1 Facile Fabrication of Multifunctional ZnO Urchins on 2 Surfaces 3

Article 1 Facile Fabrication of Multifunctional ZnO Urchins on 2 Surfaces 3 Abinash Tripathy‡,¥, Patryk Wąsik‡,ѱ, Syama Sreedharan∥, Dipankar Nandi∥, Oier Bikondoa†,£, 4 Bo Su§, Prosenjit Sen¥ and Wuge H. Briscoe‡* 5 ‡ School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, United Kingdom. 6 ¥ Centre for Nano Science and Engineering, Indian Institute of Science, Bangalore, India, 560012. 7 ѱ Bristol Centre for Functional Nanomaterials (BCFN), HH Wills Physics Laboratory, University of Bristol, 8 Tyndall Avenue, Bristol BS8 1TL, United Kingdom. 9 ∥ Department of Biochemistry, Indian Institute of Science, Bangalore, India, 560012. 10 † XMas, The UK CRG Beamline at the ESRF, The European Synchrotron, 71, avenue des Martyrs, CS 40220, 11 38043 Grenoble Cedex 9, France. 12 £ Department of Physics, University of Warwick, Gibbet Hill Road, CV4 7AL, Coventry, United Kingdom. 13 § Bristol Dental School, University of Bristol, Bristol BS1 2LY, United Kingdom. 14 *Corresponding Author: Wuge.Briscoe@bristol.ac.uk 15 16 17 Abstract: Functional ZnO nanostructured surfaces are important in a wide range of applications. 18 Here we report facile fabrication of ZnO surface structures at near room temperature with 19 morphology resembling that of sea urchins, with densely packed, μm-long, tapered nanoneedles 20 radiating from the urchin centre. The ZnO urchin structures were successfully formed on several 21 different substrates with high surface density and coverage, including silicon (Si), glass, 22 polydimethylsiloxane (PDMS), and copper (Cu) sheets, as well as Si seeded with ZnO nanocrystals. 23 Time-resolved SEM revealed growth kinetics of the ZnO nanostructures on Si, capturing the 24 emergence of “infant” urchins at the early growth stage and subsequent progressive increase in the 25 urchin nanoneedle length and density, whilst the spiky nanoneedle morphology was retained 26 throughout the growth. ε-Zn(OH)2 orthorhombic crystals were also observed alongside the urchins. 27 The crystal structures of the nanostructures at different growth time were confirmed by synchrotron 28 X-ray diffraction measurements. On seeded Si substrates, a two-stage growth mechanism was 29 identified, with a primary growth step of vertically aligned ZnO nanoneedle arrays preceding the 30 secondary growth of the urchins atop the nanoneedle array. The antibacterial, anti-reflective, and 31 wetting functionality of the ZnO urchins – with spiky nanoneedles and at high surface density – on 32 Si substrates was demonstrated. First, bacteria colonisation was found to be suppressed on the 33 surface after 24 h incubation in Gram-negative E. coli culture, in contrast to control substrates (bare 34 Si and Si sputtered with 20 nm ZnO thin film). Secondly, the ZnO urchin surface, exhibiting 35 superhydrophilic property with a water contact angle ~ 0°, could be rendered superhydrophobic 36 with a simple silanization step, characterised by a water static contact angle θ of 159°±1.4° and 37 contact angle hysteresis ∆θ < 7°. The dynamic superhydrophobicity of the surface was demonstrated 38 by bouncing-off of a falling 10 μL water droplet, with a contact time of 15.3 milliseconds (ms), 39 captured using a high-speed camera. Thirdly, it was shown that the presence of dense spiky ZnO 40 nanoneedles and urchins on the seeded Si substrate exhibited a reflectance R < 1% over the 41 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 November 2018

Here, we report facile fabrication of multifunctional 3-D ZnO urchins on silicon (Si) substrates in a solution-based synthesis at near room temperature (RT) using a one-step procedure, which could also be applied on Si substrates seeded with ZnO crystals and various other substrates.The morphology of these ZnO surface structures resembles that of sea urchins, with tapered nanoneedles of m in length radiating from a central core.The growth kinetics of the ZnO urchins was studied by examining their intermediate morphologies at different growth time intervals (0.5 -12 h).The ZnO urchin-coated surface exhibited high anti-reflectance, and superhydrophobicity after silanization as investigated by contact angle, contact angle hysteresis and drop impact analyses.They were also bacteriophobic against E. coli.The simple fabrication method for ZnO urchins could also be adapted to a variety of materials such as polydimethylsiloxane (PDMS), copper sheets and glass substrates, demonstrating its versatility.

Seeding
Seeding procedure was used to produce nucleation sites for the growth of ZnO nanostructures 31 .
Cleaned silicon substrates were dipped in a solution of zinc acetate dihydrate (Zn(CH3COO)2•2H2O, 99%, Sigma Aldrich) in ethanol (CH3CH2OH, Absolute, Sigma Aldrich) for few seconds, rinsed with clean ethanol and then dried with N2.This coating step was repeated 5 times for each seeded silicon substrate (see Figure S1).Subsequently, the substrates were heated to 300° C on a hot plate, and annealed for 30 minutes in air to thermally decompose zinc acetate crystallites to ZnO islands with (0001) planes parallel to the silicon substrate surface 31 .

ZnO Urchin growth
To grow the ZnO nanostructures, 50 mL aqueous solution of zinc nitrate hexahydrate (Zn(NO3)2•6H2O, 98%, Sigma Aldrich) solution was added dropwise to 50 mL of potassium hydroxide (KOH, 85%, Fisher Scientific) aqueous solution under constant stirring for 30 minutes.Next, the alkali solution of zincate ions was carefully poured to the glass Petri dish containing substrates (seeded Si, non-seeded Si, Cu sheet, glass and polydimethylsiloxane, separately for each type) sealed with a glass lid and a paraffin film, and kept at 20°± 2°C for 12 hours.After the synthesis, samples were rinsed with DI water and dried with N2.

Bacterial growth conditions and sample preparation
Isolated single colonies of E. coli K-12 (MG 1655) were used to prepare the pre-inoculum 52 .The culture was grown in Luria Bertani (LB) medium for 8 hrs at 37°C with constant shaking at 160 rpm.A 0.2 % pre-inoculum was added into 10 mL of LB medium and it was allowed to grow until 0.3 optical density (O.D.) at 600 nm 53 .The cells were subsequently pelleted and washed with phosphatebuffered saline (PBS).A cell suspension of 0.01 O. D. at 600 nm was used for studying the bacterial interaction with the bare, unmodified silicon, silicon with 20 nm thin film of ZnO and ZnO nanourchin surfaces.

Scanning electron microscopy (SEM)
ZnO nanostructured surfaces were imaged using field emission scanning electron microscopy (JSM-IT300 SEM (JEOL)).Dimension of the ZnO nano urchin-like structures were obtained from the SEM images using ImageJ software.For bacterial sample imaging, substrates were first washed with PBS to remove the loosely adhered cells.Substrates were then dipped in 2.5% of Glutaraldehyde to fix the cells.After that all the substrates were allowed to dry in vacuum.Thin layer of gold (15 nm) was sputtered on the samples using a Quorum sputter coater (Q150T) to avoid the charging effect while doing the SEM.Samples were scanned thoroughly using SEM (sample size 1.3 cm × 1.3 cm) and the imaging was done in triplicates for all the samples.Selecting three specimens from each type of a sample (two controls and one ZnO Urchin surface) ensured the repeatability.

Urchin dimension analysis
ImageJ 54 was used to obtain the morphological information of the nanoneedles comprising the ZnO urchin and the vertical nanoneedles using the length and angle measurement tools.

Grazing incidence X-ray diffraction (GIXRD)
GIXRD analysis of the seeded and non-seeded silicon substrates used to study the time dependence growth of ZnO nanostructures was performed at Beamline BM28 at the European Synchrotron Radiation Facility (France).Experimental parameters: radiation wavelength: 0.8856 Å, sample to detector distance: 0.24 m, detector: MAR165, calibrant: Silver behenate and ZnO powder.The diffraction patterns were reduced to one-dimensional line profiles using pyFAI, a pythonic library for 1D azimuthal / 2D radial integrations of diffraction images 55 .

Contact angle measurement and high-speed imaging
Static contact angle measurements were carried out using a Krüss Drop Shape Analyser (DSA 100).10 µL water droplet was placed gently on all the substrates to measure the contact angles.A droplet impact dynamics of water droplet on the superhydrophobic surface (salinized seeded silicon substrate with ZnO nanostructures) was captured using a high-speed camera (Photron FastCam SA4) at 10,000 fps (time resolution of 0.1 ms).The droplets were created by a micro-pipette and released from a height of 7 cm (Weber number () = 1000 ,     () = 2ℎ = 1.17 ,     () = 0.072 , and    ( ) = 0.0026 ).

Reflectance measurements
To calculate the reflectance of all the surfaces, Shimadzu MPC3600 UV-VIS-NIR Spectrometer with an absolute specular reflectance mode was used.Wavelengths ranging from 200 nm to 800 nm were used for the reflectance measurement.D2 light source was used for the range 200 nm -310 nm and Tungsten source was used for the range 310 nm -800 nm.The angle of incidence and angle of reflection were set to 5° throughout the experiment.The equipment used a photomultiplier tube (PMT) detector.The reflectance from a surface is evaluated by its refractive index profile 6 .In general, a flat surface has a high reflectance due to the discontinuous refractive index profile, whereas structured surface suppresses reflection with its graded refractive index profile 6 .

Results and Discussion
Figure 1 shows the SEM images of densely packed urchins after growth in 5 mM zincate solution on a seeded silicon substrate (Figure S2) at ~20° C for 12 h, and the simple fabrication method is described in Figure S1.XRD (Figure S11) confirmed that these urchins were ZnO.The morphology of an individual urchin (Figure 1c) reveals sharply tapered needles radiating from the centre.The length of the needles was L~ 1.65 ±0.11 µm, its width at the central base Dc ~ 156±24 nm and at the tip Dt ~ 13±7 nm, with a tapering angle  ~10 o and an average tip-to-tip spacing s ~ 504±119 nm (see Figure S3-S5 for the size distribution analysis).
Previously, ZnO nano-and micro-structures bearing resemblance of the urchin morphology have been reported.For instance, Elias et al. 48prepared µm-sized hollow spheres with a ZnO nanocolumns coating using atom layer deposition and electrodeposition, whilst the method by Shen et al. 47 used thermos-evaporation of metallic Zn powder at high temperature (~750 o C).Wahab et al. 43 fabricated ZnO nano-flowers blunt tapering via pH-controlled reactions in solution of zinc acetate dihydrate and sodium hydroxide at 90 o C. Using a similar approach, Gokarna et al. 44 synthesised ZnO urchin-like structures with columnar nanoneedles.Hieu et al. 45 sputtered zinc onto a polystyrenesphere array and subsequent oxidation at 500 o C led to columnar ZnO urchins ; and the method by Taheri et al. 46 also involved depositing zinc acetate dihydrate precursor followed by calcination at 500 o C. The solution synthesis method we report here is relatively much simpler (at RT and applicable to different surfaces as we show below) compared to these previous studies, producing spiky tapered morphology of the urchin needles with a very high density previously unreported.The presence of high density spiky ZnO urchins endows the surface with multi-functionalities as discussed below.
SEM images of the region not covered by the urchins reveal vertical ZnO nanoneedles (from the primary growth step as referred to in Figure 1d, g) with an average base diameter ~ 90±20 nm, smaller than that of the urchin needles (Figure 1d-g), and the cross-section SEM view confirms that the ZnO urchins were formed atop a layer of nanoneedle arrays (Figure 1a, b).Orthorhombic -Zn(OH)2 crystals with facet edges ~10-30 µm were also observed (Figure S7), with ZnO urchins (the secondary growth in Figure 1d) decorating the facets.The tapered or spiky nanoneedle geometry might be attributed to the concentration gradient of the zinc ions in the vicinity of the substrate where the ZnO nanocrystal seeds provided the nucleation sites for ZnO nanoneedles growth (Figure 1f, g) 56 .
Preprints To evaluate the effect of substrate seeding on the ZnO urchin morphology, the one-step fabrication procedure was performed using unseeded silicon substrates (i.e. in 5 mM zincate solution at 20° C, and 12 h growth time).SEM image in Figure 3a shows that urchin like ZnO nanostructures were also obtained with a high surface coverage (Figure 3a, c), with urchins found on orthorhombic -Zn(OH)2 crystal facets (Figure 3b).There was no significant difference in the urchin morphology (urchin size φ, needle length L, diameter D, and tip angle ) compared to the seeded substrate.However, some urchins in the bare silicon area had a smaller number of nanoneedles (Figure 3d).Comparison of the XRD patterns in Figure S11 for the 12 h growth shows that, on the unseeded Si sample, the most intense peaks correspond to the -Zn(OH)2 crystal structure, consistent with the SEM images shown in Figure 3.  the primary growth led to ZnO nanoneedles, with the ZnO urchins grown atop in the secondary growth stage.In the case of the unseeded Si sample, the most intense peaks correspond to the -Zn(OH)2 crystal structure.This is consistent with the SEM image (Figure 3b) which shows that the substrate was covered by ZnO urchins grown on orthorhombic Zn(OH)2 crystals and bare Si surface, with a significantly smaller amount of arrayed ZnO nanoneedles (Figure d).
Growth of the ZnO nanoneedles and urchin structures were also trialled on copper sheets, soft PDMS and transparent glass substrates, all seeded by dipping the substrates in a solution of zinc acetate dihydrate in ethanol (Figure S1).The ZnO nanostructures grown on these surfaces (Figure g-i) exhibited similar urchin morphologies to the those on silicon substrates (Figure 1).The growth on glass was also evident, as it became translucent after the growth (Figure d and Figure S12).This demonstrates that the facile, room temperature synthesis method for ZnO nanoneedles and urchins is adaptable to various substrates.S1).They became superhydrophobic after overnight silanization (Table S1) with a static water contact angle  ~ 159°±1.4°(Figure 5a) and a contact angle hysteresis ∆ < 7° was observed.For comparison, Table S1 shows that bare Si and Si coated with 20 nm ZnO thin film exhibited hydrophilic behaviour with a water static contact angle  ~ 36° and ~ 61°, respectively.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 November 2018
To further demonstrate the superhydrophobicity in dynamic conditions, a 10 µL water droplet was allowed to fall on the silanized ZnO urchin surface from a height of 7 cm with a Weber number  = = 49, where ρ = 1 g cm -3 is the density of water, V = 2ℎ=1.17m s -1 the impact velocity of the falling droplet ,  = 73.8mN m -1 the surface tension of water, and Dw = 2.67 mm the diameter of droplet (Table S1).The dynamic process was captured using a high-speed camera with a 10,000-fps capture rate.The water droplet bounced off the superhydrophic surface completely without leaving any visible residues, with a droplet-substrate contact time of 15.3 ms (Figure 5b and Video S1) which is close to the theoretical contact time (( ) / = 16.25 ms) for water droplet on superhydrophobic surface 58 .
In addition, the ZnO urchin coated surface also demonstrated anti-reflective behaviour.Error!Reference source not found.cshows <1% reflectance (R) over the wavelength range λ = 200-800 nm on the urchin surface, compared to two control samples (bare Si and Si with 20 nm ZnO thin film).
Different techniques such as dry etching, wet etching, metal assisted etching, nanoimprint lithography, etching using O2 plasma and inductively coupled plasma, optical lithography followed by etching etc. 66 have been used to fabricate such surfaces on silicon, polymers, and glass to achieve low reflectance.In comparison, the fabrication method we used to achieve the low reflectance (R < 1%) was simple with the synthesis undertaken at room temperature without the need for sophisticated instrumentation.nm ZnO film (with the reflection of the iPhone used to take the photo clearly visible), in contrast to the anti-reflective characteristic of the ZnO urchin surface.
To evaluate the bacterial interaction with the fabricated ZnO urchins on the seeded Si surface, it was submerged in 2 mL E. coli culture in phosphate-buffered saline (PBS) for 24 h and then imaged with SEM.This was compared with two control samples: unmodified bare Si and Si with a 20 nm sputtered ZnO film (Figure S13).SEM images in Error!Reference source not found.a(see also Table S2) show bacteria growth on the control surfaces; in contrast, no bacterium was observed on the ZnO nanostructured surface (Figure 6c, d).In addition, the SEM analysis revealed a change in the morphology of the ZnO urchins (inset in Error!Reference source not found.c):after 24 h immersion in the E. coli culture, the spiky urchin structure was transformed into the hexagonal pyramid structure (see Figure S14a).This can be attributed to the formation of sodium zinc phosphate hydrate (NaZn-PO4.H2O) 67 due to the reaction between ZnO urchins and the PBS (cf.XRD data in Figure S14b).ZnO is known to exhibit antimicrobial efficacy [15][16][17][18][19] , killing bacteria and prohibiting bacterial growth on its surface via release of Zn 2+ ions which solicits generation of reactive oxygen species (ROS).A number of naturally occurring surfaces bearing spiky nanotextures (e.g.cicada wing, dragonfly wing, and gecko skin) have been reported to exhibit bactericidal efficacy, attributed to puncturing or stretching of the bacterial membrane, although the detailed mechanisms remain to be fully understood 3,11,[68][69][70][71] .
Hence, we suggest that the ZnO urchin surfaces could prevent bacterial growth by combining synergistically the inherent chemical activity of ZnO and the spiky morphology of the urchins that inhibits the bacteria to colonise on the surface of the ZnO urchins 18,[72][73][74] .The sharp topography of the nanoneedles are particularly effective in disrupting the bacterial cell wall by imparting the localised stress on the bacterial membrane 75 .There are previous reports on reduced bacterial adhesion on superhydrophobic surfaces [76][77][78][79] where the antiwetting property of the surface plays a crucial role in the interaction of the bacteria with the surface; whereas our fabricated ZnO urchin surfaces exhibited bacteriophobic behaviour in a hydrophilic state (with a water contact angle θ ~ 0 o ).

Conclusions
In summary, ZnO urchin structures were fabricated via a simple method involving submergence of a substrate (Si, PDMS, glass, or Cu) in an alkaline aqueous zincate ion (Zn(OH)4

Figure 1 :Figure 2 :
Figure 1: (a) Scanning electron microscopy (SEM) images of ZnO urchins on a seeded silicon substrate; (b) SEM images depicting the ZnO urchin and primary vertical nanoneedle growth on the silicon substrate.SEM images show random multidirectional growth of the nanoneedles comprising the urchin (seeding concentration of zinc acetate dihydrate -5 mM, synthesis time for ZnO urchin growth -12 h, and temperature -20° C).

Figure 3 :
Figure 3: SEM images of ZnO nanostructures grown on the unseeded silicon substrate in the one-step synthesis (12 h, 20° C), with a high coverage of ZnO urchins (a) with morphology similar to that on seeded Si substrates (b, c).Orthorhombic ε-Zn(OH)2 crystals were also observed (b) decorated with urchins.Urchins with a smaller number of needles were also observed in the bare silicon area (d).

Figure
Figure further compares the XRD on both seeded and non-seeded silicon substrates after the growth in the zincate solution for 12 h.Peaks corresponding to both ZnO (PDF 36-1451) and -Zn(OH)2 (PDF 38-385) were identified, confirming their formation on the surfaces.The most intense peak in the seeded silicon sample corresponds to the ZnO (002) plane, which is the preferential growth direction of ZnO nanoneedles57 .Along with the SEM images, this confirms that on seeded Si

Figure 4 :
Figure 4: (a) Growth of ZnO urchin on seeded copper, glass slide and PDMS surfaces and the corresponding SEM images of the ZnO urchin formed on the substrates.

PreprintsFigure 5 :
Figure 5: (a) Photograph of a 10 µL water droplet on ZnO urchin-coated surface after silanization, with a contact angle θ ~ 159°±1.4°.Prior to silanization, the surface was highly hydrophilic, displaying complete wetting by water with θ ~ 0° (Table S1).(b) High-speed camera images of a 10 µL droplet bouncing off the superhydrophobic ZnO urchin surface, with a contact time of 15.3 ms.(c) Reflectance from the ZnO urchin surface compared to the two control surfaces in the wavelength range λ= 200 -800 nm.ZnO urchin surface was found to be the most anti-reflective with reflectance R < 1% over the whole wavelength range.The inset shows the highly reflective bare Si and Si with 20

PreprintsFigure 6 :
Figure 6: Representative SEM images of E. coli (false coloured Green) on different surfaces after 24 h of bacterial culture.(a) bare, unmodified silicon, (b) silicon with 20 nm thin film of ZnO and (c) & (d) ZnO urchin surface.Inset in Figure 6(c) shows the morphology of the ZnO urchin before bacterial culture on the urchin surface.

Figure S1 :
Figure S1: Process flow of the fabrication of ZnO nanostructures on seeded silicon substrates at near room temperature, Figure S2: SEM images of a silicon substrate seeded with ZnO nanoislands in 5 mM zinc acetate dihydrate solution (dipping × 5 times and annealed at 300° C for 30 minutes), Figure S3: Nanoneedle dimension measurement using ImageJ software.The values in the main text were averaged from 50 different needles, with the measurements of different parameters at each single needle repeated 5 times, Figure S4: Size distribution from ImageJ analysis of the nanoneedle tip diameter at the top Dt urchins ranging from 7-30 nm with an average diameter of Dt = 13±7 nm, Figure S5: Length of the nanoneedles L in the urchin structure vs. growth time.Length of the nanoneedle was found to increase with respect to synthesis time.Stars in figure shows that for time points 0, 0.5 and 1 h there was no formation of urchin structures, Figure S6: (a) the angled view after 9 h growth, showing ZnO urchins on the top of highly (b) oriented ZnO nanoneedles, Figure S7: Representative FESEM images of the seeded silicon substrate post 12 h synthesis in the zincate solution, Figure S8: SEM images of seeded silicon substrates after growth in zincate solution at different time intervals.Density of ZnO nano urchins was found to increase with the increase synthesis time, Figure S9: SEM images of seeded silicon substrates after growth in zincate solution at different time intervals.Zn(OH)2 crystals were observed on the substrates for synthesis time t > 3 h, Figure S10: SEM images of ZnO urchins on a non-seeded silicon substrate, taken at different time intervals: (a) 3 h, (b) 6 h, (c) 9 h, and (d) 12 h.(e-h) show enlarged views of the square regions as labelled in (a-d), respectively, Figure S11: XRD of (a)&(b) seeded and (c)&(d) unseeded silicon substrates after growth in the zincate solution at 20° C for 12 h, with the ZnO and -Zn(OH)2 peaks indicated by * and ▼, respectively, and the enlarged views shown on the right-hand side, Figure S12: Optical image showing the loss in transparency after the formation of ZnO urchin/nanoneedles on the glass substrate, Figure S13: 12 well culture plate for pouring the bacterial suspension on different samples, Figure S14: (a) SEM and (b) XRD of ZnO nanourchin surface after pouring the bacterial culture for 24 hours.Formation of sodium zinc phosphate hydrate (NaZn-PO4.H2O) was observed on the surface due to the reaction of ZnO with PBS leading to the change in morphology of the spiky ZnO nanowires, Table S1: Static contact angles of DI water droplet on different substrates, TableS2: SEM of E. coli on different substrates, Video S1: Superhydrophobic ZnO urchin.

preprints.org) | NOT PEER-REVIEWED | Posted: 22 November 2018 angle
of ~0o ; after silanization, it exhibited superhydrophobicity with a water contact angle of 159° and hysteresis smaller than 7°.In addition, its dynamic hydrophobicity was demonstrated by bouncing-off of a water drop as captured by a high-speed camera, with resident time of 15.9 ms.Furthermore, the ZnO urchin surface showed bacteriophobic behaviour, as compared to the control Si and ZnO-coated surfaces, with no bacterium colonization observed on the surface after 24 h incubation in E. coli.The facile method for preparing ZnO urchins with unique morphology of tapered nanoneedles, and its ready adaptability to different surfaces (including polymers), may open new routes for fabrication of multifunctional ZnO nanostructured surfaces.
the urchin morphology emerged at ~ 3 h reaction time with a small number of, but tapered, nanoneedles.The nanoneedle density and length then progressively increased with the reaction time.On the seeded Si substrate, a primary growth step of the vertical ZnO nanoneedles was identified preceding the secondary urchin growth.The ZnO urchin coated surface exhibited anti-reflective properties, reducing the reflectance to less than 1%.It was highly hydrophilic, with a water contact Preprints (www.