Bactericidal activity of black silicon

Abstract

Black silicon is a synthetic nanomaterial that contains high aspect ratio nanoprotrusions on its surface, produced through a simple reactive-ion etching technique for use in photovoltaic applications. Surfaces with high aspect-ratio nanofeatures are also common in the natural world, for example, the wings of the dragonfly Diplacodes bipunctata. Here we show that the nanoprotrusions on the surfaces of both black silicon and D. bipunctata wings form hierarchical structures through the formation of clusters of adjacent nanoprotrusions. These structures generate a mechanical bactericidal effect, independent of chemical composition. Both surfaces are highly bactericidal against all tested Gram-negative and Gram-positive bacteria, and endospores, and exhibit estimated average killing rates of up to ~450,000 cells min^-1 cm^-2 . This represents the first reported physical bactericidal activity of black silicon or indeed for any hydrophilic surface. This biomimetic analogue represents an excellent prospect for the development of a new generation of mechano-responsive, antibacterial nanomaterials.

ARTICLE

Received 7 Jun 2013 | Accepted 25 Oct 2013 | Published 26 Nov 2013

Elena P. Ivanova1, Jafar Hasan1, Hayden K. Webb1, Gediminas Gervinskas2,3, Saulius Juodkazis2,3, Vi Khanh Truong1, Alex H.F. Wu4, Robert N. Lamb4, Vladimir A. Baulin5, Gregory S. Watson6, Jolanta A. Watson6, David E. Mainwaring1 & Russell J. Crawford1

Black silicon is a synthetic nanomaterial that contains high aspect ratio nanoprotrusions on its surface, produced through a simple reactive-ion etching technique for use in photovoltaic applications. Surfaces with high aspect-ratio nanofeatures are also common in the natural world, for example, the wings of the dragony Diplacodes bipunctata. Here we show that the nanoprotrusions on the surfaces of both black silicon and D. bipunctata wings form hierarchical structures through the formation of clusters of adjacent nanoprotrusions. These structures generate a mechanical bactericidal effect, independent of chemical composition. Both surfaces are highly bactericidal against all tested Gram-negative and Gram-positive bacteria, and endospores, and exhibit estimated average killing rates of up to B450,000 cells min 1 cm 2. This represents the rst reported physical bactericidal activity of black silicon or indeed for any hydrophilic surface. This biomimetic analogue represents an excellent prospect for the development of a new generation of mechano-responsive, antibacterial nanomaterials.

DOI: 10.1038/ncomms3838 OPEN

Bactericidal activity of black silicon

1 Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia. 2 Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Victoria 3122, Australia. 3 Melbourne Centre for Nanofabrication, 151 Wellington Road, Clayton, Victoria 3168, Australia. 4 School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia. 5 Departament dEnginyeria Quimica, Universitat Rovira i Virgili26 Avenue dels Paisos Catalans, Tarragona 43007, Spain. 6 School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Queensland 4811, Australia. Correspondence and requests for materials should be addressed to E.P.I. (email: mailto:[email protected]

Web End [email protected] ).

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Nanomaterials that possess topographical features with high aspect ratios frequently display remarkable surface properties, such as high hydrophobicity and strong bio

logical activity on a cellular level16. The microbial contamination of surfaces remains a world wide research challenge. Recently we reported the wings of the cicada Psaltoda claripennis possessed potent bactericidal activity against Pseudomonas aeruginosa, an opportunistic human pathogen7, and showed their surfaces to be covered by an array of regularly spaced nanopillar structures. Furthermore, it was shown that the bactericidal nature of the cicada wing arose from physical phenomena based on the nanostructure of the surface, which appeared independent of the biochemical functionality of the wing8,9. On the basis of this underlying physical principle, other topologies that possess surface architectures which may exhibit similar structural characteristics were investigated. One such example is black silicon (bSi), which is a synthetic nanomaterial produced through a simple reactive-ion etching (RIE) technique to possess high aspect ratio nanoprotrusions. Black silicon was developed for photovoltaic and sensing applications1013; particularly for biomedical applications1417.

In this work, we investigate the antibacterial potential of the synthetic bSi surface, together with its related dragony Diplacodes bipunctata wing surface, and compare the antibacterial behaviour to that previously obtained for cicada wing surfaces. The physicochemical properties of bSi and native wing surfaces are chemically and structurally characterized prior to assessing their antibacterial activity against three different bacterial strains with a variety of cell wall structures. The surfaces of bSi and dragony are highly bactericidal against all tested Gram-negative and Gram-positive bacteria, and endospores, and both surfaces exhibit estimated average killing rates of up to B450,000 cells min 1 cm 2. This represents the rst reported physical bactericidal activity of bSi or indeed for any hydrophilic surface.

ResultsSurface nanoarchitecture. Scanning electron microscopy (SEM) of the upper surface of bSi and dragony forewings (Fig. 1a,b, Supplementary Fig. S1) showed disordered nanopillar systems having hierarchical topographical features that resulted from tip cluster formation through bending of the nanoprotrusions. Micrographs of surfaces tilted at an angle of 53 illustrated that the nanopillars of bSi are sharper and more distinct from one another, and approximately twice the height of those of the dragony wing. The dragony wing nanopillars tend towards a network at their bases, whereas their tips remain disconnected or form tip clusters. These differences in microstructure are also seen in their respective optical prolometry images over broader areas (see Fig. 1c,d), and in the three-dimensional (3D) reconstructions of the SEM images (Fig. 1e,f, Supplementary Movie 1).

The pillar characteristics, their spatial arrangement and nanopillar clusters present on the bSi and dragony were obtained using image analysis techniques (cicada wing surfaces were also used for comparison; Supplementary Fig. S2). Both the dragony wing and bSi surfaces clearly showed a random size, shape and spatial distribution of nano-clusters in their upper contact plane, whereas the cicada wing topology showed a regular array of pillars 5070 nm in diameter, spaced B200 nm apart, but containing 2D micro-domains due to stacking faults. Black silicon clusters spanned 2080 nm diameters with a bimodal distribution, whereas dragony clusters showed a clear sigmoidal population distribution below 90 nm, with many of these clusters below 30 nm in diameter, which suggests the presence of a greater number of more complex, ner clusters. The spatial distributions of both bSi and dragony wing clusters ranged from 200 to

1,800 nm in diameter, which arises from the randomness in their perimeters. Clusters of bSi formed a broader spatial distribution than those present on the dragony wing surfaces.

Tip cluster formation was more prevalent in the natural dragony wing surface due to the lower bending stiffness of the nanopillars, which depends upon shape, scale, respective Youngs moduli and material density18. Aizenberg and co-workers19 have shown how capillary forces induce self-reorganization under the inuence of the overall geometry and mechanical and surface properties. Such a secondary structure is also evident in the images derived from the 3D SEM reconstruction technique (Fig. 1e,f).

X-ray photoelectron spectroscopy (XPS) analysis allowed the determination of the elemental composition of the top layers of the dragony wing and bSi surface. High-resolution scans (inset) were performed in B20 eV intervals across the O 1 s and C 1 s peaks. Carbon to oxygen ratios (Supplementary Fig. S3) indicated that the dragony surface was almost exclusively composed of longer chain hydrophobic lipids, whereas the bSi surface was primarily amorphous Si with a small degree of surface oxidation arising from the processing stage. Contact angle measurements indicated that the wing surfaces were superhydrophobic, whereas the bSi surfaces were relatively lower in hydrophobicity (Table 1).

Surface bactericidal activity. To quantify the bactericidal properties of the bSi and the dragony wing surface, three species of bacteria were incubated on each surface for periods of up to 30 h and the bacterial viability monitored. The bacterial species examined represented the major prokaryotic taxa, and included Gram-negative P. aeruginosa ATCC 9027, Gram-positive Staphylococcus aureus 65.8T, and both the vegetative cells and spores of Bacillus subtilis NCIMB 3610T. The morphological appearance of all cells and spores that attached to the surface of the bSi and the dragony wings were found to be signicantly different to those attaching to planar glass and silicon surfaces, which acted as control surfaces (Fig. 2ad, il; controls given in Supplementary Fig. S4). The cellular integrity of all species appeared to be signicantly disrupted by the nanopillars present on both of these substrata despite the signicant difference in surface chemistry and hydrophobicity (Fig. 2 and Supplementary Fig. S4). Signicant deformation and engulfment can be seen with not only the three vegetative cell types but also with B. subtilis spores. We previously observed similar physical deformations of P. aeruginosa cells that attached to cicada wings, which were subsequently found to be inactivated after attachment. A viability analysis of the cells in contact with the bSi and dragony wings using confocal laser scanning microscopy conrmed that all cells and spores were also non-viable after attachment (Fig. 2eh,mp). Cell disruption was assessed using the uorescent dye propidium iodide which penetrates all ruptured cells, causing red uorescence, indicating inactivation. Notably, the bSi and dragony wing surfaces were effective not only against the Gram-negativeP. aeruginosa cells but also against Gram-positive S. aureus andB. subtilis cells. The cicada wing studies previously performed highlighted that the cicada wing substrata were effective only against the Gram-negative cells7. Gram-positive cells are generally more rigid and more resistant to mechanical lysis than Gram-negative cells due to their peptidoglycan cell wall being B45 times thicker than those of the Gram-negative bacteria20. This greater cell wall thickness requires a greater deformational stress to disrupt the cell wall, deform the inner membrane and cause cell death. In addition to being effective against vegetative bacterial cells examined, the bSi and dragony surface nanoarchitectures were clearly able to disrupt the multilayer spore coat, which controls the viability of the

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240 nm

500 nm

50 m

0

190 m

0

58.4 m

1.75 m

0

0

Figure 1 | Characterization of black silicon and D. bipunctata wings. Scanning electron micrographs of the upper surface of (a) bSi and (b) dragony forewings at 35,000 magnication demonstrate the surface patterns of the two samples. Scale bars, 200 nm. Micrographs tilted at an angle

of 53 (inset) show sharper nanopillars of black silicon distinct from one another and approximately twice the height of those of the dragony wing. Optical prolometry shows the nanoprotrusions of (c) bSi and (d) dragony forewings. Scale bars, 50 mm; inset, 2 mm. Three-dimensional reconstructions based on a displacement map technique further highlight the differences and similarities of (e) bSi and (f) dragony forewings.

Table 1 | Feature comparison of insect wings and black silicon surfaces.

Surface Cicada wing (P. claripennis)* Dragony wing (D. bipunctata) Black silicon Surface characteristics

Water contact angle 159 153 80 Chemical composition Lipids/waxes Lipids/waxes Mostly SiO2

Height of nanoprotrusions 200 nm 240 nm 500 nm

Bactericidal activity

Effectiveness Gram-negative Gram-negative

Gram-positive Spores

Gram-negative Gram-positive Spores

Efciencyw

Versus P. aeruginosa ATCC 9027 2.0 105 3.0 105 4.3 105

Versus S. aureus CIP 65.8T N/A 4.6 105 4.5 105

Versus B. subtilis NCIMB 3610T N/A 1.4 105 1.4 105

*Cicada wing values are based on previously published data79.

wValues represent the number of average killing rate (cells killed per cm 2 min 1) over 3 h.

spores21,22. The spore coating affords great durability and resistance against external stresses such as desiccation, making spores the most effective means of bacterial survival under stress

conditions. The results obtained in this study indicated that the deforming stress applied by both bSi and D. bipunctata wings to the bacterial cells is substantial, and demonstrated the signicant

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P. aeruginosa

S. aureus

B. subtilis vegetative cells

B. subtilis spores

Dragonfly wings Black silicon

Figure 2 | Cell morphology on dragony wings and black silicon. SEM images of P. aeruginosa, S. aureus, B. subtilis vegetative cells and B. subtilis spores appear to be signicantly disrupted through interaction with both the dragony wing (ad) and bSi (il). Scale bars, 200 nm. Confocal laser scanning micrographs conrm that disruption by dragony wing (eh) and bSi (mp) was lethal to the cells; non-viable bacterial cells and spores were stained with propidium iodide (red), whereas the living cells were stained with SYTO 9 (green). All cells appeared red, indicating the high efciency of surfaces in inactivating the bacteria. Scale bars, 5 mm.

potential for bSi topologies to be used in counteracting bacterial survival strategies. The interaction between the Gram-negative bacteria and the cicada wing nanopillars, leading to bactericidal activity, was previously shown to be dependent on the physical structure of the surface and cell rigidity, as recently demonstrated by the application of microwave irradiation to cells with rigid wall structures79. The nano-mechanical basis of the bactericidal effect was further conrmed by sputter-coating the bSi and dragony wing surfaces with gold. The gold modied surfaces also displayed activity against cells and spores (Supplementary Fig. S5), which is consistent with the data previously reported for gold-coated cicada wing surfaces, which exhibited activity against Gram-negative bacteria7,8.

Bactericidal efciency. The bactericidal efciency of the bSi and the D. bipunctata wing surfaces was quantitatively assessed over periods up to 30 h using a standard viability plate-count technique. This plate-count assay was adopted according to the FDA protocols for the evaluation of foodstuff sterilization, with an

adjustment made to the inoculum size to accommodate an evaluation of the antibacterial potential of surfaces rather than that of a bulk sterilization technique23. The number of colony forming units in suspensions of all cell types tested were shown to signicantly decrease over 30 h incubation (Fig. 3). On the basis of these numbers, the killing efciency of the bSi surface and dragony wings over the rst 3 h against the four cell types tested were quantied (Fig. 4). The number of cells killed by the bSi and the dragony wings was determined as the difference between the number of surviving cells and those remaining in the control after the same incubation period, in order to account for the low levels of natural cell death that occurs in low-nutrient conditions. The bactericidal activities of these topologically related surfaces appeared to be largely comparable; both surfaces had killing rates of B450,000 cells min 1 cm 2 over the rst 3 h which then declined to B50,000 cells min 1 cm 2 for S. aureus. When this killing efciency is compared with the number of cell of each type required to infect an individual, that is, the minimum infective dose (P. aeruginosa 103 cells, S. aureus 105 cells, and Bacillus

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106cfu ml1

10

50

40

30

20

106cfu ml1

10

60

40

30

20

0 0 h 3 h 18 h 24 h 30 hIncubation time

50

0 0 h 3 h 18 h 24 h 30 hIncubation time

16

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106cfu ml1

14

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8 6 4 2 0

2 0

0 h 3 h 18 h 24 h 30 h

0 h 3 h 18 h 24 h

* *

30 h Incubation time

Incubation time

Control Black silicon

Figure 3 | Bactericidal activity of black silicon surfaces. Counts of viable cells of (a) P. aeruginosa, (b) S. aureus, (c) B. subtilis and (d) B. subtilis spores remaining in suspension when incubated with bSi and control suspensions for up to 30 h. At longer time intervals (24 and 30 h) cell numbers tended to also decrease in the controls due to the nutrient-poor medium. Data for spores in suspension after 24 and 30 h are not available due to the germination of spores after 18 h. Error bars are s.d. values.

Spores

B. subtilis

P. aeruginosa

S. aureus

Dragonfly wings Black silicon

0 10 20 30

104 cells killed cm2 min1 over 3 h

40 50 60

Figure 4 | Bactericidal efciency of black silicon and dragony wings. The bactericidal efciency of both the bSi and D. bipunctata wing surfaces over the rst 3 h of incubation was calculated as the number of cells killed per square centimetre of projected sample surface area, per minute of incubation time. The number of cells killed was determined by subtracting the number of surviving cells from the number of cells remaining in controls at the corresponding time interval. The uncertainty values represent the natural variability associated with biological systems, and were calculated based on standard deviations in colony forming units. Error bars are based on s.d. values.

cereus as a representative of the non-pathogenic B. subtilis 105 cells)2426, it becomes clear that these samples would be highly effective antibacterial surfaces. For example, a sample of bSi 1 cm2 in size would be capable of killing the minimum infective dose ofS. aureus cells 810 times over 3 h, and in the case of P. aeruginosa this increases to 77,400 times the minimum infective dose. Analysis of the bactericidal activities in direct experiments with bSi surfaces in nutrient rich environments has indicated that viable cells in the infective dose range were eliminated after 6 h (Supplementary Methods and Supplementary Fig. S6).

DiscussionThere have been recent advances in both direct AFM measurement of bacterial cells, particularly with a view to examining the mechanical properties of cell walls and their internal osmotic turgor pressures27,28. For example, applying both modelling and direct AFM measurements, Deng et al.29 quantied the cell wall Youngs moduli of live E. coli as 23 and 49 MPa for axial and circumferential directions, whereas the cell turgor pressure was 29 kPa, through the analysis of the cell bulging strain under an externally imposed perturbation. As a consequence of the wall tension brought about by the indenting deformation, signicant power law stress stiffening of peptidoglycan network of the cell wall was seen to have an exponent 1.2. Here, we see that the competition between the elasticity of the cell membrane and the capillarity of the bSi and dragony wing architecture compared to cicada surfaces favors enhanced engulfment with its increased deformation and cell wall stress7, which is further increased with the longer, more pronounced nanospikes of the bSi (Supplementary Methods and Supplementary Fig. S7).

The extensive bactericidal activity displayed by the synthetic bSi provides an opportunity for a new approach to mechano-responsive microbiology, whereby the cell responds to the physical interaction forces of the surface structures (and their accompanying stresses), leading to cell deformation and death. The ability to readily fabricate such nanomaterials suggests their potential for use in the development of a wide range of bactericidal surfaces for use in biomedical and industrial applications. Our results demonstrated that (i) the nano-structured bSi and the natural D. bipunctata dragony wings are highly effective bactericidal surfaces, with the bactericidal activity being driven by mechanical and structural responses to the deformational stresses imposed by the surface nanoarchitecture on the peptidoglycan cell wall and inner membrane of bacterial cells, (ii) synthetic antibacterial nanomaterials that exhibit a similar effectiveness to the dragony wing surface structures in killing bacterial cells can be readily fabricated over

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large areas. These surfaces appear to be effective, independent of their surface chemical functionality, and (iii) such synthetic surfaces exhibit a bactericidal activity that is comparable to their natural counterparts and this may be further enhanced via optimization. These data provide a clear demonstration of the impact that mechano-responsive surfaces could play in countering microbial contamination, suggesting that novel antimicrobial nanomaterials may open the way for new applications in the eld of mechano-microbiology.

Methods

Reactive-ion beam etching. Black silicon was produced via RIE as described elsewhere3033. RIE with SF6 and O2 was performed for 5 min to produce the nanopillars on silicon wafers using an Oxford PlasmaLab 100 ICP380 instrument. A p-type boron-doped 100 mm-diameter commercial Si wafer with specic resistivity of 1020 O cm 1, (100) oriented surface and 52525 mm thickness (Atecom Ltd, Taiwan) was used as a substrate for the bSi formation. Separation between pillars was proportional to the etching time as pt0.5.

Insect wing preparation. Dragony specimens (D. bipunctata, Order Odonata) were collected from the Brisbane (Australia) parkland areas. The wings were precisely dissected using either scissors or a scalpel. These were then attached to circular discs by double sided adhesive tapes, then rinsed with copious quantities of MilliQ water (resistivity of 18.2 MO cm 1, Millipore, Billerica, MA, USA) followed by drying with 99.99% purity nitrogen gas.

Characterization of surface nanoarchitecture. High-resolution electron micro-graphs of bSi and dragony wings were recorded using a eld-emission SEM (FESEM; ZEISS SUPRA 40 VP, Oberkochen, BW, Germany) at 3 kV under

35,000, 70,000, 150,000 and 1,000,000 magnication. Prior to viewing,

the wing samples were coated with thin gold lms using a Dynavac CS300 (ref. 34). Several elds of view were recorded, including various sections of bSi samples and multiple regions of the wings, to conrm morphological consistency. The surface architecture was analysed initially using a Bruker AXS Contour GT 3D optical proling system in white light vertical scanning interferometry mode using 4,

and 230 objective lenses. Three samples were scanned to evaluate the overall

homogeneity of the surface, prior to detailed topographical proles at ve different locations on the surface. As the feature size of the nanostructures on bSi and the wing surfaces was below the optical resolution limit, their architecture was observed from the noise pattern of the optical images.

Characterization of surface chemical compositions. XPS was performed using a Kratos Axis Ultra DLD X-ray photoelectron spectrometer (Kratos Analytical Ltd., UK) equipped with a monochromatic X-ray source (Al Ka, hu 1486.6 eV)

operating at 150 W. The spectrometer energy scale was calibrated using the Au 4f7/2

photoelectron peak at binding energy of 83.98 eV. Samples were ooded with low-energy electrons during the analysis to counteract surface charging. The hydro-carbon component of the C 1 s peak (binding energy 285.0 eV) was used as a reference for charge correction. The elements present on the surfaces were determined from survey spectra in the range 01400 eV at an interval and pass energy of 1 and 160 eV, respectively. The relative atomic concentration of elements detected by XPS was quantied on the basis of the peak area in the survey spectra using sensitivity factors for the Kratos instrument. High-resolution scans were performed across each of the C 1 s, O 1 s and Si 2p peaks, which were then tted with GaussianLorentzian components after the removal of a linear background signal (Kratos Vision II software). Image analysis was carried out with Image J software (Ver. 1.47, Wayne Rasband, National Institute of Health, USA).

Surface wettability. Static water contact angles were measured on bSi and the wing using the sessile drop method7. The contact angle measurements were carried out in air using an FTA1000c equipped with a nanodispenser (First Ten ngstroms, Inc., Portsmouth, VA, USA). Volumes of the droplets were B1.0 ml.

The contact angles were measured by recording 50 images over 2 s with a Pelco model PCHM 575-4 camera and measuring contact angles after the droplet had rested on the surface for B1 s.

Bacterial growth conditions and sample preparation. Three strains were used:P. aeruginosa ATCC 9027, Staphylococcus aureus CIP 65.8T and Bacillus subtilis NCIMB 3610T. The selected strains are typical representatives of three large bacterial taxonomic lineages and were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), Culture Collection of the Institute Pasteur (CIP, Paris, France) and the National Collection of Industrial, Food and Marine Bacteria (NCIMB, Aberdeen, UK). Prior to each bacterial attachment experiment, bacterial cultures were refreshed on nutrient agar from stocks (Oxoid, Basingstoke, Hampshire, UK). Fresh bacterial suspensions were grown overnight at 37 C in

5 ml of nutrient broth (Oxoid, Basingstoke, Hampshire, UK). Bacterial cells were collected at the logarithmic stage of growth and the suspensions were adjusted to OD600 0.3. The insect wings, mounted on circular discs, were immersed in 5 ml

of the bacterial suspension for incubation intervals of either 1, 3 or 18 h. For spore formation, an aliquot from the vegetative cell suspension of B. subtilis NCIMB 3610Twas spread on to the nutrient agar (Oxoid) plate and incubated at 37 C for 7 days in depletion of oxygen. Sporulation (495%) was observed microscopically on glass slides by staining spores with malachite green and counter-staining healthy cells with safranin35. Spores were then suspended in phosphate buffered saline (PBS) solution, washed at 13,000 r.p.m. for 5 min and re-suspended. Before incubation on the insect wings, the spore suspension in PBS was adjusted to OD600 1. The insect wings were then incubated with spore suspension for 1, 3

and 18 h attachment experiments.

Cell viability analysis. Confocal laser scanning microscopy was used to visualize the proportions of live cells and dead cells using LIVE/DEAD BacLight Bacterial Viability Kit, L7012. A mixture of SYTO 9 and propidium iodide uorescent dyes (Molecular Probes, Invitrogen, Grand Island, NY, USA). SYTO 9 permeated both intact and damaged membranes of the cells, binding to nucleic acids and uorescing green when excited by a 485 nm wavelength laser. On the other hand, propidium iodide alone entered only cells with signicant membrane damage, which are considered to be non-viable, and binds with higher afnity to nucleic acids than SYTO 9. Bacterial suspensions were stained according to the manufacturers protocol, and imaged using a Fluoview FV10i inverted microscope (Olympus, Tokyo, Japan). For spore staining, a minor modication was made to this protocol36. Spores were stained with a mixture of 0.9 mmol l 1 of propidium iodide and 5 mmol l 1 of SYTO 9 for 1520 min. The samples were then imaged under the confocal microscope.

Bacterial viability plate counts. Viability assays were performed by standard plate counts37. P. aeruginosa, S. aureus and B. subtilis cells and spores were suspended in 5 ml of PBS and adjusted to OD600 0.1. Re-suspended cells were

diluted 1:10; then incubated in 3.5 cm diameter wells in triplicate with each well containing a 1 cm2 area substratum sample of either a D. bipunctata wing, bSi, smooth silicon wafer or a glass cover slip. The cell suspensions were then sampled (100 ml) at discrete time intervals (3 and 18 h), serially diluted 1:10, and each dilution spread on three nutrient agar plates. Resulting colonies were then counted, and the number of colony forming units per mL was calculated. The number of colony forming units was assumed to be equivalent to the number of live cells in suspension37. The maximum bactericidal efciency was measured as the number of inactivated cells per square centimetre of sample per minute of incubation time, calculated by subtracting the number of cells remaining in suspension from the corresponding control conditions.

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Acknowledgements

This research was supported by the Advanced Manufacturing Co-operative Research Centre (AMCRC), and was performed in part at the Melbourne Centre for Nanofabrication (MCN ANFF) and the Australian Synchrotron. We are grateful to Sasikaran Kandasamy at MCN for optimization of the RIE process, Song Ha Nguyen and Vy T.H. Pham for performing the viability experiments, Veselin Boshkovikj for performing the surface visualization and Yachong Uo for computer simulations.

Author contributions

E.P.I. conceived the project; E.P.I. and S.J. designed the experiments; J.H., G.G. and S.J. performed the experiments; E.P.I., S.J., J.H., V.A.B., R.J.C., D.E.M., R.N.L., H.K.W.,V.K.T., G.S.W., J.A.W. and A.H.F.W. analysed the data; E.P.I., H.K.W., D.E.M. and R.J.C. wrote the paper.

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How to cite this article: Ivanova, E. P. et al. Bactericidal activity of black silicon. Nat. Commun. 4:2838 doi: 10.1038/ncomms3838 (2013).

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