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Influence of TiO2 nanostructure size and surface modification on surface wettability and bacterial adhesion
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Influence of TiO2 nanostructure size and surface modification on surface wettability and bacterial adhesion Gaoqi Wanga,b, Ding Wenga, Chaolang Chena, Lei Chena, Jiadao Wanga,
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a b
State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, PR China School of mechanical engineering, University of Jinan, Jinan 250022, PR China
ARTICLE INFO
ABSTRACT
Keywords: Bacterial adhesion TiO2 Surface energy Surface morphology
The aim of the study was to explore the synergistic effects of nanostructure size and surface treatment of TiO2 coatings on its wettability and bacteria adhesion. The coatings were fabricated by TiO2 particles with a diameter ranging from 25 nm to 300 nm. The surface wettability of coatings was tuned by chemical treatment. The antiadhesion property of as-prepared coatings was evaluated with Streptococcus mutans in two types of solutions: phosphate buffer solution (PBS) and PBS with 1 wt% sucrose. The results show that particle size and surface treatment have significant influences on the wettability (with contact angles of 0–160°) and bacterial adhesion. The coating prepared by chemically modified 25 nm particles has a superhydrophobic property, and could inhibit bacterial adhesion in aqueous solutions with sucrose or not. The study also provides a facile, universal and low-cost process to fabricate anti-adhesion coating, which is applicable for diverse of substrates.
1. Introduction Bacterial adhesion on solid surfaces widely exists in nature, such as surgical tools, water filtration and ship hulls, resulting in infection or other negative impacts [1]. Generally, there are two types of antibacterial strategies: kill bacteria and reduce adhesion of bacteria. The commonly used anti-adhesion strategies contains the modification of surface morphology, changing surface wettability, and suppressing enzyme activity by natural or synthetic substances, and so on [2]. The surface morphology has been proved to have influence on the bacteria adhesion according to previous researches [3]. Quirynen et al. [4] found that rough surfaces promoted bacterial biofilm formation and maturation, and the influence of the surface roughness on plaque accumulation was more prominent than surface free energy. A further study conducted by Bollen et al. [5] indicated a smoothening below a Ra of 0.2 μm showed no further significant changes on the bacterial adhesion. In recent years, many researchers suggested surfaces with special morphology had reducing effect of bacterial adhesion [6–13]. Those micro- or nanostructures were usually fabricated by lithography, deposition, laser, electrochemical anodizing, sol-gel and so forth (Fig. 1). It is found that size of the morphology also has important effect on the bacterial adhesion. Wang et al. [14] showed adhesion of staphylococci was significantly reduced when the interpillar spacing was below 1.5 μm, which means the size of the surface patterns should be
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smaller than that of a bacterium to reduce the contact area between the bacterium and surface. Various patterns of surface morphology may show different size effects. The surface morphology not only affects the contact situation between bacterium and surface, but also changes the wettability of surfaces. Wettability, which plays an important role on bacteria adhesion, represents the degree that material surface can be wet by a liquid. Surface roughness leads to an amplification of the wetting properties of the smooth material [15], increasing the hydrophilicity or hydrophobicity of material that was naturally hydrophilic or hydrophobic. According to previous researches, in general moderate wettable surfaces prefer to attract bacteria [12]. Superhydrophobic surfaces, typically fabricated by either covalently attaching molecules with low surface energies to a roughened surface or roughening the surface of a hydrophobic material [16,17], often inhibit bacteria adhesion due to their specific self-cleaning property [18]. Additionally, some hydrophilic or superhydrophilic materials, such as poly(ethylene glycols), p (N-hydroxyethylacrylamide) and zwitterionic polymers also exhibited anti-adhesion effect due to their strong electrostatically induced hydration layers that construct a steric barrier to adhesion [19–21]. However, a few publications suggested some superhydrophobic surfaces induce more bacterial adhesion, such as poly(L-lactic acid) [22]. Therefore, it is highly needed to furtherly investigate the bacterial adhesion property of superhydrophobic surface.
Corresponding author at: State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, PR China. E-mail address: [email protected] (J. Wang).
https://doi.org/10.1016/j.colcom.2019.100220 Received 23 September 2019; Received in revised form 5 November 2019; Accepted 11 November 2019 2215-0382/ © 2019 Published by 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: Gaoqi Wang, et al., Colloid and Interface Science Communications, https://doi.org/10.1016/j.colcom.2019.100220
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Fig. 1. Anti-adhesion surfaces made by different methods [6–13]
Recently, various coatings made of particles such as TiO2 [23], Cu2O [24], ZnO [25] and Ag [26] were applied in antibacterial coatings. TiO2 was frequently used because it demonstrates nontoxic, biocompatible, inexpensive and photo induced antibacterial nature [27,28], which can kill bacteria by highly reactive species. TiO2 is mainly active with ultraviolet light and researches are carrying out in activating TiO2 using visible light and enhance photocatalytic activity by doped with metals, metal oxides, non-metal or organosilane compounds [29]. Therefore, TiO2 particle was hardly solely used to fabricate coatings, and usually used as addition incorporated into coatings to kill bacteria [30]. Few studies focused on the anti-adhesion effect of the coatings assembled with the nanoparticles. The combinational impact of particle size and surface energy on the wettability and bacterial adhesion remains to be explored. In the absence of sucrose, the sucrose-independent adhesion between bacteria and surfaces relies on bacterial surface protein [31]. While in the environment with sucrose, Gram-positive bacteria produce extracellular polymeric substances (EPS) from sucrose [32]. EPS is composed of multiple components, including polysaccharides, proteins, and extracellular DNA (eDNA) [33]. The sucrose-dependent bacterial adhesion is mainly mediated by EPS, which could enhance the adhesion between the bacteria and solid surfaces [34]. Thus, the bacterial adhesion situation on the particle assembled coatings may be different due to the existence of sucrose. In this study the synergistic effects of nanostructure and surface modification of assembled TiO2 coating on its wettability and bacteria adhesion were explored. 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (PTES) was used to modify TiO2 with lower surface energy. A typical Gram-positive Bacterium Streptococcus mutans (S. mutans) was used in the adhesion evaluation under two environments with and without sucrose. The TiO2 coating is simply-made and could be assembled on other materials, such as metal, ceramic and resin. Because the antiadhesion effect is produced mainly by the nanostructure and surface energy properties, the TiO2 also could be replaced by other particles, such as SiO2 and PTFE. The strategy provides new ideas for developing novel anti-adhesion surfaces.
modification. Each group contained three kinds of coatings with different TiO2 particle sizes of approximately 300 nm (293.0 ± 28.1 nm), 100 nm (103.1 ± 17.4 nm), and 25 nm (25.7 ± 3.8 nm), respectively (Aladdin). Specimens in group 1 were labeled as Q-300, Q-100 and Q25 according to the particle size. TiO2 of group 2 were treated with 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (PTES) (Sigma-Aldrich) before coating to reduce the surface energy of the particles and the specimens were labeled as S-300, S-100 and S-25, respectively. For group 1, TiO2 particles were placed in absolute ethanol in 5 wt%. For group 2, PTES was placed into absolute ethanol in 1 wt%, and the solution was mechanically stirred for 2 h before 5 wt% TiO2 particles was added in the solution. All the TiO2 suspensions were sonicated for 10 min, and mechanically stirred for 24 h. 2 g of glue (EVO-STIK serious glue) was added into 100 g absolute ethanol, and the solution was mechanically stirred for 10 min. The diluted glue were dropwise added onto the glasses, and dried in a drying stove in 70 °C for 3 h. The TiO2 suspensions were sprayed on the surface of glasses for 6 s with a distance of 400 mm under pressure of 0.8 MPa. In order to evaluate the thickness of coating on the wettability and antiadhesion, spraying times of 3 s, 6 s and 9 s were used in fabricating S-25 specimen. Then the glasses were dried in air for 10 min, and then dried in 100 °C for > 2 h. Cover glass (G group) and PTES-treated cover glasses (S-G group), which were immersed into 1 wt% PTES ethanol solution for 24 h, were set as control groups. 2.2. Characterization of the coatings Surface morphology of the coatings was determined using a Field Emission Scanning Electron Microscopy (SEM, JEOL) and Atom Force Microscope (AFM, Asylum Research) in tapping mode. The surface roughness parameters were also obtained by AFM. Energy dispersive xray spectrometry (EDX, Oxford) analysis was conducted to identify the chemical constituents of the coatings. White light interferometer (Nexview, Zygo) was employed to measure the thickness of the coatings after the coatings were scratched by a needle. Thickness was obtained by the height difference between coating surfaces and substrates (shown in Fig. 3). In order to characterize the wettability, the contact angles and roll-off angles of pure water were measured at room temperature via the sessile-drop method using an optical contact angle meter (Dataphysics). 4 μL and 10 μL drops were used in contact angle and roll-off angle tests, respectively. The measurements were repeated 3 times for each sample.
2. Materials and methods 2.1. Fabrication of coatings The specimens were divided into two groups based on surface 2
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2.3. Bacterial adhesion experiment
3.2. Bacteria adhesion
S. mutans ATCC 25715 was used in the adhesion experiments. A colony from a brain heart immersion (BHI) agar plate was inoculated into 10 mL of BHI broth, cultured for 24 h at 37 °C. Then it was inoculated a second culture grown for 16 h in 200 mL BHI broth. Bacteria were harvested by centrifugation (2400 g) for 10 min, and washed twice in sterile phosphate buffer solution (PBS). Bacteria were re-suspended to a concentration of 1× 107 CFU/mL in sterile PBS which was used as mediums in the adhesion experiments. The bacteria concentration was obtained by measuring the nephelometric turbidity unit (NTU) (based on a calibration curve of NTU vs CFU/mL). Bacterial aggregates were broken by mild sonication for 60 s. The specimens were put into 6 well cell culture clusters which were full of bacterial suspension and incubated for 3 h. In order to compare bacterial adhesion in the presence and absence of sucrose, 1 wt% sucrose was added in half of the samples. After incubation the specimens were picked up with a sterile forcep, mildly rinsed in sterile water to remove the loosely adhered bacteria. To quantify viable adherent bacteria, surfaces were subjected to a sonication in 10 mL of 0.9% PBS solution, serial dilutions performed and 10 μL from dilution were cultured in BHI agar in triplicate following the drop plate method [35]. Culture plates were incubated at 37 °C for 48 h and colony forming units (CFU) were counted. The entire bacterial adhesion experiment was repeated in 6 independent assays. The adhesion amounts were analyzed with two-way analysis of variance (ANOVA), followed by Tukey's HSD test at a significance level of α = 0.05 (SPSS Statistics ver.25, SAS, USA). In order to investigate the morphology of adhered surfaces, one specimen in each group was soaked in 2.5 wt% glutaraldehyde for 24 h. Then, they were dehydrated in 30 wt% sucrose water solution for 24 h, and were dehydrated in saturated sucrose water solution for 24 h. After coated with Pt, the specimens were observed with SEM.
The adhesion amounts of bacteria are shown in Fig. 5 with bar graph. Meanwhile, the contact angles of the surfaces are also exhibited with scatter and line. Two-way ANOVA revealed that adhesion quantity was significantly affected by sucrose and type of surfaces (p < .05). Adhesion quantities in PBS with sucrose were larger than that without sucrose. In both solutions superhydrophobic S-25 groups showed hardly any S. mutans in both environments, and Q-25 groups, which had the least contact angle, remained the most bacteria on the surfaces. However, in the presence of sucrose there were no significant differences between G, Q-300, Q-100 and their corresponding hydrophobic groups (S-G, S-300, S-100), respectively. Similar phenomenon was observed in the absence of sucrose, except a reduction of adhesion amount of S-100 compared with Q-100. For specimens without surface treatment the adhesion amount increased with the increase of wettability in both environments, but the PTES treated surfaces didn't show the similar results. For S-25 coatings with different thickness, no significant difference was found. The representative surfaces after adhesion are shown in Fig. 6. In the presence of sucrose EPS around the bacterial cells was observed, and the distribution of adhered bacteria was concentrated because EPS enhanced the interaction among bacteria. However, in the absence of sucrose the adhered bacteria were distributed scattered and no obvious EPS were detected. 4. Discussion As indicated in previous study [36] that the size of the morphology has an effect on the bacterial adhesion, we fabricated coatings with different size of TiO2 particles from 25 nm to 300 nm. The largest size of the TiO2 particles is approximately equal to the diameter of S. mutans. The chemical treatment combined with nanostructure produced a large range of wettability, which can be represented by contact angles ranging from 0 to 160°, leading to a different bacterial adhesion performance. Glass and TiO2 are naturally hydrophilic and have high surface energy. PTES can reduce their surface energy by its chemical contents. The PTES treatment on flat glass raised its contact angle from 15.1° to 62.3°. Attributed to the amplification effect of morphology, the 25 nm and 100 nm TiO2 particles made the Q-25 (contact angle is 0) and Q100 (11.9°) coatings more hydrophilic, and increased the hydrophobicity of S-25 (160°) and S-100 (130.6°) coatings. Especially S-25 coating was superhydrophobic and meanwhile had roll-off angle of < 1°. However, Q-300 (90.3°) didn't show the expected hydrophilicity as Q-25 and Q-100 did. It is speculated the nanostructure fabricated from 300 nm particles made the surface in mix wettability modes of Wenzel and Cassie [37], in which small amount of air pockets remained trapped within the texture, leading to the water being partly suspended above the surface (Fig. 7a). While droplets on Q-25 and Q100 coatings were in Wenzel state (Fig. 7b), in which the droplets penetrated into the surface asperities and completely wet the surface. In the solution containing sucrose, S. mutans produce EPS [32], which enhances the adhesion between the bacteria and coatings [33]. The interaction between EPS and solid surface plays the main role during the bacterial adhesion. While in no-sucrose solution, the interaction between bacterial surface protein and solid surface affords the main adhesion force, which is much smaller than the adhesion force between EPS and solid surface [33]. That is confirmed by the phenomenon in the present study that larger amount of bacterial adhesion can be seen in the sucrose-contained solution compared with no-sucrose environment. Possessing the same surface structures, untreated Glass (G) and 300 nm (Q-300) groups showed no significant differences on the adhesion amount with their corresponding chemically treated groups (S-G and S-300). It indicates surface energy alone has limited effect on the S. mutans adhesion. That is similar to the previous study [38,39], which
3. Results 3.1. Characterization of the coatings Fig. 2 presents typical surface morphology of the specimens observed by SEM, in which uniform surfaces were obtained. According to the EDX results in Fig. 2, Q-25 and S-25 had less Si (content of glass) and C (content of the glue) contents indicating they were more compact than other specimens. In hydrophobic groups S-25 specimens had the most content of F element, indicating it adsorbed the most hydrophobic grouping because the 25 nm particles have the largest specific surface area. The three-demensional surface morphology of the coatings obtained by AFM is shown in Fig. 3. Meanwhile, the surface roughness Ra was listed in Table 1. There were no significant differences of Ra between corresponding hydrophilic and hydrophobic groups with the same TiO2 diameter. Moreover, it can be seen that Ra increased with the decrease of TiO2 diameter. Thickness of the coatings is also listed in Table 1, in which 300 nm coatings have larger thickness than 100 nm and 25 nm coatings. A longer spraying time made the coating thicker, but didn't have significant influence on the surface roughness, contact angle and roll-off angle. The contact angle and roll-off angle of the specimens were listed in Table 1. Obviously, the PTES-treated surfaces exhibited larger contact angles than the non-treated surfaces. The smaller particle size enhanced the hydrophilicity and hydrophobicity for the two groups respectively. The Q-25 group had a superhydrophilic surface with contact angle of approximate 0 (Fig. 4A). The S-25 group had contact angle of 160° (Fig. 4B) and roll-off angle of < 1° (Fig. 4C), indicating a superhydrophobic performance. S-300 and S-100 group also showed hydrophobicity, but were sticky at the same time. Q-300 had a contact angle of 90.3°, indicating the roughness structure was not help to enlarge the hydrophilic property of TiO2. 3
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Fig. 2. Surface morphology and element content of the coatings.
means that the bacteria adhesion could not be prevented by only change of surface energy without morphology. For 25 nm and 100 nm surfaces, the apparent difference in adhesion amount between hydrophilic and hydrophobic groups is attributed to the wettability enlarged
significantly by the synergistic effect of surface morphologies and chemical modification. Nanostructure made of 25 nm TiO2 combined with PTES treatment (S-25) has a superhydrophobic property, which can inhibit S. mutans adhesion in solutions with sucrose or not. Previous
Fig. 3. Three-dimensional surface morphology of (a) Q-300, (b) Q-100, and (c) Q-25 obtained by AFM and measurement of coating thickness by white light interferometer. 4
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Table 1 Surface roughness, contact angle and roll-off angle of the coatings. Non-treated G
Surface roughness (nm) Thickness (μm) Contact angle (°) Roll-off angle (°) a
– – 15.1 ± 0.3 –
PTES-treated Q-300
86.5 ± 6.8 1.91 ± 0.11 90.3 ± 1.2 –
Q-100
148.7 ± 11.3 1.59 ± 0.06 11.9 ± 0.2 –
Q-25
163.5 ± 13.8 1.41 ± 0.09 0 –
S-G
S-300
– – 62.3 ± 0.9 –
79.9 ± 6.6 1.84 ± 0.13 135.7 ± 3.4 –
S-100
153.8 ± 10.5 1.56 ± 0.06 130.6 ± 3.8 –
S-25a 3s
6s
9s
163.7 ± 14.2 0.88 ± 0.04 159.3 ± 0.5 < 1
171.1 ± 12.4 1.45 ± 0.11 160.0 ± 0.7 < 1
170.3 ± 13.7 1.79 ± 0.11 160.7 ± 0.7 < 1
S-25 coatings with spraying time of 3 s, 6 s and 9 s are measured. Others were prepared with spraying time of 6 s.
Fig. 4. Contact angle of (A) Q-25 and (B) S-25 and (C) roll-off angle of S-25.
researches suggested some superhydrophilic polymer materials can reduce bacterial adhesion because the hydration layers prevented proteins or EPS from contacting [19]. However, superhydrophilic surfaces assembled with 25 nm particles (Q-25) didn't show the inhibition of adhesion in both solutions. That is because the superhydrophilic property of the coating is formed by the enlargement of surface roughness. TiO2 itself does not have such high surface energy that can produce strong hydration effect. It is generally acknowledged smooth surfaces are not conducive to bacterial adhesion because a rough surface, possessing greater surface area, provides more favourable sites for bacterial colonization, and the depressions have certain protecting effects for the bacteria [4]. Ra of the 6 groups of coatings were all < 0.2 μm, which was regarded as a threshold below which the bacterial adhesion would not be significantly influenced [5]. However, there were some difference in the adhesion between flat surfaces (G) and rough surfaces (Q-100, Q-25). The larger adhesion amount on the rougher surfaces indicates the assembled 25 nm and 100 nm coatings without surface treatment is failed to reduce bacterial adhesion. No obvious change from Q-300 to G is reasoned to be some air existing between Q-300 coatings and solution reduces the contact between the bacteria and surfaces. For the chemically treated coatings, the situations are more complex. It has been proved by the previous study that S. mutans have slightly larger adhesion force with hydrophobic surface because its surface protein has many kinds of hydrophobic groups [31]. But at the same time the air trapped within hydrophobic coatings tends to reduce the bacteria adhesion. Therefore, the quantity of bacteria on the hydrophobic coatings depends on the air amount which associated with wettability. The anti-adhesion mechanism of the coatings is illustrated in Fig. 7(c-e). Superhydrophobic S-25 surface has hardly any adhesion because the air trapped between solution and the coating produces a shelter that prevents the bacteria cell from contacting with the coating.
Fig. 5. Adhesion amounts of bacteria in (A) PBS and (B) PBS with 1 wt% sucrose. G: glass, Q-300~Q-25: 300 nm~25 nm TiO2 coatings; S-G: glass treated with PTES, S-300~S-25: 300 nm~25 nm TiO2 coatings treated with PTES. Same uppercase letters (without sucrose) or lowercase (with sucrose) letters represent no significant differences among different surfaces in the same solution (p > .05). S-25 coatings with spraying time of 3 s, 6 s and 9 s are displayed, and others were prepared with spraying time of 6 s.
S-300 surface, with larger particle size and less hydrophobicity than S25, has less quantity of air trapped in the tri-phase interface, providing more contact opportunity for bacteria and the coating. Superhydrophilic Q-25 coating has more contact area with bacteria than hydrophobic coatings because there is no air between the bacteria and coating. Consequently, the anti-adhesion effect can be achieved by ultra-low wettability surfaces which obtained by both surface 5
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Fig. 6. Bacterial adhesion in (A) PBS and (B) PBS with 1 wt% sucrose.
morphologies and chemical modification. Different from the commonly-used rod shape or bump surface structures, this study introduces an anti-adhesion nanostructured surface fabricated by assembly of TiO2 particles which has a simpler process than the previous nanostructure fabrication techniques, such as lithography, laser, electrochemical anodizing. The coating made by the process is water-resistant due to the water-resistant property of the glue. The process is capable of other substrate materials, and it is speculated TiO2 also can be replaced by other nanoparticles that achieve the similar effect. Moreover, based on the anti-adhesion mechanism, the assembled coatings can inhibit adhesion of other bacteria. It's worth noting that in some specific situation, the air may disappear from a superhydrophobic solid/liquid interface and change solid/liquid contact mode from Cassie's mode to Wenzel's mode. This may influence the anti-adhesion performance of the superhydrophobic coatings. In addition, adhesion amount could not fully represent adhesion strength between bacteria and coatings because of the interfacial air. Therefore, the adhesion strength and duration of air existence of the coatings should be studied in future.
5. Conclusions We have fabricated an anti-adhesion coating and explored the synergistic effects of nanostructure size and surface modification on bacteria adhesion. Within the limitations of this study, the following conclusions are drawn: 1) Uniform coatings made of different size of TiO2 were obtained by the assemble process. The coatings have different wettability with contact angles from 0 to 160°. The 25 nm TiO2 particle coating combined with chemical treatment has superhydrophobic property. 2) Surface energy treatment and surface morphology have synergistic effects on the adhesion of S. mutans. The superhydrophobic 25 nm TiO2 particle coating exhibits the best anti-adhesion performance in both cultivation environments with or without sucrose. 3) The anti-adhesion effect of the superhydrophobic surfaces is attributed to the air existed between solution and the coating produced a shelter that prevented the bacteria cell from contacting. The anti-adhesion function of the hydrophobic coatings depends on the air amount which associated with wettability.
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Fig. 7. Wetting behavior of a droplet on coatings (a) Q-300 Cassie's and Wenzel's mix mode (b) Q-100 Wenzel's mode, and mechanism of the bacterial adhesion on (c) S-25 superhydrophobic, (d) S-300 hydrophobic, and (e) Q-25 superhydrophilic coatings.
Funding
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Colloid and Interface Science Communications journal homepage: www.elsevier.com/locate/colcom
Influence of TiO2 nanostructure size and surface modification on surface wettability and bacterial adhesion Gaoqi Wanga,b, Ding Wenga, Chaolang Chena, Lei Chena, Jiadao Wanga,
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a b
State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, PR China School of mechanical engineering, University of Jinan, Jinan 250022, PR China
ARTICLE INFO
ABSTRACT
Keywords: Bacterial adhesion TiO2 Surface energy Surface morphology
The aim of the study was to explore the synergistic effects of nanostructure size and surface treatment of TiO2 coatings on its wettability and bacteria adhesion. The coatings were fabricated by TiO2 particles with a diameter ranging from 25 nm to 300 nm. The surface wettability of coatings was tuned by chemical treatment. The antiadhesion property of as-prepared coatings was evaluated with Streptococcus mutans in two types of solutions: phosphate buffer solution (PBS) and PBS with 1 wt% sucrose. The results show that particle size and surface treatment have significant influences on the wettability (with contact angles of 0–160°) and bacterial adhesion. The coating prepared by chemically modified 25 nm particles has a superhydrophobic property, and could inhibit bacterial adhesion in aqueous solutions with sucrose or not. The study also provides a facile, universal and low-cost process to fabricate anti-adhesion coating, which is applicable for diverse of substrates.
1. Introduction Bacterial adhesion on solid surfaces widely exists in nature, such as surgical tools, water filtration and ship hulls, resulting in infection or other negative impacts [1]. Generally, there are two types of antibacterial strategies: kill bacteria and reduce adhesion of bacteria. The commonly used anti-adhesion strategies contains the modification of surface morphology, changing surface wettability, and suppressing enzyme activity by natural or synthetic substances, and so on [2]. The surface morphology has been proved to have influence on the bacteria adhesion according to previous researches [3]. Quirynen et al. [4] found that rough surfaces promoted bacterial biofilm formation and maturation, and the influence of the surface roughness on plaque accumulation was more prominent than surface free energy. A further study conducted by Bollen et al. [5] indicated a smoothening below a Ra of 0.2 μm showed no further significant changes on the bacterial adhesion. In recent years, many researchers suggested surfaces with special morphology had reducing effect of bacterial adhesion [6–13]. Those micro- or nanostructures were usually fabricated by lithography, deposition, laser, electrochemical anodizing, sol-gel and so forth (Fig. 1). It is found that size of the morphology also has important effect on the bacterial adhesion. Wang et al. [14] showed adhesion of staphylococci was significantly reduced when the interpillar spacing was below 1.5 μm, which means the size of the surface patterns should be
⁎
smaller than that of a bacterium to reduce the contact area between the bacterium and surface. Various patterns of surface morphology may show different size effects. The surface morphology not only affects the contact situation between bacterium and surface, but also changes the wettability of surfaces. Wettability, which plays an important role on bacteria adhesion, represents the degree that material surface can be wet by a liquid. Surface roughness leads to an amplification of the wetting properties of the smooth material [15], increasing the hydrophilicity or hydrophobicity of material that was naturally hydrophilic or hydrophobic. According to previous researches, in general moderate wettable surfaces prefer to attract bacteria [12]. Superhydrophobic surfaces, typically fabricated by either covalently attaching molecules with low surface energies to a roughened surface or roughening the surface of a hydrophobic material [16,17], often inhibit bacteria adhesion due to their specific self-cleaning property [18]. Additionally, some hydrophilic or superhydrophilic materials, such as poly(ethylene glycols), p (N-hydroxyethylacrylamide) and zwitterionic polymers also exhibited anti-adhesion effect due to their strong electrostatically induced hydration layers that construct a steric barrier to adhesion [19–21]. However, a few publications suggested some superhydrophobic surfaces induce more bacterial adhesion, such as poly(L-lactic acid) [22]. Therefore, it is highly needed to furtherly investigate the bacterial adhesion property of superhydrophobic surface.
Corresponding author at: State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, PR China. E-mail address: [email protected] (J. Wang).
https://doi.org/10.1016/j.colcom.2019.100220 Received 23 September 2019; Received in revised form 5 November 2019; Accepted 11 November 2019 2215-0382/ © 2019 Published by 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: Gaoqi Wang, et al., Colloid and Interface Science Communications, https://doi.org/10.1016/j.colcom.2019.100220
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Fig. 1. Anti-adhesion surfaces made by different methods [6–13]
Recently, various coatings made of particles such as TiO2 [23], Cu2O [24], ZnO [25] and Ag [26] were applied in antibacterial coatings. TiO2 was frequently used because it demonstrates nontoxic, biocompatible, inexpensive and photo induced antibacterial nature [27,28], which can kill bacteria by highly reactive species. TiO2 is mainly active with ultraviolet light and researches are carrying out in activating TiO2 using visible light and enhance photocatalytic activity by doped with metals, metal oxides, non-metal or organosilane compounds [29]. Therefore, TiO2 particle was hardly solely used to fabricate coatings, and usually used as addition incorporated into coatings to kill bacteria [30]. Few studies focused on the anti-adhesion effect of the coatings assembled with the nanoparticles. The combinational impact of particle size and surface energy on the wettability and bacterial adhesion remains to be explored. In the absence of sucrose, the sucrose-independent adhesion between bacteria and surfaces relies on bacterial surface protein [31]. While in the environment with sucrose, Gram-positive bacteria produce extracellular polymeric substances (EPS) from sucrose [32]. EPS is composed of multiple components, including polysaccharides, proteins, and extracellular DNA (eDNA) [33]. The sucrose-dependent bacterial adhesion is mainly mediated by EPS, which could enhance the adhesion between the bacteria and solid surfaces [34]. Thus, the bacterial adhesion situation on the particle assembled coatings may be different due to the existence of sucrose. In this study the synergistic effects of nanostructure and surface modification of assembled TiO2 coating on its wettability and bacteria adhesion were explored. 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (PTES) was used to modify TiO2 with lower surface energy. A typical Gram-positive Bacterium Streptococcus mutans (S. mutans) was used in the adhesion evaluation under two environments with and without sucrose. The TiO2 coating is simply-made and could be assembled on other materials, such as metal, ceramic and resin. Because the antiadhesion effect is produced mainly by the nanostructure and surface energy properties, the TiO2 also could be replaced by other particles, such as SiO2 and PTFE. The strategy provides new ideas for developing novel anti-adhesion surfaces.
modification. Each group contained three kinds of coatings with different TiO2 particle sizes of approximately 300 nm (293.0 ± 28.1 nm), 100 nm (103.1 ± 17.4 nm), and 25 nm (25.7 ± 3.8 nm), respectively (Aladdin). Specimens in group 1 were labeled as Q-300, Q-100 and Q25 according to the particle size. TiO2 of group 2 were treated with 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (PTES) (Sigma-Aldrich) before coating to reduce the surface energy of the particles and the specimens were labeled as S-300, S-100 and S-25, respectively. For group 1, TiO2 particles were placed in absolute ethanol in 5 wt%. For group 2, PTES was placed into absolute ethanol in 1 wt%, and the solution was mechanically stirred for 2 h before 5 wt% TiO2 particles was added in the solution. All the TiO2 suspensions were sonicated for 10 min, and mechanically stirred for 24 h. 2 g of glue (EVO-STIK serious glue) was added into 100 g absolute ethanol, and the solution was mechanically stirred for 10 min. The diluted glue were dropwise added onto the glasses, and dried in a drying stove in 70 °C for 3 h. The TiO2 suspensions were sprayed on the surface of glasses for 6 s with a distance of 400 mm under pressure of 0.8 MPa. In order to evaluate the thickness of coating on the wettability and antiadhesion, spraying times of 3 s, 6 s and 9 s were used in fabricating S-25 specimen. Then the glasses were dried in air for 10 min, and then dried in 100 °C for > 2 h. Cover glass (G group) and PTES-treated cover glasses (S-G group), which were immersed into 1 wt% PTES ethanol solution for 24 h, were set as control groups. 2.2. Characterization of the coatings Surface morphology of the coatings was determined using a Field Emission Scanning Electron Microscopy (SEM, JEOL) and Atom Force Microscope (AFM, Asylum Research) in tapping mode. The surface roughness parameters were also obtained by AFM. Energy dispersive xray spectrometry (EDX, Oxford) analysis was conducted to identify the chemical constituents of the coatings. White light interferometer (Nexview, Zygo) was employed to measure the thickness of the coatings after the coatings were scratched by a needle. Thickness was obtained by the height difference between coating surfaces and substrates (shown in Fig. 3). In order to characterize the wettability, the contact angles and roll-off angles of pure water were measured at room temperature via the sessile-drop method using an optical contact angle meter (Dataphysics). 4 μL and 10 μL drops were used in contact angle and roll-off angle tests, respectively. The measurements were repeated 3 times for each sample.
2. Materials and methods 2.1. Fabrication of coatings The specimens were divided into two groups based on surface 2
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2.3. Bacterial adhesion experiment
3.2. Bacteria adhesion
S. mutans ATCC 25715 was used in the adhesion experiments. A colony from a brain heart immersion (BHI) agar plate was inoculated into 10 mL of BHI broth, cultured for 24 h at 37 °C. Then it was inoculated a second culture grown for 16 h in 200 mL BHI broth. Bacteria were harvested by centrifugation (2400 g) for 10 min, and washed twice in sterile phosphate buffer solution (PBS). Bacteria were re-suspended to a concentration of 1× 107 CFU/mL in sterile PBS which was used as mediums in the adhesion experiments. The bacteria concentration was obtained by measuring the nephelometric turbidity unit (NTU) (based on a calibration curve of NTU vs CFU/mL). Bacterial aggregates were broken by mild sonication for 60 s. The specimens were put into 6 well cell culture clusters which were full of bacterial suspension and incubated for 3 h. In order to compare bacterial adhesion in the presence and absence of sucrose, 1 wt% sucrose was added in half of the samples. After incubation the specimens were picked up with a sterile forcep, mildly rinsed in sterile water to remove the loosely adhered bacteria. To quantify viable adherent bacteria, surfaces were subjected to a sonication in 10 mL of 0.9% PBS solution, serial dilutions performed and 10 μL from dilution were cultured in BHI agar in triplicate following the drop plate method [35]. Culture plates were incubated at 37 °C for 48 h and colony forming units (CFU) were counted. The entire bacterial adhesion experiment was repeated in 6 independent assays. The adhesion amounts were analyzed with two-way analysis of variance (ANOVA), followed by Tukey's HSD test at a significance level of α = 0.05 (SPSS Statistics ver.25, SAS, USA). In order to investigate the morphology of adhered surfaces, one specimen in each group was soaked in 2.5 wt% glutaraldehyde for 24 h. Then, they were dehydrated in 30 wt% sucrose water solution for 24 h, and were dehydrated in saturated sucrose water solution for 24 h. After coated with Pt, the specimens were observed with SEM.
The adhesion amounts of bacteria are shown in Fig. 5 with bar graph. Meanwhile, the contact angles of the surfaces are also exhibited with scatter and line. Two-way ANOVA revealed that adhesion quantity was significantly affected by sucrose and type of surfaces (p < .05). Adhesion quantities in PBS with sucrose were larger than that without sucrose. In both solutions superhydrophobic S-25 groups showed hardly any S. mutans in both environments, and Q-25 groups, which had the least contact angle, remained the most bacteria on the surfaces. However, in the presence of sucrose there were no significant differences between G, Q-300, Q-100 and their corresponding hydrophobic groups (S-G, S-300, S-100), respectively. Similar phenomenon was observed in the absence of sucrose, except a reduction of adhesion amount of S-100 compared with Q-100. For specimens without surface treatment the adhesion amount increased with the increase of wettability in both environments, but the PTES treated surfaces didn't show the similar results. For S-25 coatings with different thickness, no significant difference was found. The representative surfaces after adhesion are shown in Fig. 6. In the presence of sucrose EPS around the bacterial cells was observed, and the distribution of adhered bacteria was concentrated because EPS enhanced the interaction among bacteria. However, in the absence of sucrose the adhered bacteria were distributed scattered and no obvious EPS were detected. 4. Discussion As indicated in previous study [36] that the size of the morphology has an effect on the bacterial adhesion, we fabricated coatings with different size of TiO2 particles from 25 nm to 300 nm. The largest size of the TiO2 particles is approximately equal to the diameter of S. mutans. The chemical treatment combined with nanostructure produced a large range of wettability, which can be represented by contact angles ranging from 0 to 160°, leading to a different bacterial adhesion performance. Glass and TiO2 are naturally hydrophilic and have high surface energy. PTES can reduce their surface energy by its chemical contents. The PTES treatment on flat glass raised its contact angle from 15.1° to 62.3°. Attributed to the amplification effect of morphology, the 25 nm and 100 nm TiO2 particles made the Q-25 (contact angle is 0) and Q100 (11.9°) coatings more hydrophilic, and increased the hydrophobicity of S-25 (160°) and S-100 (130.6°) coatings. Especially S-25 coating was superhydrophobic and meanwhile had roll-off angle of < 1°. However, Q-300 (90.3°) didn't show the expected hydrophilicity as Q-25 and Q-100 did. It is speculated the nanostructure fabricated from 300 nm particles made the surface in mix wettability modes of Wenzel and Cassie [37], in which small amount of air pockets remained trapped within the texture, leading to the water being partly suspended above the surface (Fig. 7a). While droplets on Q-25 and Q100 coatings were in Wenzel state (Fig. 7b), in which the droplets penetrated into the surface asperities and completely wet the surface. In the solution containing sucrose, S. mutans produce EPS [32], which enhances the adhesion between the bacteria and coatings [33]. The interaction between EPS and solid surface plays the main role during the bacterial adhesion. While in no-sucrose solution, the interaction between bacterial surface protein and solid surface affords the main adhesion force, which is much smaller than the adhesion force between EPS and solid surface [33]. That is confirmed by the phenomenon in the present study that larger amount of bacterial adhesion can be seen in the sucrose-contained solution compared with no-sucrose environment. Possessing the same surface structures, untreated Glass (G) and 300 nm (Q-300) groups showed no significant differences on the adhesion amount with their corresponding chemically treated groups (S-G and S-300). It indicates surface energy alone has limited effect on the S. mutans adhesion. That is similar to the previous study [38,39], which
3. Results 3.1. Characterization of the coatings Fig. 2 presents typical surface morphology of the specimens observed by SEM, in which uniform surfaces were obtained. According to the EDX results in Fig. 2, Q-25 and S-25 had less Si (content of glass) and C (content of the glue) contents indicating they were more compact than other specimens. In hydrophobic groups S-25 specimens had the most content of F element, indicating it adsorbed the most hydrophobic grouping because the 25 nm particles have the largest specific surface area. The three-demensional surface morphology of the coatings obtained by AFM is shown in Fig. 3. Meanwhile, the surface roughness Ra was listed in Table 1. There were no significant differences of Ra between corresponding hydrophilic and hydrophobic groups with the same TiO2 diameter. Moreover, it can be seen that Ra increased with the decrease of TiO2 diameter. Thickness of the coatings is also listed in Table 1, in which 300 nm coatings have larger thickness than 100 nm and 25 nm coatings. A longer spraying time made the coating thicker, but didn't have significant influence on the surface roughness, contact angle and roll-off angle. The contact angle and roll-off angle of the specimens were listed in Table 1. Obviously, the PTES-treated surfaces exhibited larger contact angles than the non-treated surfaces. The smaller particle size enhanced the hydrophilicity and hydrophobicity for the two groups respectively. The Q-25 group had a superhydrophilic surface with contact angle of approximate 0 (Fig. 4A). The S-25 group had contact angle of 160° (Fig. 4B) and roll-off angle of < 1° (Fig. 4C), indicating a superhydrophobic performance. S-300 and S-100 group also showed hydrophobicity, but were sticky at the same time. Q-300 had a contact angle of 90.3°, indicating the roughness structure was not help to enlarge the hydrophilic property of TiO2. 3
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Fig. 2. Surface morphology and element content of the coatings.
means that the bacteria adhesion could not be prevented by only change of surface energy without morphology. For 25 nm and 100 nm surfaces, the apparent difference in adhesion amount between hydrophilic and hydrophobic groups is attributed to the wettability enlarged
significantly by the synergistic effect of surface morphologies and chemical modification. Nanostructure made of 25 nm TiO2 combined with PTES treatment (S-25) has a superhydrophobic property, which can inhibit S. mutans adhesion in solutions with sucrose or not. Previous
Fig. 3. Three-dimensional surface morphology of (a) Q-300, (b) Q-100, and (c) Q-25 obtained by AFM and measurement of coating thickness by white light interferometer. 4
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Table 1 Surface roughness, contact angle and roll-off angle of the coatings. Non-treated G
Surface roughness (nm) Thickness (μm) Contact angle (°) Roll-off angle (°) a
– – 15.1 ± 0.3 –
PTES-treated Q-300
86.5 ± 6.8 1.91 ± 0.11 90.3 ± 1.2 –
Q-100
148.7 ± 11.3 1.59 ± 0.06 11.9 ± 0.2 –
Q-25
163.5 ± 13.8 1.41 ± 0.09 0 –
S-G
S-300
– – 62.3 ± 0.9 –
79.9 ± 6.6 1.84 ± 0.13 135.7 ± 3.4 –
S-100
153.8 ± 10.5 1.56 ± 0.06 130.6 ± 3.8 –
S-25a 3s
6s
9s
163.7 ± 14.2 0.88 ± 0.04 159.3 ± 0.5 < 1
171.1 ± 12.4 1.45 ± 0.11 160.0 ± 0.7 < 1
170.3 ± 13.7 1.79 ± 0.11 160.7 ± 0.7 < 1
S-25 coatings with spraying time of 3 s, 6 s and 9 s are measured. Others were prepared with spraying time of 6 s.
Fig. 4. Contact angle of (A) Q-25 and (B) S-25 and (C) roll-off angle of S-25.
researches suggested some superhydrophilic polymer materials can reduce bacterial adhesion because the hydration layers prevented proteins or EPS from contacting [19]. However, superhydrophilic surfaces assembled with 25 nm particles (Q-25) didn't show the inhibition of adhesion in both solutions. That is because the superhydrophilic property of the coating is formed by the enlargement of surface roughness. TiO2 itself does not have such high surface energy that can produce strong hydration effect. It is generally acknowledged smooth surfaces are not conducive to bacterial adhesion because a rough surface, possessing greater surface area, provides more favourable sites for bacterial colonization, and the depressions have certain protecting effects for the bacteria [4]. Ra of the 6 groups of coatings were all < 0.2 μm, which was regarded as a threshold below which the bacterial adhesion would not be significantly influenced [5]. However, there were some difference in the adhesion between flat surfaces (G) and rough surfaces (Q-100, Q-25). The larger adhesion amount on the rougher surfaces indicates the assembled 25 nm and 100 nm coatings without surface treatment is failed to reduce bacterial adhesion. No obvious change from Q-300 to G is reasoned to be some air existing between Q-300 coatings and solution reduces the contact between the bacteria and surfaces. For the chemically treated coatings, the situations are more complex. It has been proved by the previous study that S. mutans have slightly larger adhesion force with hydrophobic surface because its surface protein has many kinds of hydrophobic groups [31]. But at the same time the air trapped within hydrophobic coatings tends to reduce the bacteria adhesion. Therefore, the quantity of bacteria on the hydrophobic coatings depends on the air amount which associated with wettability. The anti-adhesion mechanism of the coatings is illustrated in Fig. 7(c-e). Superhydrophobic S-25 surface has hardly any adhesion because the air trapped between solution and the coating produces a shelter that prevents the bacteria cell from contacting with the coating.
Fig. 5. Adhesion amounts of bacteria in (A) PBS and (B) PBS with 1 wt% sucrose. G: glass, Q-300~Q-25: 300 nm~25 nm TiO2 coatings; S-G: glass treated with PTES, S-300~S-25: 300 nm~25 nm TiO2 coatings treated with PTES. Same uppercase letters (without sucrose) or lowercase (with sucrose) letters represent no significant differences among different surfaces in the same solution (p > .05). S-25 coatings with spraying time of 3 s, 6 s and 9 s are displayed, and others were prepared with spraying time of 6 s.
S-300 surface, with larger particle size and less hydrophobicity than S25, has less quantity of air trapped in the tri-phase interface, providing more contact opportunity for bacteria and the coating. Superhydrophilic Q-25 coating has more contact area with bacteria than hydrophobic coatings because there is no air between the bacteria and coating. Consequently, the anti-adhesion effect can be achieved by ultra-low wettability surfaces which obtained by both surface 5
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Fig. 6. Bacterial adhesion in (A) PBS and (B) PBS with 1 wt% sucrose.
morphologies and chemical modification. Different from the commonly-used rod shape or bump surface structures, this study introduces an anti-adhesion nanostructured surface fabricated by assembly of TiO2 particles which has a simpler process than the previous nanostructure fabrication techniques, such as lithography, laser, electrochemical anodizing. The coating made by the process is water-resistant due to the water-resistant property of the glue. The process is capable of other substrate materials, and it is speculated TiO2 also can be replaced by other nanoparticles that achieve the similar effect. Moreover, based on the anti-adhesion mechanism, the assembled coatings can inhibit adhesion of other bacteria. It's worth noting that in some specific situation, the air may disappear from a superhydrophobic solid/liquid interface and change solid/liquid contact mode from Cassie's mode to Wenzel's mode. This may influence the anti-adhesion performance of the superhydrophobic coatings. In addition, adhesion amount could not fully represent adhesion strength between bacteria and coatings because of the interfacial air. Therefore, the adhesion strength and duration of air existence of the coatings should be studied in future.
5. Conclusions We have fabricated an anti-adhesion coating and explored the synergistic effects of nanostructure size and surface modification on bacteria adhesion. Within the limitations of this study, the following conclusions are drawn: 1) Uniform coatings made of different size of TiO2 were obtained by the assemble process. The coatings have different wettability with contact angles from 0 to 160°. The 25 nm TiO2 particle coating combined with chemical treatment has superhydrophobic property. 2) Surface energy treatment and surface morphology have synergistic effects on the adhesion of S. mutans. The superhydrophobic 25 nm TiO2 particle coating exhibits the best anti-adhesion performance in both cultivation environments with or without sucrose. 3) The anti-adhesion effect of the superhydrophobic surfaces is attributed to the air existed between solution and the coating produced a shelter that prevented the bacteria cell from contacting. The anti-adhesion function of the hydrophobic coatings depends on the air amount which associated with wettability.
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Fig. 7. Wetting behavior of a droplet on coatings (a) Q-300 Cassie's and Wenzel's mix mode (b) Q-100 Wenzel's mode, and mechanism of the bacterial adhesion on (c) S-25 superhydrophobic, (d) S-300 hydrophobic, and (e) Q-25 superhydrophilic coatings.
Funding
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