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High Aspect Ratio Nanostructures Kill Bacteria via Storage and Release of Mechanical Energy Denver P. Linklater, Michael De Volder, Vladimir A Baulin, Marco Werner, Sarah Jessl, Mehdi Golozar, Laura Maggini, Sergey Rubanov, Eric Hanssen, Saulius Juodkazis, and Elena P. Ivanova ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01665 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018
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30 µm VACNT
c)
a)
Vertically Aligned CNT (VACNT) Forest
e) L= 1 µm
Dead cell
1 µm
L= 2 µm L= 3 µm L= 10 µm L= 30 µm
c)
a)
b)
d)
e) 10 µm
1 µm
1 µm VACNT
b)
d)
10 µm
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High Aspect Ratio Nanostructures Kill Bacteria via Storage and Release of Mechanical Energy Denver P. Linklater1,3, Michael De Volder2, Vladimir A. Baulin4, Marco Werner,4 Sarah Jessl2, Mehdi Golozar2, Laura Maggini2, Sergey Rubanov5, Eric Hanssen5, Saulius Juodkazis3 and Elena P. Ivanova1,6* 1
Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, VIC 3122,
Australia 2
Institute for Manufacturing, Department of Engineering, University of Cambridge, Cambridge CB3
0FS, UK 3
Centre for Micro-Photonics and Industrial Research Institute Swinburne, Faculty of Science,
Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, 3122, Australia 4
Department d’Enginyeria Quimica, Universitat Rovira, Virgili, 26 Av. dels Paisos Catalans, 43007
Tarragona, Spain 5
Advanced Microscopy Facility, Bio21 Institute, University of Melbourne, 30 Flemington Rd, 3010,
Victoria, Australia 6
School of Science, College of Science, Engineering and Health, RMIT University, GPO Box 2476,
Melbourne, Victoria 3001, Australia
KEYWORDS: Carbon nanotubes, vertically aligned carbon nanotubes, mechanobactericidal mechanism, storage of elastic energy, interface interactions, nanoscale mechanics
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ABSTRACT
The threat of a global rise in the number of untreatable infections caused by antibiotic resistant bacteria calls for the design and fabrication of a new generation of bactericidal materials. Here, we report a concept for the design of antibacterial surfaces, whereby cell death results from the ability of the nanofeatures to deflect when in contact with attaching cells. We show, using three-dimensional transmission electron microscopy, that the exceptionally high aspect ratio (100 – 3000) of vertically aligned carbon nanotubes (VACNTs) imparts extreme flexibility, which enhances the elastic energy storage in CNTs as they bend in contact with bacteria. Our experimental and theoretical analyses demonstrate that, for high aspect ratio structures, the bending energy stored in the CNTs is a substantial factor for the physical rupturing of both Gram-positive and Gram-negative bacteria. The highest bactericidal rates (99.3% for Pseudomonas aeruginosa and 84.9% for Staphylococcus aureus) were obtained by modifying the length of the VACNTs, allowing us to identify the optimal substratum properties to kill different types of bacteria efficiently. This work highlights that the bactericidal activity of high aspect ratio nanofeatures can outperform both natural bactericidal surfaces and other synthetic nanostructured multifunctional surfaces reported in previous studies. The present systems exhibit the highest bactericidal activity of a CNT-based substratum against a Gram-negative bacterium reported to date, suggesting the possibility of achieving close to 100% bacterial inactivation on VACNT-based substrata.
Bactericidal surface modifications have long been used to prevent the formation of bacterial biofilms on a variety of surfaces, including medical tools and implants.1 The control of the physico-chemical properties on the micro-nano scales has led to the development of anti-biofouling surfaces and greater regulation of bacterial attachment and biofilm formation.2 These surfaces generally rely on a chemical coating which slowly releases a biocidal agent and kills bacteria that come into contact. However, this ii ACS Paragon Plus Environment
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approach has some draw-backs since the bacteria deeper within a biofilm are not exposed to the biocide and may develop resistance over prolonged periods of exposure, leading to the increase in antibiotic resistance in pathogenic strains of bacteria as the biocidal agents in the coating deplete.3 Following the discovery of the highly bactericidal nature of insect wings surfaces,4 the bactericidal activity of nanostructured surfaces has recently become a subject of intense investigation and promises to play a large role in combatting the emerging worldwide epidemic of bacterial resistance to antibiotics.4-6 For example, studies of cicada wings have shown that they are covered with arrays of pillars of approximately 200 nm in height and 60 nm in diameter.7 The mechanism of the antibacterial activity of these surfaces has been described in terms of purely physical mechano-bactericidal effects, which are understood to be the cause of bacterial cell death.8 The corresponding bactericidal mechanism involves the stretching and rupture of the cell membrane between the pillars (at the point subjected to the highest mechanical stress), and the pillars are not expected to substantially deflect upon contact with bacteria. This process has been studied using both experimental
5
and theoretical
6
approaches to define the
events occurring at the cell-substrate interface that eventually lead to cell death. Physical killing of Gram-negative bacterial cells, such as Pseudomonas aeruginosa, was initially observed on cicada wings; 4-5 whereas Gram-positive bacteria (e.g Staphylococcus aureus), have been observed to resist this stretching process better, which might be due to differences in the mechanical properties of the cell membrane.9 Subtle changes in the wing nanoarchitecture observed across dragonfly species lead to considerable changes in bactericidal activity.10 Likewise, synthetic surfaces patterned to mimic natural specimens also exhibit similar variations in bactericidal activity.9,
11-12
The bactericidal efficiency of
nanostructured surfaces is influenced by the spacing and dimensions of the nanofeatures; suggesting that optimization of the nanopattern can thus be used to increase the bactericidal activity against a wider range of bacteria, including Gram-positive and bacterial spores.13
Finally, surface chemistry and
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hydrophobicity have been shown to have negligible impact on the bactericidal mechanism of these nanostructured substrates.8, 13 Carbon nanotubes (CNTs) are very attractive nanomaterials for biotechnology due to their unique geometric (high aspect ratio, uniform diameter), physical (strength and stiffness of individual nanotubes, electrical and thermal conductivity), and chemical (controllable surface moieties) characteristics.14-18 CNTs have been tested in several biotechnology applications, including monitoring the electrical activity of electrogenic cells,19-20 as scaffolds for cell growth,21 and as antimicrobial agents.22-23 Most current studies on the antimicrobial properties of CNTs (Table S1) are performed on CNTs dispersed in suspension, whereby CNT ‘needles’ are hypothesized to act as darts that pierce bacterial cells, however, the physical interaction between bacterial cells in suspension compared to incubation on a nanostructured surface is quite different, as shown by this study. In media, CNTs and bacterial cells are proposed to physical piercing leading to a loss of cell viability;23-26 moreover, chemical functionalization of CNTs by plasma treatment was found to enhance cell-CNT contacts through greater attractive forces between the cell and CNTs.27-29 Using CNT suspensions has some significant downsides compared to solid substrata for the testing of antimicrobial activity. For instance, high concentrations of CNTs are required in order to form aggregates with the cells and CNTs often aggregate with each other rather than with bacterial cells.24 Therefore, decreased performance of CNTs in solution has motivated the chemical functionalization of CNTs to enhance cell - CNT contact and to increase the bactericidal effects. Furthermore, for many antibacterial applications, antibacterial surfaces are preferred over suspensions. For these applications, arrays of vertically aligned carbon nanotube (VACNT) forests have been proposed.30 For example, a VACNT nanoarray with Ag nanoparticle ‘tips’ was used to enhance the bactericidal properties of multiwalled CNTs. Ag-functionalized VACNTs inactivated 93 % of E. coli in comparison to 42 % by pristine VACNTs.31 VACNT membranes have also been used for water purification, exhibiting high water flux and increased antibiofouling properties iv ACS Paragon Plus Environment
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compared to polymer membranes.32 The mechanism of VACNT-mediated killing has, however, not previously been identified, and therefore, there is a need to investigate the bactericidal mechanism of VACNTs, which is determined by their unique physico-chemical properties. The aim of this work was to design surfaces with high aspect ratio nanotubes in order to investigate their antibacterial activity and determine the particular mechanism contributing to bactericidal efficiency. Here, we report the discovery of another class of mechanobactericidal surfaces, whose activity is associated with the elastic properties of densely grafted VACNTs. While the VACNTs possess similar functionalities to dispersed CNTs, their immobilization onto a surface and their vertical alignment provide them with distinctive anisotropic mechanical properties, high tensile strength, extraordinary flexibility and resilience. By modifying both the length and the surface chemistry of the CNTs, our VACNTs allow for an effective control over the elastic energy stored in the nanotubes and their interaction with bacteria. The bactericidal effect of VACNTs is elucidated by in-depth cell viability analysis, along with focused ion beam – scanning electron microscopy (FIB-SEM), scanning transmission electron microscopy (STEM) tomography, and mechanical modelling studies. Results and Discussion Characterization of VACNT nanoarrays VACNTs consist of high aspect ratio (>1000) nanotubes whose bending stiffness can be easily controlled by their length.15,
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To determine the optimal VACNT height for bacterial killing, we
synthesised VACNTs of different height (see materials and methods). The height of the multi-walled VACNTs was controlled by their synthesis time: a growth time of 20 s resulted in forests of nanotubes of approximately 1 µm in height (Figure 1a), whereas CNT heights of approximately 30 µm were obtained with a growth time of 60 s (Figure 1b, Table 1, Figure S1). An advantage of VACNTs over CNT powders is that their surface can be easily modified by plasma treatment, which allows for the v ACS Paragon Plus Environment
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modification of the surface chemistry and hence the wettability of the CNT arrays while preserving their vertical alignment.35-37 Substratum hydrophobicity/hydrophilicity is known to affect the attachment of bacterial cells. Here, pristine VACNT forests, due to their surface density and specific alignment, showed a water contact angle of approximately 150° (Table 1). After oxygen plasma treatment of the VACNT arrays for 1 and 10 min, carbonyl and other oxygen groups are introduced on the CNT sidewalls,36 resulting in contact angles of 21° and 45°, respectively (Table 1). Following plasma treatment, the VACNTs showed some bundling of the CNT tips (Figure S1b), in agreement with previous results.38-39 To increase the hydrophobicity of the VACNT surfaces, CF4 plasma treatment was used to attach fluorine atoms to the sidewalls of the CNTs through C-F bonds.40-41 As shown in Table 1, the CF4 plasma-treated VACNTs show contact angles of approximately 137° for 1 µm high and 147° for 30 µm high CNTs. Some authors reported subtle changes in the morphology of CNTs after exposure to CF4 plasma for long periods of time;41 however, the present VACNT surfaces were exposed to CF4 plasma for periods of only 10 or 40 s, and no morphological changes were observed in the SEM images (Figure S1).
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Figure 1. SEM images contrasting the heights of the high aspect ratio nanotubes (a) 1 µm and (b) 30 µm VACNTs. False colour SEM images of (c) S. aureus and (d) P. aeruginosa attached onto VACNT surfaces, revealing the bending of the CNTs and deformation of the bacterial cell membrane. All scale bars are 1 µm, unless otherwise indicated.
To determine whether treatment with CF4 plasma induced any changes in the graphitic structure of the CNT nanoarrays, we performed Raman and XPS measurements of the treated nanoarrays (Figure S6, Table S2). The Raman spectra (Figure S6) showed that the ID/IG ratio increased from 0.58 (30 µm pristine samples) to 0.90 (30 µm CF4 plasma-treated samples) to 1.41 (30 µm O2 plasma-treated samples), indicating an increase in the number of defects in the graphitic structure of the treated CNTs. The same trend was observed for the 1 µm CNT samples, whose ID/IG ratio increased from 0.27 (pristine) to 1.02 (CF4 plasma-treated) to 1.31 (O2 plasma-treated). The increase in the ID/IG ratio for the tall CNT nanoarrays was accompanied by a reduction in the intensity of the 2D peak. These trends were confirmed by XPS analysis which showed a decrease in the ratio of sp2- to sp3-bonded carbon atoms, upon both O2 and CF4 plasma-treatment (Figures S6 (c, d) and S7-9). Bactericidal activity of VACNT arrays To assess the effects of nanotube height and surface treatment (and corresponding hydrophilic properties of the substratum) on bacterial attachment and bactericidal efficiency, tall and short VACNTs with either hydrophobic or hydrophilic surfaces, as identified by the analyses described in the previous section, were tested against Gram-negative P. aeruginosa and Gram-positive S. aureus bacterial cells. The bacterial cell viability of different substrates was analysed over an incubation period of 18 h. The results highlight a high bactericidal activity for all VACNT arrays towards both Gram-negative and Grampositive bacteria. Confocal laser scanning microscopy (CLSM) confirmed cell inactivation through the
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use of two dyes: Propidium iodide and Syto 9®. Syto 9® is a green fluorescing membrane permeable stain that binds to nucleic acids, whereas Propidium iodide fluoresces red and can only enter the cell and bind to nucleic acids upon significant membrane perturbation. CLSM micrographs in Figure 2 show a large percentage of red (dead) cells for P. aeruginosa cells incubated on both 1 µm and 30 µm VACNTs. CLSM micrographs of S. aureus incubated on 30 µm VACNTs show significant amounts of green (viable) cells, confirming, as expected, that Gram-positive bacteria are more resistant to mechanical rupturing. A quantitative analysis of cell viability is analysed in the next section. Finally, we used electron microscopy (SEM and TEM) to examine the morphology of bacteria on the VACNTs, following cell fixation and critical point drying (see methods). The analysis showed that the nanotubes did not encapsulate the bacteria (especially for S. aureus) (Figure 2). Due to the nature of the VACNT fabrication process and high surface density of the resultant nanotubes, the voids present in the CNT surfaces are much smaller than the size of both P. aeruginosa and S. aureus bacteria, which prevents the cells from penetrating deeper into the CNT surface. The nanotubes are clearly observed to interact with P. aeruginosa cells, and an altered cell morphology is evident in the SEM micrographs in (Figure 2 a, c and e) due to stretching of the cell compared with the cell interacting with plain silicon controls (Figure S1). These results suggest that cell death is likely to have occurred due to elastic forces exerted by CNTs on bacterial cells (see below). Effect of VACNT height and surface hydrophobicity on bactericidal activity and bacterial attachment Rates of cell inactivation and quantification of cellular attachment were obtained through CLSM and SEM pictures and image analysis. VACNT surfaces possessed notable selectivity towards the two bacterial species and a significant difference in bactericidal efficacy was noted for 1 µm high as compared to 30 µm high VACNTs (Figure 3). The analysis of the bactericidal activity of VACNT surfaces indicated that S. aureus was more efficiently killed by 1 µm VACNTs (Figure 3c, graphs viii ACS Paragon Plus Environment
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reporting the percentage of non-viable cells), i.e., for pristine CNTs, shorter nanotube arrays killed approximately 67% of attached bacteria compared to approximately 17% for 30 µm VACNTs. This trend was also detected for plasma treated VACNTs (CF4 30 µm – 15%/CF4 1 µm– 85% and O2 30 µm – 9%/O2 1µm – 50%). Statistical analysis using a standard T-test showed that there was a statistically significant difference between the percentage of damaged S. aureus cells on short and tall VACNT arrays, respectively, however less difference was observed between damaged P. aeruginosa cells for all surfaces; substantial elimination of P. aeruginosa cells was achieved on all types of VACNT arrays (Figure 3a). While 1 µm pristine VACNT arrays had slightly lower bactericidal activity towards P. aeruginosa (75%) than 30 µm arrays, the latter possessed considerable antibacterial activity against these bacteria, attaining 99.36% inactivation. No significant variation within the error bars for the bactericidal efficacy of the VACNT arrays was observed after treatment with CF4 plasma, however due to the change in surface morphology after O2 plasma processing (Figure S1), there was a slight drop in the bactericidal efficiency of both 1 µm and 30 µm CNT surfaces against S. aureus. The ability of bacteria to attach to the VACNT surface was not reduced by plasma treatment; in fact, there was a substantial increase of attached cells, in particular in the case of S. aureus. Chemical functionalization of the surface using either O2 or CF4 plasma resulted in an increased bacterial attachment density compared to pristine CNT arrays (Figure 3d). No such effect was observed for P. aeruginosa, which showed a similar density of attached cells on most VACNT surfaces. These results are in agreement with a study by Yick et al., who showed an increase in cell attachment to plasmatreated VACNT arrays for some Gram-positive bacteria (B. subtilis and S. epidermidis).42 O2 plasma treatment reduced the density of P. aeruginosa attachment in comparison to the other two surfaces, possibly due to a lack of available attachment points because of the bundling of CNT tips following plasma treatment (SEM images Figure 2C). This phenomenon may also have contributed to the lesser killing efficiency observed for S. aureus on 1 µm VACNT arrays (SEM images Figure 2f). It is ix ACS Paragon Plus Environment
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hypothesised that degradation of the nanoarray for surfaces with shorter nanotubes allowed S. aureus cells to preferentially settle between the nanotubes, proceeding to proliferate. A different bactericidal efficacy against Gram-negative and Gram-positive bacteria (S. aureus and E. coli, respectively) was previously reported for acid-treated SWCNTs coated onto paper.43 As shown in Table S1, the authors reported greater antibacterial activity against Gram-positive (S. aureus) than Gram-negative bacteria (E. coli); they also observed morphological changes in both bacterial strains as a result of direct contact with acid-treated SWCNTs, and cytoplasmic material was detected in the surrounding medium as a result of membrane disruption.43 However, in this study, Gram-negative bacteria were more susceptible to the killing effects of CNTs than Gram-positive bacteria, as is evident from quantification of non-viable cells of S. aureus and P. aeruginosa. It was also previously reported that the chemical functionalization of SWCNTs (dispersed in solution) does not have a critical influence on the bactericidal efficiency,44 which is consistent with the present finding that the VACNT geometry, rather than the surface chemistry, plays a key role in the bactericidal efficacy.
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Figure 2. Representative CLSM (top) and SEM (bottom) images of P. aeruginosa and S. aureus bacterial cells on VACNT array surfaces: a-b) pristine, c-d) O2 plasma-treated, and e-f) CF4 plasmatreated arrays. The SEM images highlight the altered bacterial morphology at the CNT interface. Scale bars are 1 µm and 100 nm in the SEM images and the corresponding inset, respectively, and 5 µm in the CLSM images. xi ACS Paragon Plus Environment
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Figure 3. Bactericidal activity (a, c) and attachment (b, d) of P. aeruginosa and S. aureus bacterial cells, respectively, for VACNT nanoarrays with different post-fabrication surface modifications. Statistical significance is denoted by ** (p < 0.05). Error bars span one standard deviation.
Characterization of the cell-substratum interface Next, a detailed FIB-SEM analysis of the cells/VACNT interfaces was performed to further elucidate the cell-substratum interaction (Figure S5). FIB-SEM measurements were performed on VACNT forests xii ACS Paragon Plus Environment
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incubated with bacteria for 18 h, then fixed and critically point-dried to preserve the morphology of both the cells and the substrata, and to visualize static snapshots of the interface in a liquid environment. The most commonly reported mechanism of bacterial cell death on nanostructured surfaces is stressinduced rupturing due to membrane absorption onto a nanopillared, or similar arrays.13 In this study, SEM images of the substrata exposed by FIB milling confirmed direct contact between the cell membrane and the CNTs and deformation of the cell membrane upon attachment of the bacterial cell onto the substratum. Upon attachment, it is clearly observed in SEM and TEM images, that subsequent bending of the VACNTs occurred (Figure 4 S5). ‘Capturing’ of the both bacterial species by the CNTs is evident at the cell - substratum interface and the loss of a clearly defined cellular membrane for P. aeruginosa suggests that the membrane has been damaged during the attachment process, resulting in an altered cell morphology and loss of cell integrity. It is well recognised that cellular adsorption onto a nanostructured surface induces stretching of the cell membrane, however, the mechanism by which cell stretching and rupture is initiated may differ between nanostructured topographies. For nanopillared surfaces, rupturing occurs due to stretching forces acting upon the membrane suspended between adjacent pillars.6 Here, there are no obvious free-standing pillars, therefore, stretching of the cell membrane is proposed to result from a distinct process: (i) cell attach onto the VACNT array, (ii) in response to cell attachment, the CNTs deflect, (iii) retract, (iv) stretch and (v) tear the adsorbed bacterial cell. FIB milling was also used to create thin (~200 nm) sections of the same samples, and TEM images (Figures 4c, d and S11) of these sections were then recorded. The TEM and STEM images also showed direct contact between bacterial cells and CNTs, in agreement with the FIB-SEM images. It is likely that once the bacteria are attached to the VACNT forest surface, their membrane is subjected to progressive stretching and tearing, as the flexibility of the CNTs enables their lateral displacement in aqueous conditions. The result of progressive stretching and tearing is visualized most clearly in topxiii ACS Paragon Plus Environment
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view SEM images where compromised S. aureus (Figure 4a) and P. aeruginosa (Figure 4b) cell morphology is clearly observed. Investigation of the biointerface by 3D TEM tomography showed the initial contact between P. aeruginosa bacterial cells and the VACNT surface (Figure S12), revealing bending of the tips of the nanotubes in direct contact with the cell. Subsequently, following further interaction between bacteria and CNTs, the membrane is significantly deformed: this was evidenced by SEM images following FIB milling of the substratum - cell interface, and the TEM images showing the internalization of some of the nanotube structures (Figure 4, S11). The internalization of the carbon nanotubes suggests that the membrane became increasingly deformed until it was ruptured, allowing the CNTs to penetrate within the interior of the cell. The CNTs do not appear to encapsulate the cell but come into direct contact and then bend. Further interaction between the bacterial cell and the VACNTs leads to additional damage, as seen in the STEM micrograph in (Figure 4d), which illustrates the direct contact between the VACNTs and P. aeruginosa bacterial cell, whose cell membrane is severely damaged or ruptured. These findings suggest that bacterial cell death on VACNT forests occurs because of the physical perturbation of the membrane, which leads to loss of cell viability. This conclusion is consistent with the hypothesis that bacterial cell death results from the stress exerted on the cell wall by the CNTs, as discussed in the next section.
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Figure 4. Biointerface of S. aureus and P. aeruginosa cells and VACNTs. Top-view SEM of (a) S. aureus and (b) P. aeruginosa showing altered cellular morphology due to the interaction with the VACNT nanoarray. FIB-SEM of bacterial cells, (c) S. aureus and (d) P. aeruginosa, compromised by the flexible motion of CNTs leading to internalization of CNTs and cell death and TEM micrographs showing (e) cross-sectional profile and (f) lateral cross section of P. aeruginosa cell interacting with CNT. A sequence of 2D sections of a 3D image is shown in Figure S12, illustrating the attachment of a xv ACS Paragon Plus Environment
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P. aeruginosa cell onto the VACNT forest and the subsequent deflection of the nanotubes. Region of affected CNTs due to contact with bacteria at the bottom (blue, radius Ra) and at the top (yellow, radius Rb). Red arrows pinpoint areas of VACNT attachment and stretching/loss of integrity of bacterial membrane. Storage of elastic energy in bent CNTs The VACNT forests are made of dense multiwalled carbon nanotubes with a high aspect ratio (1003000) between their length (microns) and diameter (approximately 10 nm). Owing to this geometry, the CNTs are flexible and can easily deform in contact with the bacterial cells and subsequently release the stored energy. The experiments conducted in this study showed that short CNTs (1 µm) have a greater ability to inactivate both Gram-positive and Gram-negative bacteria. To estimate the possible elastic forces of VACNTs on bacterial cells, we applied the Euler-Bernoulli beam theory.45 We show that the amount of elastic energy that can be stored within 1 µm CNT as opposed to 30 µm CNT for the same horizontal deflection is greater, subsequently allowing the storage of more elastic energy. To determine the elasticity of a single CNT of different length, we assumed that nanotubes in VACNTs only differ in length, while other geometrical parameters, such as the shape of CNTs, their diameter, grafting density, Young modulus are the same for all samples studied in this work. However, in reality, the nanotubes in VACNTs are not perfectly straight, and touch each other over the height of the forest, which makes them more stiff than freestanding nanotubes.46 For our model using Euler-Bernoulli’s beam theory, we considered a force P (load) acting on the tip of a CNT parallel to the substratum.
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1 µm 2 µm 3 µm 10 µm
30 µm
Figure 5. Stored and released energy U in kT of CNTs of different lengths vs. tip deflection δ in µm for different nanotube lengths L. As the nanotube length increases, the same tip deflection allows to store (and release) less energy. Similarly, shorter nanotubes can store larger amounts of energy while undergoing less bending.
The beam theory predicts a deflection of the tip δ as:
δ
(1)
where L is the length of the pillar, E is the material’s Young modulus, and I the area moment. According to equation (1). it indicates that the same force P results in a larger deflection for the longer CNTs, and the same applies for the energy stored in the bent CNTs, the external energy
, which is balanced by
. Note that for a given deflection δ, both the force P and elastic energy U are
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moment, I, as well as a typical value for the Young’s modulus of a multiwalled CNT, E~1TPa,47-50 let us illustrate the relation between deflection and elastic energy in Figure 5. Following the same tip displacement, 1 µm CNTs exert stronger forces than 30 µm CNTs on attached cells and, as a result, can induce more damage to the cell membrane leading to a greater degree of cell death. However, the nanotubes in VACNTs are not perfectly straight, and touch each other over the height of the forest, which makes them more stiff than freestanding nanotubes.46 Furthermore, linear beam theory is not applicable for those CNTs showing deflections on the length scale of the CNT itself. Nevertheless, to have an idea of the range of elastic forces and energies involved in the attachment of cells, we estimated typical deflections for S. aureus and P. aeruginosa attached to 1 µm (Figures 4 d and e, respectively) and 30 µm (Figures 2 a and b, respectively) VACNTs by comparing the lateral length scales of bacteria and effective substratum areas where the bacterium is linked to CNTs. For short pillars, typical elastic energies per CNT are found in a range of several 100 to several 1000 kT, with k being the Boltzmann’s constant and T the absolute temperature. Effective tensions imposed on the bacterium cell wall have been estimated as dUtot/dAb, with Utot being the total elastic energy per bacterium, and Ab its projected area. For short VACNTs we find values between the order of 1 mN/m and several 10 mN/m depending on lateral pillar distance, fraction of attached pillars, as well as – most importantly – the exact diameter of the pillar entering to the 4th power in the area moment (Table 2). In contrast, in 30 µm VACNT forests, the elastic contributions resulting from the vertical alignment seem to play a minor role only for the bactericidal effect as it is underlined experimentally by the observed decline of bactericidal effects for the long VACNTs. We estimate the maximum of stored energy to be found typically in the orders between 0.1 and 10 kT. Maximal lateral surface tensions imposed will not exceed the order of 0.01 mN/m. These arguments support experimental evidence that shorter VACNTs nanoarrays are more efficient bactericidal nanopatterns than very tall VACNTs, for the deflections observed in our micrographs. Recently, the differential bactericidal activity, delivered by changes in height, as opposed xviii ACS Paragon Plus Environment
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to other geometric parameters, was also investigated. Super high aspect ratio black silicon nanopillars (7 µm) were shown to exceed the bactericidal performance of shorter, blunter nanopillars (2 µm). Additionally, it was found that dependence of antibacterial efficiency on topographical factors such as density was lost for nanopillars larger than 3 µm in height.51 This corroborates the findings in this study showing long pillars have greater bactericidal effect. The above model therefore, further confirms and builds upon what has been found during experimental work; that the release of elastic energy particularly stored in CNTs with intermediate aspect ratio may contribute to rupture bacteria cell walls of both types (Gram-positive and Gram-negative) of medically relevant, pathogenic bacteria. Elastic effects might be complemented by effects observed in previous studies showing oxidative stress, disruption of metabolic routes, and physical damage to the cell membrane;25,
52-54
including by piercing the cells leading to a loss of cellular viability.26 Kang et al.
reported irreversible damage to E. coli cells upon contacting SWCNTs deposited onto a membrane filter, inactivating up to 87.6% of all cells.22 Other CNT membranes, used for water filtration, have been reported to possess similar efficiency in removing microbes from the environment 32, 55-57 (Table S1). It was also reported that the physicochemical properties of the CNTs, including diameter,23 length,23, 25, 58 surface functionality,59-60 and conductivity,53 may affect their bactericidal efficiency. Liu et al. confirmed through atomic force microscopy that physical puncturing of bacterial cell membranes by CNTs caused cell death, and that ‘softer’ cells were more vulnerable to the action of dispersed CNT ‘nanodarts’ than more rigid cells.61 The potential of this nanomaterial for use in biomedical applications is promising based on its ability to inactivate a high percentage of Gram-positive and Gram-negative bacteria. It was recently demonstrated that mammalian cells are able to probe the thickness of a compliant substratum by sensing deformational resistance with traction-mediated mechano-sensing via actomyosin interactions through focal adhesion.62 This finding poses implications for the ability of bacteria to be able to ‘sense’ their xix ACS Paragon Plus Environment
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physical environment, responding to surface thickness or compliance, as well as structural changes. It is well known currently that bacteria can respond appropriately to physical cues in their environment and preferentially choose to settle in between grooves or at the base of features which are larger than themselves.63-65 Conclusions Here, we report a mechanism of bactericidal activity, different from that observed for bacterial cells in suspension with dispersed CNTs and for cells attached onto the nanopillar-arrayed surfaces of insect wings, such as cicada and dragonfly wings, and synthetic substrates such as black silicon. The currently well-studied black silicon surfaces possess structures which are pyramidal in shape, causing them to be completely inflexible and incapable of deflection upon the forces exerted during bacterial attachment. Similar occurrences have been observed for cicada wings, where the nanopillars are not perceived to deflect substantially. Further corroboration of the bactericidal mechanism of nanopillared surfaces illustrated the physical killing of bacteria as a process of adsorption of the cell membrane on to the nanopillars. Continued adsorption places increasing stress on the cellular membrane as it is stretched beyond its elastic limit, causing permanent deformation through rupturing of the membrane. In this work we have revealed a mechanism of physical rupturing of bacterial cells using flexible, high aspect ratio VACNTs. We fabricated highly bactericidal VACNTs arrays with different aspect ratios and surface chemistries, and investigated their capacity to inactivate both Gram-negative (P. aeruginosa) and Gram-positive (S. aureus) bacterial cells. Plasma treatment of the 30 µm VACNTs with either O2 or CF4 plasma did not noticeably affect the bactericidal efficiency; however, the nanotube height had a significant impact on the bactericidal efficiency against S. aureus, with 1 µm CNTs achieving markedly higher rates of inactivation than 30 µm CNTs. Oxygen plasma treatment caused some lessening of bactericidal efficiency for 1 µm VACNTs as compared to CF4 plasma treated and pristine arrays, which may have been due to bundling of the CNT tips as a result of this process. The rates of bacterial xx ACS Paragon Plus Environment
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inactivation measured for the VACNT substrata are among the highest reported in the literature for both natural and synthetic bactericidal nanostructured surfaces. The bactericidal mechanism reported here, reflects the ability of the high aspect ratio nanostructures to store elastic energy due to their flexibility. Release of the elastic energy previously stored in the CNTs upon contact with a bacterial cell and bending of the CNTs results in physical perturbation (i.e., stretching) of the membrane and cell death. The modulation of the CNT characteristics is also expected to further enhance our understanding of mechanobactericidal mechanisms and will be the focus of future work. Materials and methods Growth of CNTs The VACNTs (Figure S1) were synthesized by chemical vapor deposition (CVD) under atmospheric pressure at 800 ºC. The CNTs were grown from a catalyst layer (10 nm Al2O3 and 1 nm Fe) deposited on a silicon wafer using physical vapor deposition (PVD). The catalyst was annealed for 15 min under helium (100 sccm) and hydrogen (400 sccm) gas stream. Next, hydrogen (20 sccm), ethylene (20 sccm), and helium (560 sccm) were introduced into the furnace. The growth time under the He/H2/ethylene gas stream in the furnace was 20 s for the short forests and 60 s for the long forests. After growth, the substrates were rapidly cooled in the same helium/hydrogen/ethylene gas stream. Oxygen plasma modification Short and long VACNT forests were treated for 1 and 10 min, respectively, in oxygen plasma at 0.8 mbar in a Diener plasma cleaner. CF4 plasma modification Short and long VACNT forests were treated for 20 and 40 s in a CF4 plasma barrel etcher.
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Wettability Static water contact angle measurements of the CNT samples were performed in air using a 1000C analyzer (First Ten Angstroms Inc.), which dispensed approximately 1.0 µL of water onto the substrate surface via a nanodispenser tip. Images were taken with a Pelco Model PCHM 575-4 camera within 1–2 s of the drop being placed on the surface. Cell viability Pseudomonas aeruginosa ATCC 9721 and Staphylococcus aureus CIP 65.8T were obtained from American Type Culture Collection and Culture Institute Pasteur (France), respectively, and refreshed with nutrient agar (Oxoid) for 24 h at 37 °C. Bacterial cell suspensions of optical density (OD)600 = 0.1 were prepared by diluting one loopful of bacteria in nutrient broth (Oxoid) until the correct optical density was achieved, as determined by UV-Vis spectrophotometry (Dynamica HALO RB-10) at a wavelength of 600 nm. Silicon wafer samples were used as control surfaces to monitor cell attachment, morphology, and viability (Figure S1). Further details on the sample handling are included in the Supplementary Information, Figure S2. The CNT samples were stored in sterile 24-well plates (Invitrogen) and incubated in duplicate in 1 mL of bacterial suspension in a UV-sterilized 24-well plate (In Vitro Technologies), under dark and static conditions at 25 °C for 18 h. At least two technical replicates were conducted to confirm the results. Confocal laser scanning microscopy (CLSM) was used to assess the viability of bacterial cells attached to the CNT surfaces after 18 h of incubation. The attached cells were stained for 25 min at room temperature and under dark conditions using a L7012 LIVE/DEAD Baclight bacterial viability kit (Molecular Probes, Life Technologies). CLSM imaging was performed using a Fluoview FV10i inverted microscope (Olympus). Images were taken at 8–10 different fields of view to generate statistically reliable data. In addition, at least two independent technical replicates were performed to
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reconfirm the results. Cell attachment and viability were assessed using the CellC module of the MatLab software. SEM measurements To assess the cell morphology, CNT samples with attached bacterial cells were observed by SEM (Raith150 Two, Raith GmbH). Prior to imaging, the surfaces were gently rinsed twice with phosphatebuffered saline (PBS, pH 7.4) and fixed with 2.5% glutaraldehyde. The surfaces were then dehydrated with ethanol in 30, 50, 70, 90 and 100% concentrations. The CNT surfaces were kept in 100% ethanol and transferred to a critical point dryer (Polaron E3100, Quorum Technologies Ltd). The samples were immersed in liquid CO2 via a series of ethanol/liquid CO2 exchanges, and the liquid CO2 was then heated to supercritical temperature and pressure (31.1°C and 1000 psi). The dried samples were then sputtered with gold for 2 min using a NeoCoater MP-19020NCTR sputtering machine. XPS analysis XPS measurements were conducted using a Kratos Axis Nova instrument (Kratos) with a monochromatic Al Kα source (source energy 1486.69 eV) at a power of 150 W. Elemental identification was carried out using survey spectra collected at a pass energy of 160 eV with 1 eV steps. A Shirley algorithm was used to measure the background core-level spectra, and chemically distinct species in the high-resolution regions of the spectra were fitted with synthetic Gaussian–Lorentzian components after removing the background (using the CasaXPS software, v. 2.3.15). The relative atomic concentrations of the elements were quantified based on the peak area in the selected high-resolution region, determined using the appropriate sensitivity factors for the instrument. High-resolution XPS scans were performed in the C 1s, O 1s, and F 1s regions. Raman spectroscopy Raman spectra of the vertically aligned CNT arrays were obtained using a Renishaw Invia confocal Raman microscope with a 50× objective lens and 514 nm laser excitation. xxiii ACS Paragon Plus Environment
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Focused ion beam milling FIB milling of cells incubated on the CNT surfaces was performed using an FEI Nanolab 200 dualbeam FIB system. The samples were fixed with 2.5% glutaraldehyde for 45 min and then washed three times at room temperature with 0.1 M cacodylate buffer containing 2 mM calcium chloride. The samples were stained with 2% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h at room temperature and then washed extensively. A 10-min incubation followed, with thiocarbohydrazide used as a mordant. A second staining was completed using 2% osmium tetroxide for 30 min. The samples were en bloc stained overnight at 4 °C using 1% uranyl acetate, and then further stained with lead aspartate at 60 °C for 30 min. After ethanol dehydration over ice and drying using a CPD system, the sample surfaces were finally coated with a Pt protection layer using an e-beam Pt deposition process prior to milling. Transmission electron microscopy TEM sectioning was performed using FIB until lamellas of approximately 100 nm were obtained (Figure S10). The sections were welded to a micromanipulator using Pt deposition and then transferred to copper grids. Imaging was carried out on an FEI Tecnai F20 microscope in TEM and STEM modes (Figure S11). The STEM tomography was observed on an FEI Tecnai F30 microscope. REFERENCES 1. Hasan, J.; Crawford, R. J.; Ivanova, E. P., Antibacterial Surfaces: The Quest for a New Generation of Biomaterials. Trends Biotechnol. 2013, 31, 295-304. 2. Liu, Y.; Yang, S.-F.; Li, Y.; Xu, H.; Qin, L.; Tay, J.-H., The Influence of Cell and Substratum Surface Hydrophobicities on Microbial Attachment. J. Biotechnol. 2004, 110, 251-256. 3. Anderl, J. N.; Franklin, M. J.; Stewart, P. S., Role of Antibiotic Penetration Limitation in Klebsiella Pneumoniae Biofilm Resistance to Ampicillin and Ciprofloxacin. Antimicrob. Agents Chemother. 2000, 44, 1818-1824. 4. Ivanova, E. P.; Hasan, J.; Webb, H. K.; Truong, V. K.; Watson, G. S.; Watson, J. A.; Baulin, V. A.; Pogodin, S.; Wang, J. Y.; Tobin, M. J.; Löbbe, C.; Crawford, R. J., Natural Bactericidal Surfaces: Mechanical Rupture of Pseudomonas Aeruginosa Cells by Cicada Wings. Small 2012, 8, 2489-2494. 5. Hasan, J.; Webb, H. K.; Truong, V. K.; Pogodin, S.; Baulin, V. A.; Watson, G. S.; Watson, J. A.; Crawford, R. J.; Ivanova, E. P., Selective Bactericidal Activity of Nanopatterned Superhydrophobic Cicada Psaltoda Claripennis Wing Surfaces. Appl Microbiol Biotechnol 2012, 97, 9257-9262. xxiv ACS Paragon Plus Environment
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6. Pogodin, S.; Hasan, J.; Baulin, Vladimir A.; Webb, Hayden K.; Truong, Vi K.; Phong Nguyen, The H.; Boshkovikj, V.; Fluke, Christopher J.; Watson, Gregory S.; Watson, Jolanta A.; Crawford, Russell J.; Ivanova, Elena P., Biophysical Model of Bacterial Cell Interactions with Nanopatterned Cicada Wing Surfaces. Biophys. J. 2013, 104, 835-840. 7. Abid, M. I.; Wang, L.; Chen, Q.-D.; Wang, X.-W.; Juodkazis, S.; Sun, H.-B., Angle-Multiplexed Optical Printing of Biomimetic Hierarchical 3d Textures. Laser Photonics Rev. 2017, 11, 1600187. 8. Linklater, D. P.; Juodkazis, S.; Rubanov, S.; Ivanova, E. P., Comment on “Bactericidal Effects of Natural Nanotopography of Dragonfly Wing on Escherichia Coli”. ACS Appl. Mater. Int. 2017. 9. Linklater, D. P.; Nguyen, H. K. D.; Bhadra, C. M.; Juodkazis, S.; Ivanova, E. P., Influence of Nanoscale Topology on Bactericidal Efficiency of Black Silicon Surfaces. Nanotechnology 2017, 28, 245301. 10. Mainwaring, D. E.; Nguyen, S. H.; Webb, H.; Jakubov, T.; Tobin, M.; Lamb, R. N.; Wu, A. H. F.; Marchant, R.; Crawford, R. J.; Ivanova, E. P., The Nature of Inherent Bactericidal Activity: Insights from the Nanotopology of Three Species of Dragonfly. Nanoscale 2016, 8, 6527-6534. 11. Bhadra, C. M.; Khanh Truong, V.; Pham, V. T. H.; Al Kobaisi, M.; Seniutinas, G.; Wang, J. Y.; Juodkazis, S.; Crawford, R. J.; Ivanova, E. P., Antibacterial Titanium Nano-Patterned Arrays Inspired by Dragonfly Wings. Sci. Rep. 2015, 5, 16817. 12. Wang, X.; Bhadra, C. M.; Yen Dang, T. H.; Buividas, R.; Wang, J.; Crawford, R. J.; Ivanova, E. P.; Juodkazis, S., A Bactericidal Microfluidic Device Constructed Using Nano-Textured Black Silicon. RSC Adv. 2016, 6, 26300-26306. 13. Ivanova, E. P.; Hasan, J.; Webb, H. K.; Gervinskas, G.; Juodkazis, S.; Truong, V. K.; Wu, A. H. F.; Lamb, R. N.; Baulin, V. A.; Watson, G. S.; Watson, J. A.; Mainwaring, D. E.; Crawford, R. J., Bactericidal Activity of Black Silicon. Nat. Comm. 2013, 4. 14. Pang, M.; Zhang, Y. Q.; Chen, W. Q., Transverse Wave Propagation in Viscoelastic SingleWalled Carbon Nanotubes with Small Scale and Surface Effects. J. Appl. Phys. 2015, 117, 024305. 15. De Volder, M. F. L.; Tawfick, S. H.; Baughman, R. H.; Hart, A. J., Carbon Nanotubes: Present and Future Commercial Applications. Science 2013, 339, 535-539. 16. Baughman, R. H., Carbon Nanotubes--the Route toward Applications. Science 2002, 297, 787792. 17. Martin, C. R.; Kohli, P., The Emerging Field of Nanotube Biotechnology. Nat. Rev. Drug Discovery 2003, 2, 29-37. 18. Harrison, B. S.; Atala, A., Carbon Nanotube Applications for Tissue Engineering. Biomat. 2007, 28, 344-353. 19. Cools, J.; Copic, D.; Luo, Z.; Callewaert, G.; Braeken, D.; De Volder, M., 3d Microstructured Carbon Nanotube Electrodes for Trapping and Recording Electrogenic Cells. Adv. Funct. Mater. 2017, 1701083. 20. Lovat, V.; Pantarotto, D.; Lagostena, L.; Cacciari, B.; Grandolfo, M.; Righi, M.; Spalluto, G.; Prato, M.; Ballerini, L., Carbon Nanotube Substrates Boost Neuronal Electrical Signaling. Nano Lett. 2005, 5, 1107-1110. 21. Correa-Duarte, M. A.; Wagner, N.; Rojas-Chapana, J.; Morsczeck, C.; Thie, M.; Giersig, M., Fabrication and Biocompatibility of Carbon Nanotube-Based 3d Networks as Scaffolds for Cell Seeding and Growth. Nano Lett. 2004, 4, 2233-2236. 22. Kang, S.; Pinault, M.; Pfefferle, L. D.; Elimelech, M., Single-Walled Carbon Nanotubes Exhibit Strong Antimicrobial Activity. Langmuir 2007, 23, 8670-8673. 23. Kang, S.; Herzberg, M.; Rodrigues, D. F.; Elimelech, M., Antibacterial Effects of Carbon Nanotubes: Size Does Matter! Langmuir 2008, 24, 6409-6413. 24. Yang, C.; Mamouni, J.; Tang, Y.; Yang, L., Antimicrobial Activity of Single-Walled Carbon Nanotubes: Length Effect. Langmuir 2010, 26, 16013-16019. xxv ACS Paragon Plus Environment
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25. Chen, H.; Wang, B.; Gao, D.; Guan, M.; Zheng, L.; Ouyang, H.; Chai, Z.; Zhao, Y.; Feng, W., Broad-Spectrum Antibacterial Activity of Carbon Nanotubes to Human Gut Bacteria. Small 2013, 9, 2735-2746. 26. Le, T. T. A.; McEvoy, J.; Khan, E., Mitigation of Bactericidal Effect of Carbon Nanotubes by Cell Entrapment. Sci. Total Environ. 2016, 565, 787-794. 27. Ahmad, M. N.; Xie, J.-y.; Ma, Y.-h.; Yang, W.-t., Surface Functionalization of Single-Walled Carbon Nanotubes Using Photolysis for Enhanced Dispersion in an Organic Solvent. Carbon 2010, 48, 3004. 28. Pasquini, L. M.; Hashmi, S. M.; Sommer, T. J.; Elimelech, M.; Zimmerman, J. B., Impact of Surface Functionalization on Bacterial Cytotoxicity of Single-Walled Carbon Nanotubes. Environ. Sci. Technol. 2012, 46, 6297-6305. 29. Zardini, H. Z.; Amiri, A.; Shanbedi, M.; Maghrebi, M.; Baniadam, M., Enhanced Antibacterial Activity of Amino Acids-Functionalized Multi Walled Carbon Nanotubes by a Simple Method. Colloids Surf., B 2012, 92, 196-202. 30. Mocan, T.; Matea, C. T.; Pop, T.; Mosteanu, O.; Buzoianu, A. D.; Suciu, S.; Puia, C.; Zdrehus, C.; Iancu, C.; Mocan, L., Carbon Nanotubes as Anti-Bacterial Agents. Cell. Mol. Life Sci. 2017, 74, 3467-3479. 31. Akhavan, O.; Abdolahad, M.; Abdi, Y.; Mohajerzadeh, S., Silver Nanoparticles within Vertically Aligned Multi-Wall Carbon Nanotubes with Open Tips for Antibacterial Purposes. J. Mater. Chem. 2011, 21, 387-393. 32. Baek, Y.; Kim, C.; Seo, D. K.; Kim, T.; Lee, J. S.; Kim, Y. H.; Ahn, K. H.; Bae, S. S.; Lee, S. C.; Lim, J.; Lee, K.; Yoon, J., High Performance and Antifouling Vertically Aligned Carbon Nanotube Membrane for Water Purification. J. Membr. Sci 2014, 460, 171-177. 33. Xu, M.; Futaba, D. N.; Yamada, T.; Yumura, M.; Hata, K., Carbon Nanotubes with TemperatureInvariant Viscoelasticity from -196 to 1000 c. Science 2010, 330, 1364-1368. 34. Hata, K., Water-Assisted Highly Efficient Synthesis of Impurity-Free Single-Walled Carbon Nanotubes. Science 2004, 306, 1362-1364. 35. Felten, A.; Bittencourt, C.; Pireaux, J. J.; Van Lier, G.; Charlier, J. C., Radio-Frequency Plasma Functionalization of Carbon Nanotubes Surface O2, Nh3, and Cf4 Treatments. J. Appl. Phys. 2005, 98, 074308. 36. Xu, T.; Yang, J.; Liu, J.; Fu, Q., Surface Modification of Multi-Walled Carbon Nanotubes by O2 Plasma. Appl. Surf. Sci. 2007, 253, 8945-8951. 37. Karousis, N.; Tagmatarchis, N.; Tasis, D., Current Progress on the Chemical Modification of Carbon Nanotubes. Chem. Rev. 2010, 110, 5366-5397. 38. Zhao, B.; Zhang, L.; Wang, X.; Yang, J., Surface Functionalization of Vertically-Aligned Carbon Nanotube Forests by Radio-Frequency Ar/O2 Plasma. Carbon 2012, 50, 2710-2716. 39. Hou, Z.; Cai, B.; Liu, H.; Xu, D., Ar, O2, Chf3, and Sf6 Plasma Treatments of Screen-Printed Carbon Nanotube Films for Electrode Applications. Carbon 2008, 46, 405-413. 40. Valentini, L.; Puglia, D.; Armentano, I.; Kenny, J. M., Sidewall Functionalization of SingleWalled Carbon Nanotubes through Cf4 Plasma Treatment and Subsequent Reaction with Aliphatic Amines. Chem. Phys. Lett. 2005, 403, 385-389. 41. Valentini, L.; Macan, J.; Armentano, I.; Mengoni, F.; Kenny, J. M., Modification of Fluorinated Single-Walled Carbon Nanotubes with Aminosilane Molecules. Carbon 2006, 44, 2196-2201. 42. Yick, S.; Mai-Prochnow, A.; Levchenko, I.; Fang, J.; Bull, M. K.; Bradbury, M.; Murphy, A. B.; Ostrikov, K., The Effects of Plasma Treatment on Bacterial Biofilm Formation on Vertically-Aligned Carbon Nanotube Arrays. RSC Adv. 2015, 5, 5142-5148. 43. Deokar, A. R.; Lin, L.-Y.; Chang, C.-C.; Ling, Y.-C., Single-Walled Carbon Nanotube Coated Antibacterial Paper: Preparation and Mechanistic Study. J, Mater. Chem. B 2013, 1, 2639. xxvi ACS Paragon Plus Environment
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64. Anselme, K.; Davidson, P.; Popa, A. M.; Giazzon, M.; Liley, M.; Ploux, L., The Interaction of Cells and Bacteria with Surfaces Structured at the Nanometre Scale. Acta Biomater. 2010, 6, 3824-3846. 65. Lorenzetti, M.; Dogša, I.; Stošicki, T.; Stopar, D.; Kalin, M.; Kobe, S.; Novak, S., The Influence of Surface Modification on Bacterial Adhesion to Titanium-Based Substrates. ACS Appli. Mater. & Int. 2015, 7, 1644-1651. Author contributions M.D.V, S.J, M.G and L.G fabricated the CNT samples and performed the plasma treatments. D.L performed cell viability tests, surface characterizations, chemical characterizations, and FIB milling procedures, and prepared the TEM samples. E.H and S. R performed TEM and tomography analyses. V.A.B and M.W developed the bactericidal model. All authors contributed to writing the manuscript and approved its final version. Additional Information The authors declare no competing financial interests Funding Sources MDV and SJ acknowledge support from the ERC Starting Grant HIENA 337739. V.A.B. M.W. and E.P.I. acknowledge funding from Marie Curie Actions under EU FP7 Initial Training Network SNAL 608184. Acknowledgments The authors would like to acknowledge Bio21 Advanced Microscopy facility for assistance with the FIB milling procedures, STEM tomography and TEM, Armandas Balcytis for assistance with the Raman spectroscopy measurements, Dr. David Beesley for assistance with the CF4 plasma etching, and Dr. Deming Zhu for assistance with the XPS analysis. Associated Content Supporting Information The supporting information is available free of charge on the ACS publications website at DOI: [insert DOI here] xxviii ACS Paragon Plus Environment
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The supporting information contains figures of pristine and plasma-treated VACNT arrays, bacteria on control surfaces, the effects of capillary forces on VACNT morphology, FIB-SEM cross-sectional profiles of the biointerface, Raman and XPS data, TEM lamella fabrication by FIB-milling and resultant TEM micrographs of bacteria on short and tall nanotubes (PDF). 3D transmission electron tomography movie showing the bending of CNTs in contact with bacterial cells (AVI)
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Table 1. Physicochemical characteristics of the vertically aligned CNT arrays
Type of VACNT Height (µm)
Tall
Short
32 ± 2
1 ± 0.6
50–100
50–100
Spacing between CNTs (nm) Plasma Pristine
CF4
O2
Pristine
CF4
O2
147.93
45.5
149.5
137.1
21.4
± 5.8
± 4.0
± 3.1
± 1.5
± 0.9
0.90
1.41
0.27
1.02
1.31
treatment Water
contact 148.6
angle, θ (°)
± 4.3
Raman intensities ratio 0.58 (ID/IG)
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Table 2. Estimates of VACNT forest rigidity Type of VACNT
Tall (32 µm)
Short (1µm)
Affected area at the bottom,* A_a, (µm2)
0.76
0.29
Affected area at the top,* A_b, (µm2)
0.26
0.05
Resulting deflection, r = A_a/A_b
3.03
5.06
Lower bound estimate: CNT inner diameter 2 nm, outer diameter 5 nm, distance between CNTs 0.10 µm, active fraction 0,30 Maximum stored elastic energy (kT) Tension (mN/m)
0.6
~5000
0
-1.2
Upper bound estimate: CNT inner diameter 0 nm, outer diameter 10 nm, distance between CNTs 0,05 µm, active fraction 0,80 Maximum stored elastic energy (kT) Tension (mN/m)
10
~79000
-0.03
-210
(*from Figure 4, S. aureus)
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