Black silicon spacing effect on bactericidal efficacy against gram-positive bacteria

The morphology of regular and uniform arrays of black silicon structures was evaluated for bactericidal efficacy against gram-positive, non-motile Staphylococcus epidermidis (S. epidermidis). In this study, uniform and regular arrays of black silicon structures were fabricated using nanosphere lithography and deep reactive ion etching. The effects of nanomorphology on bacterial killing were systematically evaluated using silicon nanostructures with pitches ranging from 300 to 1400 nm pitch on spherical cocci approximately 500 to 1000 nm in diameter. Our results show that nanostructure morphology factors such as height and roughness do not directly determine bactericidal efficacy. Instead, the spacing between nanostructures plays a crucial role in determining how bacteria are stretched and lysed. Nanostructures with smaller pitches are more effective at killing bacteria, and an 82 ± 3% enhancement in bactericidal efficacy was observed for 300 nm pitch nanoneedles surface compared to the flat control substrates.

Recent nanofabrication approaches, including 3D printing [25], nanosphere lithography [26], and reactive ion etching [27,28], have been able to demonstrate bio-inspired antibacterial surfaces that either (1) reduce bacteria adhesion [29,30] or (2) mechanically kill bacteria cells.The first approach uses a combination of surface nano topography and surface functionalization to reduce bacteria adhesion [31,32].The second antibacterial approach was inspired by cicada (Psaltoda claripennis) wing surfaces, which have been shown to be mechanically bactericidal to gram-negative Pseudomonas aeruginosa (P.aeruginosa) cells [33][34][35][36][37][38].However, cicada wing structures have demonstrated limited effect on Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence.Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.rigid, gram-positive bacteria cells [39].Biophysical models of bacterial cell/surface interactions have suggested that this is due to the cell rigidity of gram-positive bacteria [40].The cell walls of gram-negative bacteria are generally only 5-10 nm thick, consisting of a single peptidoglycan layer [41].In contrast, the thickness of the cell walls of gram-positive bacteria is around 20-80 nm, which consists of several layers of peptidoglycan.Both black silicon and dragonfly wings (Diplacodes bipunctata) havedemonstrated bactericidal properties against gram-negative P. aerouginosa, grampositive Staphylococcus aureus (S. aureus), and the vegetative cells and spores of Bacillus subtilis [39,[42][43][44][45].
Hazell et al employed black silicon and diamond-coated black silicon substrates with various surface morphologies and tested those surfaces against both gram-negative Escherichia coli (E.coli) and gram-positive Streptococcus gordonii bacteria cells [46].However, killing was only observed for gramnegative E. coli.Furthermore, other studies suggest that nanopatterned arrays may be selectively bactericidal towards bacteria that are motile as opposed to those that are non-motile.Diu et al showed that nanopatterned surfaces were effectively bactericidal against motile bacteria (gram-negative P. aeruginosa, gram-negative E. coli, and gram-positive Bacillus subtilis), while ineffective against non-motile bacteria (gram-positive S. aureus, gram-positive Enterococcus faecalis, and gram-negative Klebsiella pneumoniae) [47].
In this paper, we systematically study the effect of the morphology of different uniform and regular black silicon arrays on bactericidal efficacy.The black silicon is fabricated using a nanosphere lithography approach, which allows us to create a hexagonal array of regularly spaced nanoneedles.Previous studies on mechanically bactericidal nanostructures have focused on non-uniform and irregular nanostructures in cicada wings and black silicon.Such randomness complicates the analysis of how specific changes in surface morphology affect bactericidal efficacy [48][49][50].These nanoneedles are tested against Staphylococcus epidermidis (S. epidermidis), which consists of cocci approximately 500-1000 nm in diameter, arranged in clusters [51,52].S. epidermidis is a gram-positive, low-motility bacterium, a common infection source on indwelling medical devices such as catheters.The combination of both thick cell walls as a gram-positive bacteria and low motility suggest that S. epidermidis is challenging to mechanically kill and no surfaces in the literature have demonstrated a mechanical bactericidal effect against S. epidermidis.Studying uniform black silicon arrays on spherical bacteria allows for a more systematic analysis.This study evaluates regular and uniformly arrayed black silicon with pitches ranging from 300 to 1400 nm, which spans the range of Staphylococcus epidermidis (S. epidermidis) diameters.
Our findings indicate that while surface parameters such as height and surface roughness do not directly influence bactericidal efficacy, pitch is the key determinant.Scanning electron microscope images provide insight into how the bacteria interact with various surfaces and why smaller pitch nanostructures lead to higher bactericidal efficacy.Black silicon arrays with larger pitches of 800 and 1400 nm do not demonstrate any killing of bacteria.This is because S. epidermidis cells can deform and fit into the spaces between nanoneedle structures.In contrast, 500 and 300 nm pitch black silicon samples demonstrate bactericidal efficacy, where the bacteria cells sit on top of the nanoneedle structures.At the three-hour time point, the 500 nm pitch black silicon demonstrates 30 ± 3% bactericidal efficacy.In contrast, the 300 nm pitch black silicon demonstrates the highest bactericidal efficacy against S. epidermidis compared to the control.These small pitch nanoneedle arrays provide the highest killing because bacteria cells are deformed the most.This stretching of the cell wall results in tearing where cytoplasmic materials discharge from the cells, and are lysed.

Experimental procedure
Fabrication of black silicon substrates P-type boron-doped (100) silicon wafers served as substrates.These substrates were cleaned sequentially with acetone, methanol, and isopropanol before being dried with nitrogen gas.Subsequently, polystyrene (PS) nanospheres were patterned self-assembled at the air-water interface [26,[53][54][55].This monolayer was then transferred to the substrate and allowed to dry at room temperature.Reactive ion etching (RIE) with oxygen was used to reduce the diameter of the PS nanospheres.The pressure was set at 25 mTorr and the RF power was set at 25 W. The flow rate of oxygen was 25 sccm, which yielded an etch rate of 80 nm per min.By adjusting the etch duration, the diameters of the nanosphere mask could be modified.Inductively coupled plasma reactive ion etching (ICP-RIE) was then used to etch the silicon, with the PS nanospheres acting as a mask.SF 6 was used as the etching gas and C 4 F 8 was used as the passivation gas.The ratio of etching gas to passivation gas was 33 sccm to 82 sccm.The etch duration was varied to obtain nanoneedles of different heights.Tapering effects were observed on the nanoneedles, with varying tip and base diameters for all structures.Finally, the PS nanospheres were removed by ultrasonication in acetone for 5 min.

Bacterial culture preparation
Single colonies of S. epidermidis were isolated by streaking from frozen glycerol stocks onto Petri dishes with peptone yeast (PY) agar.The agar was comprised of 15 g l −1 peptone (BD Difco), 1 g l −1 yeast extract (BD Difco), 15 g l −1 bacto agar (BD Difco), and 0.25% dextrose (Fisher Scientific).After streaking, the dishes were incubated at 37 °C overnight.A single colony from these dishes was selected to inoculate 5 ml of PY broth, which contains the same constituents as the PY agar, excluding the agar.This culture was incubated overnight at 37 °C with agitation at 250 rpm.A 50 ml culture was then initiated using 2 ml of the overnight culture, with 48 ml of peptone yeast-extract dextrose (PYD) in a 250 ml baffled flask.After an 18-h incubation at 37 °C with 250 rpm agitation, the culture achieved an Optical Density at 560 nm (OD560) of 1.49 absorbance units.The 50 ml culture wastransferred to a 50 ml conical tube and centrifuged at 5000 × g.The supernatant was discarded, and the bacterial pellet was resuspended and diluted 1:10 in 0.01 M Phosphate-Buffered Saline (PBS), resulting in a culture with an OD of 0.1 absorbance units.PBS was chosen not as a growth medium but as a buffer, given its lack of nutrients essential for bacterial proliferation.Nonetheless, it provides suitable conditions to maintain the structural integrity of the bacterial cells for further experiments.

Bactericidal efficiency experiments
For each silicon sample, 10 μl of the 1:10 S. epidermidis:PBS culture was aliquoted onto each1.5 cm by 1.5 cm substrate.Four replicates of each of the five distinct samples were evaluated at three different time points: 0, 1, and 3 h.60 samples were evaluated in total.Matlab 2021a was used for conducting statistical analyses and generating corresponding plots.For the 0 h time, the bacteria culture was dropped and immediately withdrawn from the substrates.At the one-hour and three-hour time points, the substrates were kept inside a humidity-controlled environment after the culture was aliquoted for the specified amount of time.The relative humidity was maintained at nearly 100% to avoid evaporation.Given the difficult in completely recovering the 10 μl culture, an additional 90 μl PBS was added.This was mixed well by pipetting, after which100 μl was withdrawn.Subsequently, the bacterial culture underwent a 10-fold serial dilution in 270 μl of 1× PBS.From the E-4 dilution, 100 μl was plated on PYD agar and spread with sterile glass beads.The bactericidal effect was evaluated in triplicate for both the bare silicon control and black silicon arrays.After incubating the plates at 37 °C for 24 h CFU/mL counts were determined.Scanning electron microscopy (SEM; Zeiss Sigma 500 VP) was used to evaluate the surface of the used black silicon samples.

Results and discussion
Four different black silicon surfaces were fabricated, evaluated, and then compared to a flat control silicon sample.Figure 1 presents SEM images, tilted at a 70 • angle from overhead, illustrating varying pitches of (a) 1400, (b) 800, (c) 500, and (d) 300 nm.SEM was used to characterize the physical morphologies of the four different black silicon surfaces.The pitch of each sample was determined by the diameter of the close-packed PS nanospheres used.Thus, the range of pitches studied spans the size of the interacting bacteria.Table 1 summarizes the morphology and bactericidal efficacy evaluations of the four black silicon samples.Different ICP RIE times were employed to vary the height of the samples.The four black silicon samples were etched for 32, 17, 11, and 7 min, respectively, to achieve heights of 6.0, 2.3, 2.0, and 1.0 μm, respectively.The roughness was calculated by the ratio of the surface area of the etched substrate to the flat control surface.
The four black silicon samples and the control sample were then subjected to bactericidal efficacy experiments.Figure 2(a) displays bacterial counts at various time points for all five different silicon samples, including the control and the four black silicon samples.For the zero-hour time point data, the bacterial culture was aliquoted and retrieved immediately.In these experiments, the bacteria did not have sufficient time to interact with the black silicon surface.This step was utilized to evaluate the effect of the aliquoting process and subsequent withdrawal of bacteria.At the 0 h time point, no statistically significant difference in S. epidermidis counts was observed across any of the evaluated samples.
The bactericidal effect for a specific sample was quantified by subtracting the number of surviving cells on it from the number of cells remaining on the bare silicon control at the corresponding time point.At the one-hour time point, the bactericidal effect was observed only for the 300 nm pitch samples.20 ± 6% killing was observed for these samples.A two sample t-test against the control samples yielded a pvalue of 0.006.There was some variation among the other types of samples, but there was no statistically significant killing of bacteria.Two sample t-tests against the control samples produced p-values of 0.35, 0.43, 0.66 respectively for the 1400, 800, and 500 nm pitch black silicon substrates, respectively.
At the three-hour mark, the 1400 and 800 nm pitch samples had no significant bactericidal effect withp-values of 0.12 and 0.16, respectively.The 500 nm pitch samples demonstrate killing (p-value of 0.003), where 30 ± 3% bacteria are killed.The 300nm pitch samples performed best with a p-value <0.00001.These samples significantly reduced the number of viable cells and the killing was measured to be 82 ± 3%. Figure 2(ii) displays representative agar plates from the three-hour time point, with the most pronounced bactericidal effect observed on the 300 nm pitch samples characterized by nanoneedles with minimal height and spacing.Various reports on bactericidal effects of nanostructured surfaces suggest that the nanostructures' pitch significantly influences bactericidal performance, with variations in pitch correlating with efficacy.Theoretical studies have posited that larger pitches can boost bactericidal efficiency [56].However, this model treated bacteria as a semi-infinite, thin elastic layer.It assumed bacteria to be considerably larger than both the nanostructures and their spacing.Dickson et al studied E. coli on the uniform, nanopatterned PMMA surfaces [57].These studies report that more tightly packed nanopillars are more effective at killing bacteria.However, E. coli is a gram-negative bacteria.Linklater et al [58] also studied various black silicon surfaces on gram-positive S. aureus and gram-negative P. aeruginosa, but these surfaces were non-uniform.They found that surfaces with denser nanoneedles were more effective at killing bacteria.Hazell et al studied various black silicon surfaces on E. coli and S. gordonii [46].The black silicon surfaces were nonuniform, but higher areal density was found to result in the greater killing of E. coli.However, the surfaces were ineffective against gram-positive S. gordonii.
To gain mechanistic insights into bactericidal efficiency differences, SEM images were taken of S. epidermidis on various black silicon samples at the 3 h mark.Figure 3 shows these results.Figure 3(a) and (b) show results for the 1400 and 800 nm pitch black silicon samples.These samples showed no statistically significant bactericidal effect.S. epidermidis has a diameter of 0.5-1 μm.The larger-pitch black silicon surfaces are ineffective at killing S. epidermidis, as their larger pitches allowed bacteria to simply settle in spaces between the nanoneedles.For the800 nm pitch, despite being smaller than some S. epidermidis cocci, bacteria can deform and nevertheless, squeeze into areas between the nanoneedles, resulting in no significant bactericidal effect.Figure 3(c) shows SEM images of S. epidermidis on top of the 500 nm pitch black silicon samples.Some deformation can be seen in the bacteria cells, though they largely remain spherical.As discussed above, 30 ± 3% bacteria are killed.In contrast, the 300 nm pitch black

Conclusion
Black silicon substrates, fabricated using nanosphere lithography and deep reactive ion etching, exhibited significant bactericidal effects on S. epidermidis, a gram-positive, nonmotile bacterium.By controlling the height, pitch, and top diameter of these substrates, the interaction between the sharp nanoneedles of black silicon and bacterial cells was evident.
When bacterial cells come into contact with these nanoneedles, their cell walls and membranes can sustain damage.Notably, gram-positive bacteria with their thicker cell walls, are often more resistant to mechanical destruction.However, our SEM images revealed pronounced bacterial deformation, particularly on substrates with a smaller pitch.Samples with a 300 nm pitch were particularly effective, exhibiting an 82% bactericidal rate at the three-hour mark.Deformation on the black silicon surface results in compromised cellular integrity, causing cellular contents to leak, which ultimately leads to cell death.As bacterial resistance to drugs continues to rise, our results highlight the potential of black silicon substrates as an innovative approach for creating bactericidal surfaces.

Figure 2 .
Figure 2. Colony-forming unit (CFU) studies of bacteria on different pitch nanoneedle surfaces compared to control.(I) CFU/mL plotted against time in hours.Error bars indicate standard error.(II) Representative optical images of plates at the 3 h time point following a 24 h incubation period: control, and black silicon nanoneedle surfaces with pitches of (a) 1400, (b) 800, (c) 500, and (d) 300 nm.

Table 1 .
Morphology parameters of black silicon substrates, as characterized under SEM.