study the influence mechanism of wettability regulated bacteria adhesion on structured surfaces, which still is a burning question needed to be urgently answered up to date. To circumvent the unexpected influence from the varied topographies, a series of bioinspired hierarchical structures with tunable wettability and same topographies are DOU ET AL.
constructed by duplicating rose petal topographies onto supramolecular gelator G1 [bis(2-(2-hydroxyethoxy)-ethyl)2,2 0 -(terephthaloylbis(azanediyl))bis(3-phenylpropanoate)] and gelator G2 [dihexyl 2,20 -(terephthaloylbis(azanediyl))bis(3-phenylpropanoate)] based films (Figure 1a).31 When different functional groups are introduced into the gelators, the interfacial chemical composition of the self-assembled films can be tuned via noncovalent interactions32,33 between the gelators (Figure 1b), ensuring to vary surface wettability. Second, choosing hydrogels as substrates can simplify the process to duplicate rose petal structures (just like “stamping”, Figure 1c) and enable us to prepare surfaces with same hierarchical structures. Typically, the inherent magnification property of the hierarchical structures may guarantee the preparation of superwettable surfaces.31 The study of Gram-positive bacteria adhesion reveals that the bacteria have different interaction mechanisms with different wettable surface structures (Figure 1d). To explore this, the dynamic adsorption of peptidoglycan (PGN) on the wettable surfaces is studied, since PGN is one of the main components of Gram-positive bacterial cell envelope34,35 and acts as an effective factor for regulating their adhesion. The response VOL. XXX
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[email protected]. Received for review February 10, 2015 and accepted October 2, 2015. Published online 10.1021/acsnano.5b04231 C XXXX American Chemical Society
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ARTICLE Figure 1. (a) The chemical structures of supramolecular gelators G1 and G2. (b) SEM images of the xerogels with a skin layer on the surface and numerous nanofibers in the bulk. (c) The “stamping” method to duplicate rose template onto xerogel surfaces. (d) Schematic demonstration of the patterned surfaces with tunable wettability from superhydrophilic to superhydrophobic by changing surface chemical composition and further wettability regulated bacteria adhesion study.
of PGN to wettable surfaces is found in good agreement with bacteria adhesion behaviors. This study demonstrates that the adhesion behaviors of Grampositive bacteria on wettable surfaces are crucially mediated by PGN through direct interaction with the surfaces, but with different interaction mechanisms on different wettable surfaces. RESULTS AND DISCUSSION The 1,4-benyldicarbonxamide-phenylalanine based supramolecular gelators were respectively coupled with hydrophilic diethylene glycol (G1) and hydrophobic n-hexyl (G2). The parallel orientation of two hydrogenbonding amide moieties provided strong and uniaxial intermolecular interactions, which were necessary to enforce one-dimensional (1D) self-assembly (Figure S2 and S3).3640 As characterized by scanning electronic microscopy (SEM), a homogeneous skin layer was achieved on the top of xerogels due to the significant drying effects;41 however, numerous nanofibers were formed in the bulk of the xerogels (Figure 1b). Ultravioletvisible (UVvis) spectra of both diluted G1 and G2 nanofibrous gels gave rise to the absorbance maximum at the wavelength of 250258 nm, indicative of ππ* transitions in the benzene groups (Figure S4).42 Circular Dichroism (CD) spectra show distinct trough near 200 nm (ππ* transition, characteristic peak of random coil) and less pronounced trough near 240 nm (nπ* transition), suggesting β-turn-like arrangements DOU ET AL.
(Figure 2a).43 The Small Angle X-ray Scattering (SAXS) patterns of G1 and G2 xerogels (Figure 2b) reveal the crystalline nature of the fibrillar networks and polymorph arrangements in the fibers.44 Two SAXS peaks at 3.27 and 3.26 nm1 are observed from G1 and G2, which correspond to d-spacing of 19.20 and 19.26 Å, respectively. These were comparable with the calculated molecular length of G1 and G2 (20.48 Å for G1, 20.77 Å for G2, Figure S5a,b). Here, both gelators may adopt a conformation in which the phenyl moieties fold inward, shielding the amide moieties from the aqueous environment and thus allowing the formation of nanofibers through hydrogen-bonding interactions (Figure S5c).45 Absence of peak in the smaller angle region suggests no higher order lamellar arrangements in the gel state.46 The preparation of hierarchical rose petal-like structures on self-assembled films was carried out by a “stamping” technique through solvent-evaporationdriven imprint pattern transfer process (Figures 1c and S6). The remarkable micro/nano hierarchical structures could be perfectly duplicated onto the dried gel surfaces. Moreover, such a simple duplication process can be applied to different precursors (e.g., mixture of G1 and G2), which is not restricted by chemical component or interface properties (Figure S8). The closely packed arrays of approximately hemispherical micropapillaes were observed (diameter, ∼20 μm; height, ∼10 μm) on the duplicated G1, G2, and mixture films VOL. XXX
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Figure 2. (a) CD spectra of G1 and G2. (b) SAXS patterns of G1 and G2.
Figure 3. SEM images of (a) original red rose's petal and rose duplicated films: (b) G1, (c) G1:G2 = 3:1, (d) G1:G2 = 1:1, (e) G1:G2 = 1:3, (f) G2. (g) The water contact angles of the unduplicated substrates (black line) and rose structured substrates (red line) with different mass ratios of G1 and G2. Schematic illustrations of a drop of water under the (h) Wenzel's state, (i) Cassie's state, and (j) Cassie impregnating wetting state.
(mass ratio of G1:G2 from 1:3 to 3:1), which were similar to periodic microstructures of original rose petal. The magnified SEM images in Figure 3af clearly show that these micropapillaes exhibit cuticular folds in the nanometer scale on the top, keeping the remarkable hierarchical topographies of rose petals. The roughness of these duplicated surfaces is from 5 to 7 μm, which is similar to that of original rose petal (6.3 μm). The wettability of unduplicated and duplicated gel surfaces was evaluated by static water contact angles (CA). For unduplicated surfaces, the CA values of G1 and G2 were 39.4° ( 1.2° and 92.3° ( 1.6°, respectively (Figure S9), which was attributed to the different surface chemical composition as characterized by X-ray photoelectron spectroscopy (XPS, Figure S10). Although both G1 and G2 were built up of carbon atoms with a binding energy of 284.6 eV, the percentage of carbonoxygen single bonds of G1 surface was higher than that of G2 surface, demonstrating relative higher surface energy of G1.47 Because of the effect of chemical composition, the dried gel film containing both G1 and G2 gelators could increase the surface hydrophobicity with the increase of the DOU ET AL.
hydrophobic G2 in the mixed films (black line in Figure 3g). After duplicating rose hierarchical structures onto G1 and G2 films, great changes in CA values were observed in Figure 3g (red line). The duplicated G1 surfaces showed superhydrophilicity (CA = 5.4° ( 1.8°), while, the superhydrophobic G2 surfaces were achieved with the CA value of 150.1° ( 2.3° (Figure S11). From Wenzel model48 (cos θw = r cos θy, where θy is the Young contact angles on the flat surface and r is the roughness ration, defined as the ratio of true area of surface to its projected area, the state is shown in Figure 3h) and CassieBaxter model49 (cos θc = rf cos θy þ f 1, where θc and θy are the CassieBaxter angle and the Young contact angle, respectively, r is the ration of the actual area to the projected area of the solid surface that is wetted by the liquid, and f is the area fraction of the projected wet area, the state is shown in Figure 3i), it is clear that the true CA will be lower with the increase of roughness (r) if the surface is originally hydrophilic; otherwise, it will be higher for originally hydrophobic surface.11 The reason is that the roughness effect can amplify the intrinsic wettability of the substrate VOL. XXX
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ARTICLE Figure 4. (a) Schematic diagram of the procedure to apply bacteria on diverse surfaces. After 2 h and rinsing by PBS, they were imaged by Olympus IX73 Inverted Fluorescent Microscope. M. purpurea adhesion on various wettable surfaces after 2 h: (b) CA = 5°; (c) CA = 31°; (d) CA = 54°; (e) CA = 130°; and (f) CA = 150°. Scale bar: 20 μm.
(magnification effect).5052 Therefore, the hydrophilic G1 films became superhydrophilic and the hydrophobic G2 films became superhydrophobic after duplicating rose hierarchical structures onto them. Typically, for superhydrophobic G2 films, the water droplets maintained the sphere shape when the surface was facing up or even when it was turned upside down (Figure S12), implying a high contact angle hysteresis and a CassieBaxter impregnating wetting state (Figure 3j).31,53 In this state, water droplets are expected to enter into microscale grooves of the surface but not into nanoscale ones. It could be understood that water sealed in micropapillae would cling to the superhydrophobic G2 surface. Due to the magnification effect of surface microand nanocomposite structures on wettability54,55 after duplication, the CA values were decreased from 47.3° ( 1.8° to 31.1° ( 2.3° for the mixed hydrophilic films (G1:G2 = 3:1) and from 65.4° ( 2.9° to 54.3° ( 3.1° for the mixed films (G1:G2 = 1:1). In contrast, the same rose petal structures could induce a large increase of CA from 88° ( 2.5° to 130.2° ( 2.7° for the hydrophobic films (G1:G2 = 1:3). Here, if a droplet of water was freely dispensed on the hydrophobic films, it was found that the contact area kept constant with the evaporation, clearly indicating the occurrence of the Wenzel state (Figure S13).56,57 With increasing hydrophilic component G1 from 0% to 25% in the mixed films, the surface energy were obviously enhanced and water molecules preferred to stay in the nanoscale structures for achieving minimum energy state. This may induce the transition of the droplet from metastable CassieBaxter impregnating wetting state to stable Wenzel state.57 Therefore, the CA of the resulting structured films were able to be tailored over a wide range by varying the mass ratio of G1 and G2. Substrates with varied wettabilities (from superyhydrophilicity (CA e 5°), DOU ET AL.
hydrophilicity (CA = 565°), hydrophobicity (CA = 65150°) to superhydrophobicity (CA g 150°)) and identical topographies were facilely fabricated for studying and tuning bacteria adhesion. The bacteria adhesion on the structured surface was subsequently tested by using Gram-positive bacteria Micromonospora purpurea (Figure 4a). Bacterial adhesion behaviors within 2 h were investigated, since surface wettability could alter the physicochemical interactions between bacteria and substrate in initial phase58 and further regulate bacteria adhesion on wettable surfaces. Figure 4bf presents the fluorescent images of the adsorbed M. purpurea stained with DNA-binding dye 40 ,6-diamidino-2-phenylindole (DAPI). The investigation of M. purpurea on structured substrates with CA between 5° and 150° suggested the highest level of bacteria adhesion occurred in the region of 54°130°. Significant decrease of M. purpurea adhesion was observed on superwettable G1 (CA = 5°) and G2 (CA = 150°) surfaces, though the films possessed the different interface properties. The adhered amount of M. purpurea was decreased by 83.4% on G1 and by 88.6% on G2 surfaces compared to the mixed hydrophobic surface (G1:G2 = 1:3, CA = 130°). Similar adhesion phenomenon was also found for other Grampositive bacteria, e.g., Serinicoccus chungangensis, Kocuria marina, and Bacillus licheniformis (blue lines in Figure S14). As PGN is a major surface component of Grampositive bacteria, the dynamic adsorption behavior of PGN was then investigated to explore the mechanism of Gram-positive bacteria adhesion (Figure 5a). After 60 min, PGN adsorption reached equilibrium state for all substrates. With tuning surface wettability from superhydrophilicity to superhydrophobicity, the adsorbed PGN increased at first and was followed by a decrease. VOL. XXX
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ARTICLE Figure 5. (a) PGN adsorption on different surfaces within 180 min. (b) Pseudo-first-order kinetic analysis of PGN adsorption: the LFO plots of ln(1 qt/qe) vs time. The slope of line represents k1. (black line, G1, CA = 5°; red line, G1:G2 = 3:1, CA = 31°; blue line, G1:G2 = 1:1, CA = 54°; green line, G1:G2 = 1:3, CA = 130°; pink line, G2, CA = 150°) (c) The curves of PGN adsorption (red line) on various wettable films. (d) The curves of M. purpurea adhesion (black line) on various wettable films. Notes: statistical analysis of bacteria adhesion was counted at least 3 pictures by using ImageJ.
The maximum PGN adsorption occurred on hydrophobic surfaces with CA 130° (2.12 ng/cm2). Much less amount of PGN was adsorbed on the superhydrophilic G1 (CA = 5°, 0.32 ng/cm2) and superhydrophobic G2 (CA = 150°, 0.11 ng/cm2) surfaces, decreased by 82.8% and 94.8% compared to that on hydrophilic (G1:G2 = 1:1, CA = 54°, 1.86 ng/cm2) and hydrophobic (G1:G2 = 1:3, CA = 130°, 2.12 ng/cm2) surfaces, respectively. The PGN adsorption rate constants (k1) were further determined by using Lagergren's pesudo-first order (LFO) equation (for calculation details, see Supporting Information part 9).59 The calculated k1 of PGN adsorption on the superwettable G1 and G2 surfaces were both extremely low, 0.0264 and 0.0193 min1, respectively, while the k1 values on the moderate hydrophilic or hydrophobic surfaces (G1:G2 = 1:33:1, CA = 31130°) were in the range from 0.0629 to 0.0970 min1 (Figure 5b, Table S1) and several times higher than those on superwettable surfaces. This suggested significantly unfavorite interaction for the superwettable surfaces toward PGN, while PGN adsorbed more readily onto moderate hydrophilic or hydrophobic surfaces, which may be closely related with H-bonding and hydrophobic interactions with the wettable surfaces.60,61 According to the PGN adsorption (Figure 5c) and Grampositive bacteria adhesion fitting curves (Figure 5d and black lines in Figure S13), the varying trend of adsorbed PGN was basically consistent with that of the adhered Gram-positive bacteria (the similar adsorption trends of PGN and Gram-positive bacteria were also found on the flat films (Figure S15)), which demonstrated that Gram-positive bacteria adhesion on the surfaces may be mainly mediated by PGN through the interactions between PGN and the materials surfaces. DOU ET AL.
It is known that PGN only exists in Gram-positive bacteria shell, but not in Gram-negative bacteria outer wall. As control, two kinds of Gram-negative bacteria (Escherichia coli Top 10 and E. coli OH5R) without PGN shell were selected to further demonstrate the important mediation role of PGN during Gram-positive bacteria adhesion. The varying adhesion trends of E. coli Top 10 and E. coli OH5R were similar to those of Grampositive bacteria (M. purpurea). However, the trends of Gram-negative bacteria adhesion were not consistent with that of adsorbed PGN (Figure S16). The possible reason is that Gram-negative bacteria shells are surrounded with lipopolysaccharide but not with PGN, and the adhesion behavior of Gram-negative bacteria is closely related to the lipopolysaccharide shell. Attenuated total internal reflectance transform infrared (ATR-FTIR) spectra were used to deeply study the interaction mechanism between PGN and substrates (Figure S18). The spectrum of PGN exhibited strong and broad band centered at around 3269 cm1, coming from the OH stretching vibrations of functional groups engaged in hydrogen bonds (H-bonds). Superhydrophilic G1 exhibited a strong band centered at 32793280 cm1, which was also assigned to the OH stretching vibrations. A narrow band at 3280 cm1 was observed on G1 surface with the adsorbed PGN (G1@PGN), implying the weak H-bonded driving force between PGN and the superhydrophilic film, which should be ascribed to the repulsive forces between PGN and G1. The tightly bond water layer adjacent to G1 interface was responsible for the repulsive force (Figure 6a).62,63 With the decrease of surface wettability from superhydrophilicity to moderate hydrophilictiy, the peak above 3000 cm1 became broad, indicating the increased H-bonding interaction between VOL. XXX
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ARTICLE Figure 6. Schematic depiction of PGN mediated Gram-positive bacteria adhesion on (a) superhydrophilic, (b) moderate hydrophilic, (c) moderate hydrophobic, and (d) superhydrophobic surfaces.
PGN and substrate (Figure 6b). In contrast, for hydrophobic substrate with the adsorbed PGN ('G1:G2 = 1:30 @PGN), the spectrum did not show broad band above 3000 cm1 and this may be due to the lack of H-bonds between substrate and PGN. The PGN adsorption mechanism may switch from mainly H-bonding interaction to hydrophobic interaction with the decrease of surface wettability from hydrophilicity to hydrophobicity (Figure 6c). No characteristic peaks of PGN in the spectrum of G2@PGN indicated that no PGN adsorption occurred on the superhydrophobic surfaces. The resistance to PGN on superhydrophobic surface is attributed to the specific hierarchical structures, leading to the air trapped into the nanoscale interstices of superhydrophobic surface (Figure 6d), enabling minimal hydrophobic interaction area between PGN and substrate.64,65 Although the determination of the exact mechanism of bacterial adhesion is beyond the scope of this publication, it offers a new perspective to predicate and control bacteria adhesion through investigating interaction between bacterial outer glycoproteins and wettable surfaces. CONCLUSION In conclusion, with circumventing the influence induced by the varied topographies, we can carry out
EXPERIMENTAL SECTION Materials. All chemicals used in the synthesis of G1 and G2 were purchased from Aladdia and used without further purification. The gelators G1 and G2 were synthesized with high yields through conventional liquid phase reaction in three steps according to Scheme S1 and ref 32. Preparation of Gel Film. G1, G2, or mixture of G1 and G2 gelators were initially added in xylene (2 mg/mL). The solution of xylene was heated until gelators were dissolved. The gel formed after cooling the solution to room temperature. The gels (the height is about 5 mm) placed on the substrates (glasses or silicon slices) were dried in the air at room temperature, and the gel film formed. Transmission Electron Microscope (TEM). TEM images were obtained with an analytical transmission electron microscope (JEM-2010/INCA OXFORD, working voltage of 200 kV). One drop of sample (0.1 wt % gelators) was placed onto a copper grid, and
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the systematic study of wettability regulated Grampositive bacteria adhesion on bioinspired hierarchical structures. The bacteria adhesion is found to be crucially mediated by PGN, which has the different interaction mechanisms with the varied wettable surfaces. The study makes it possible to systematically study the mechanism of wettability regulated bacteria adhesion on structured surfaces. Thanks to the merit of the tunable chemical composition and strong selfassembly ability of C2 based gelators, this research not only paves a new way to make the bioinspired topographies with various wettability for studying wettability regulated bioadhesion, but also directly bridges the research gap between wettability and bacterial adhesion. Due to the generality of the study, it may broaden bacteria adhesion related researches, rather than only on rose-petal like surfaces, e.g, carnation-petal like structures or even manmade hierarchical structures.66 Considering more complex component of Gram-negative bacterial wall than those of Gram-positive ones, the complicated interactions with wettable surfaces can be expected. Nevertheless, it should also be closely related with the interfacial property of major surface components, which will be followed in our group.
a thin film was produced by blotting off the redundant liquid with filter paper. Scanning Electron Microscopy (SEM). SEM samples (0.2 wt %) were prepared as the TEM samples. Samples placed on the silicon slices were dried in air at room temperature and visualized with an FEI QUANTA 250 scanning electron microscope using an accelerating voltage of 10 kV and working distance of 10 mm. To view the inner structure of gel, a piece of cover glass was lightly put on the gel. After the gel had dried, the cover glass was quickly removed. Part of the outside skin layer was torn off, and the inside structure was exposed. Atomic Force Microscopy (AFM). AFM samples (0.1 wt % gelators) were prepared as the SEM samples. Samples were placed on the mica plates. Images were obtained using a Vecco NanoScope IIIa atomic force microscope and MikroMasch NSC11 cantilevers/tips (radius of curvature less than 10 nm). The fiber diameters were analyzed by the software BinOffline5000609.
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Conflict of Interest: The authors declare no competing financial interest. Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b04231. (1) Synthic routes of G1 and G2; (2) morphological study; (3) UVvis and SAXS studies; (4) XPS spectra; (5) bacteria and PGN adsorption; (6) ATR-FITR spectra (PDF)
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Acknowledgment. Financial support came from the National Science Foundation of China (51273111, 51173105), the National Basic Research Program of China (973 Program2012CB933803), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. Research Fund for the Doctoral Program of Higher Education of China, SRF for ROCS, SEM. Shanghai Jiaotong Medical/Engineering Foundation (YG2012MS29).
REFERENCES AND NOTES
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Small Angle X-ray Scattering (SAXS) Study. The SAXS patterns were obtained from the G1 and G2 xerogels. The SAXS patterns were recorded on an Anton Paar SAXSess Instrument. UltravioletVisible (UVVis) and Circular Dichroism (CD) Spectroscopy. A JASCO J-815 CD spectrometer was used to collect UVvis and CD data. Deionized water was used to dissolve the sample by heating to 100 °C. All the concentrations of gelators were 5 104 wt %. Spectra were measured in a 1 mm path length cell at room temperature. X-ray Photoelectron Spectroscopy (XPS). G1 and G2 hydrogels were dried on silicon slices to prepare the samples. XPS spectroscopy was carried out using a Kratos AXIS ULTRA DLD instrument with an Al (Mono) and a 1000.0 meV step. Fabrication of Surface with Hierarchical Structures. Poly(vinyl alcohol) (PVA, MW = 22 000 g/mol) water solution (10 wt %) was poured onto the fresh surface of a red rose petal and exposed to air at room temperature. When water was evaporated, the PVA film was peeled off, which imprinted the inverse petal structures. The PVA film was used as a “stamp” and pressed on G1, G2, or mixture of G1 and G2 xylene gel. After an air drying step and peeling of PVA film, G1, G2, and mixture of G1 and G2 coating glass with petal structures were obtained. The roughness of surface was tested by the Carl Zeiss confocal laser scanning microscope (LSM 700). Static Contact Angle Measurements. The static contact angle measurements were done using a JC2000D optical contact angle meter (Zhongchen, Shanghai). The water droplet used in each experiment was 2 μL. The reading of the contact angle was done after 30 s when the droplet had been steady. The measurements were performed for at least three trials at different areas of the sample surface and were replicated in three more samples. Attenuated Total Internal Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The ATR spectra were measured by using a Nicolet 6700 Instrument. The hydrogel was placed on the Ti substrate and dried for ATR test. Adsorption of PGN. PGN was purchased from Shanghai Xinyu Biotechnology Co. Ltd. A 100 μL solution of PGN (20 ng/mL) was dropped on the glass (diameter: 1 cm) coated G1 and G2 sample (0.02 mg) for different times (from 0 to 180 min) at room temperature. The adsorbed PGN on different films was tested by PGN Elisa Kit (bought from Shanghai Xinyu Biotechnology Co. Ltd.). The absorbance of PGN was measured with a Tecan Infinite 200 Pro spectrometer using λ = 450 nm and correlating these intensity values to a calibration curve. Bacterial Adhesion. All the bacteria were a gift from Shuangjun Lin group, School of Biomedical Engineering of Shanghai Jiaotong University. A single isolated bacteria colony was inoculated in 5 mL Tryptic Soy Broth (TSB) overnight at 35 °C. The bacterial culture was centrifuged at 3000 rpm for 10 min, and the bacteria pellet was resuspended in TSB. The optical density of suspension was adjusted to 0.5 at 600 nm. All samples were individually placed in a 24 well-plate (Corning). A volume of 100 μL of bacterial culture was added in each well, and the plate was then statically incubated at 37 °C for 2 h. The samples were removed from bacterial culture, washed with PBS and deionized water twice (to remove the detached or unadhered bacteria), and stained with 100 μL of DAPI dye solution (10 μg/mL) for 20 min. After a washing step with PBS twice, the samples were imaged using Olympus IX73 Inverted Fluorescent Microscope under 400 objective. The amount of attached bacterial cells was expressed as the mean of bacteria ( standard deviation of three images. Statistical analysis was done using Software ImageJ. All the chemicals used in the bacterial adhesion test such as TSB, DAPI, PBS, and so on were purchased from Shanghai YESEN Biotechnology Co. Ltd.
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