High Aspect Ratio Nanostructures Kill Bacteria via Storage and

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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*,†,# †

Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia Institute for Manufacturing, Department of Engineering, University of Cambridge, Cambridge CB3 0FS, United Kingdom § Centre for Micro-Photonics and Industrial Research Institute Swinburne, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia ∥ Department d’Enginyeria Quimica, Universitat Rovira, Virgili, 26 Av. dels Paisos Catalans, 43007 Tarragona, Spain ⊥ Advanced Microscopy Facility, Bio21 Institute, University of Melbourne, 30 Flemington Rd, Parkville, Victoria 3010, Australia # School of Science, College of Science, Engineering and Health, RMIT University, GPO Box 2476, Melbourne, Victoria 3001, Australia ‡

S Supporting Information *

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 threedimensional 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. KEYWORDS: carbon nanotubes, vertically aligned carbon nanotubes, mechanobactericidal mechanism, storage of elastic energy, interface interactions, nanoscale mechanics

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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

actericidal 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 physicochemical properties on the micro and nano scale has led to the development of antibiofouling 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 approach has some drawbacks since the bacteria deeper within a biofilm are not exposed to the biocide and may develop resistance over prolonged © XXXX American Chemical Society

Received: March 4, 2018 Accepted: May 31, 2018 Published: May 31, 2018 A

DOI: 10.1021/acsnano.8b01665 ACS Nano XXXX, XXX, XXX−XXX

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Cite This: ACS Nano XXXX, XXX, XXX−XXX

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ACS Nano 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.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 experimental5 and theoretical6 approaches to define the events occurring at the cell−substratum 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 have lead to considerable changes in bactericidal activity.10 Likewise, synthetic surfaces patterned to mimic natural specimens have also exhibited similar variations in bactericidal activity.9,11,12 The bactericidal efficiency of nanostructured surfaces is therefore 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 bacteria and bacterial spores.13 Finally, surface chemistry and hydrophobicity have been shown to have negligible impact on the bactericidal mechanism of these nanostructured substrata.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 growth21 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 are proposed to physically pierce the bacterial cells, 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 Escherichia 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 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 physicochemical 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 modeling 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.15,33,34 To determine the optimal VACNT height for bacterial killing, we synthesized VACNTs of different height (see Materials and Methods). The height of the multiwalled 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 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 B


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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 Grampositive S. aureus bacterial cells. The bacterial cell viability of different substrates was analyzed over an incubation period of 18 h. The results highlight a high bactericidal activity for all VACNT arrays toward both Gram-negative and Gram-positive bacteria. Confocal laser scanning microscopy (CLSM) confirmed cell inactivation through the 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 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 analyzed 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 Materials and 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 2a,c,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 toward the two bacterial species, and a significant difference in bactericidal efficacy was noted for 1 μm as compared to 30 μm 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 reporting the percentage of nonviable cells), that is, for pristine CNTs,

Figure 1. SEM images contrasting the heights of the high aspect ratio nanotubes (a) 1 μm and (b) 30 μm VACNTs. False color 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.

only 10 or 40 s, and no morphological changes were observed in the SEM images (Figure S1). 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 treatments (Figures S6c,d and S7− S9). Bactericidal Activity of VACNT Arrays. To assess the effects of nanotube height and surface treatment (and corresponding hydrophilic properties of the substratum) on

Table 1. Physicochemical Characteristics of the Vertically Aligned CNT Arrays

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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 hypothesized 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 nonviable 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. 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 incubated with bacteria for 18 h and 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 stress-induced rupturing due to membrane adsorption onto a nanopillared, or similar, array.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 and Figure S5). “Capturing” of 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 was damaged during the attachment process, resulting in an altered cell morphology and loss of cell integrity. It is well recognized 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

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. Regions of affected CNTs due to contact with bacteria at the bottom (blue circles, projected area Aa) and at the top (yellow circles, projected area Ab).

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 1 μm, 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 toward 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 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 D


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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 postfabrication surface modifications. Statistical significance is denoted by ** (p < 0.05). Error bars span 1 standard deviation.

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) cells 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 (Figure 4c,d and Figure 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 top-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 and Figure S11). The internalization of the CNTs 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. Storage of Elastic Energy in Bent CNTs. The VACNT forests are made of dense multiwalled CNTs with a high aspect ratio (100−3000) 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 Grampositive 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, and Young modulus are the same for all samples studied in this work.46 For our model using Euler− E


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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 (Figure 4e,d, respectively) and 30 μm (Figure 2a,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 the projected contact 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 the inner and outer diameters of the pillar entering to the fourth 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 1 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 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: The release of elastic energy particularly stored in CNTs with intermediate aspect ratio may contribute to rupturing 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 environment32,55−57 (Table S1). It was also reported that the physicochemical properties of the CNTs,

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 of S. aureus and (f) lateral cross section of P. aeruginosa cell interacting with CNTs. A sequence of 2D sections of a 3D image is shown in Figure S12, illustrating the attachment of a P. aeruginosa cell onto the VACNT forest and the subsequent deflection of the nanotubes. Regions of affected CNTs due to contact with bacteria at the bottom (blue arrows, projected area Aa) and at the top (yellow arrows, projected area Ab). Red arrows pinpoint areas of VACNT attachment and stretching/loss of integrity of bacterial membrane.

Bernoulli’s beam theory, we considered a force P (load) acting on the tip of a CNT parallel to the substratum. The beam theory predicts a deflection of the tip δ as δ=

PL3 3EI

(1)

where L is the length of the pillar, E is the material’s Young modulus, and I the area moment. According to eq 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, U =

3EIδ 2 , 2L3

which is

Pδ . 2

Note that for a given balanced by the external energy W = deflection δ, both the force P and elastic energy U are inversely proportional to L3. Assuming a full cylinder of a diameter of 10 nm for calculating the area moment, I, as well as a typical value F


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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 for the storage (and release) of less energy. Similarly, shorter nanotubes can store larger amounts of energy while undergoing less bending.

Table 2. Estimates of VACNT Forest Rigidity

a

From Figure 2b and Figure 4e, S. aureus.

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 physical environment, responding to surface thickness or compliance, as well as structural changes. It is wellknown 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 nanopillararrayed surfaces of insect wings, such as cicada and dragonfly G


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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 Supporting 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 UVsterilized 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 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 phosphate-buffered 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. Focused Ion Beam Milling. FIB milling of cells incubated on the CNT surfaces was performed using an FEI Nanolab 200 dual-beam 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

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 rigid and deflect little 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 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 °C. 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. 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 H


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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. 2013, 97, 9257−9262. (6) Pogodin, S.; Hasan, J.; Baulin, V. A.; Webb, H. K.; Truong, Vi K.; Phong Nguyen, H.; Boshkovikj, V.; Fluke, C. J.; Watson, G. S.; Watson, J. A.; Crawford, R. J.; Ivanova, E. 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. Interfaces 2017, 9, 29387. (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. Commun. 2013, 4, 2838. (14) Pang, M.; Zhang, Y. Q.; Chen, W. Q. Transverse Wave Propagation in Viscoelastic Single-Walled 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, 787−792. (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. Biomaterials 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, 27, 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.

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.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b01665. 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)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Denver P. Linklater: 0000-0003-1433-3685 Michael De Volder: 0000-0003-1955-2270 Vladimir A. Baulin: 0000-0003-2086-4271 Marco Werner: 0000-0001-5433-8443 Elena P. Ivanova: 0000-0002-5509-8071 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. Funding

M.D.V. and S.J. 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. Notes

The authors declare no competing financial interest.

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. 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 I


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High Aspect Ratio Nanostructures Kill Bacteria via Storage and

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