Learning from Nature: Fighting Pathogenic Escherichia coli Bacteria Using Nanoplasmonic Metasurfaces

Bioinspired nanoplasmonic 3D crossed surface relief gratings and metasurfaces are fabricated on azobenzene molecular glass thin films to create effective antibacterial surfaces. A synergetic mechanical and photothermal interaction at the interface between the nanostructures and the Escherichia coli (E. coli) bacteria results in a significant decrease in the viable bacterial population. In particular, combined exposure to the interfacial nanospikes as well as the evanescent blue and red electromagnetic fields induced by the nanoplasmonic metasurface, results in a 97% reduction of the viable E. coli in only 25 min, when illuminated with a low‐power white light.


DOI: 10.1002/admi.202300269
surfaces via etching. Jang et al. [7] created a rough surface containing nanopores and nanospikes on stainless steel 316L by electrochemical etching, and effectively inhibited bacterial adhesion on its surface. Also, Silva et al. [8] reported on a plasma etching process in the presence of silver and copper to create conical nanostructures on poly(lactic acid) (PLA) surfaces which could ultimately prevent the adhesion and growth of E. coli biofilm by ≈95% after 24 h of incubation. Other tactics to create biomimetic nanostructures include direct replication of natural structures through molding, [9] or using a variety of lithography techniques on photoresists or photoactive azobenzene materials. [10,11] Azobenzene-based materials contain photoactive molecules that go through fast cyclic cis-trans isomerizations upon exposure to a laser beam. [12] This isomerization culminates in a photomechanical mass movement of molecules which can be utilized to create various types of metasurfaces depending on the interference pattern and polarization of the incident inscribing laser beam. [13] The resulting nanostructured metasurfaces can be easily and rapidly created on azobenzene-based thin films in the forms of linear, [14] crossed [15] or circular diffraction gratings, [16] as well as photonic crystals, quasicrystals, [17] and Moiré gratings [18] with many potential applications in photonics, optoelectronics, and sensors. [19] Therefore, photolithography approaches have been used to fabricate bioinspired nanostructures on azobenzene-based thin films. For instance, it has been shown that some of the fabricated nanostructures on azobenzene molecular glass thin films such as crossed surface relief gratings (CSRG) and Moiré gratings, resemble the surface patterns of cicada's wings [20] or Peruvian lily petals. [18] Altogether, by learning from nature, researchers were able to fabricate bioinspired antibacterial surfaces by manipulating the shape, size, and arrangement of synthetic nanostructures, to alter the surface texture of materials and to control the interactions between the surface and bacteria, thus achieving antibacterial properties through mechanical effects. [21] One such mechanobactericidal effect is the reduced adhesion of bacteria to a surface resulting from a high contact angle due to nanotextures on the surface. Alternatively, nanostructures can induce mechanical rupture of bacteria by puncturing their membranes similar to natural surfaces. [21] In addition to the mechano-bactericidal effects which could be created by bioinspired surfaces, a second tool to fight www.advancedsciencenews.com www.advmatinterfaces.de bacteria is to illuminate the surfaces with a specific wavelength of light which can effectively reduce the population of pathogenic microorganisms. In nature, this happens all the time by sunlight. Therefore, researchers have tested a variety of ultraviolet (UV), blue, green, red, or near-infrared (NIR) wavelengths and they have shown their photo-induced or photothermal antibacterial effects, also called as phototherapy. [22] UV and blue lights, for instance, have been used to reduce the spread of COVID-19 (SARS-CoV-2) or other pathogens in healthcare facilities or kitchen surfaces, leading to a reduction in hospital-associated infections and food-borne illnesses. [23] UV and blue light work mostly through mutagenic DNA destructive mechanisms, [24,25] while green, red, and NIR wavelengths have more photochemical or photothermal effects on microorganisms. [26,27] Furthermore, a third approach is the use of nanomaterials for antibacterial purposes. [28] Thin films of randomly dispersed nanoparticles or nanocomposites of gold, [29] silver, [30] copper, [31] titania, [32] carbon nanomaterials, [33] polymethyl methacrylate (PMMA), [34] and silicon, [35] have been shown to effectively kill bacteria. The use of quantum dots has also been reported to inhibit bacterial growth. [36] These antibacterial properties may be due to the release of ions, produced by localized chemical/photochemical effects of free radicals in nanoparticles, or by localized thermo-plasmonic effects caused by free electron oscillations at the interfaces between the nanoparticles and the surrounding dielectric media. [28,37,38] Tan et al. [39] for instance, chemically coated glass substrates with thin films of plasmonic gold nanoparticles with an average width of ≈73 nm and inhibited the growth of E. coli by shining white light on the surface.
A fourth method is being presented in this paper, which combines all the approaches mentioned before in killing bacteria. The idea is that nanostructured surfaces can be engineered to exhibit mechano-bactericidal, photo-bactericidal, and plasmonic effects simultaneously on a large surface area. This provides an opportunity to exploit the photonic properties of nanostructured surfaces in order to establish a connection between bioinspired nanostructured surfaces and light-induced bactericidal properties. Inspired from natural surfaces, the synthetic nanostructured surfaces contain periodic or semi-periodic features such as ridges, grooves, pits, and pores that can act as light-trapping elements in metasurfaces. [40] In certain conditions these metasurfaces can be designed to generate standing or propagating surface plasmon resonance (SPR) waves, which are collective oscillation of electrons at the interface between a metallic surface and a dielectric material, leading to interfacial emissions. [41] This paper presents a novel perspective that creates a bridge between the mechano-bactericidal properties of bioinspired nanostructures and their photo-bactericidal plasmonic behavior. This could be beneficial in developing smart surfaces for a range of applications in optical, medical, or food processing industries, where the ability to quickly and efficiently eliminate bacteria is vital. The presented nanostructured metasurfaces in this work were fabricated using laser interference lithography on azobenzene molecular glass thin films and they were ultimately used as plasmonic antibacterial surfaces under low-power white light illumination.

Results and Discussion
Among natural surfaces, cicada wings are known to have nanostructures that provide them with unique hydrophobic and antireflective properties. They are made up of ridges and grooves that form a regular pattern, each with a depth of ≈300 nm. [4] These nanostructured ridges act as diffraction gratings that give the cicadas the ability to hide, self-clean, attract mates, and repel water to remain clean and dry. [42] Although the nanostructures on cicada wings are incredibly complex, it is possible to replicate them synthetically to create materials with similar properties, such as improved hydrophobicity and antibacterial behavior. [9] Figure 1a is an atomic force microscopy (AFM) image taken from a region of a Cicada Neotibicen canicularis wing, seen in Figure 1a inset. The surface of the wing is composed of needle-shaped nanostructures with an average tip-to-tip distance of 152 nm and a maximum peak depth of 350 nm. The zoomed-in 1 μm × 1 μm 2D and 3D AFM images of this wing in Figure 1b show more clearly the needle-shaped nanostructures which are dispersed in a quasiordered pattern.
To mimic the nanostructures on Neotibicen canicularis cicada wings, nanostructured crossed surface relief gratings (CSRG) were inscribed onto Disperse Red-1 azobenzene molecular glass (gDR1) thin films using a Lloyd's mirror interferometry, as described in the Experimental Section. Figure 2a is a 5 μm × 5 μm 2D AFM image of a sample labeled CSRG1, which consist of nanostructured orthogonal gratings with a pitch value of 306 nm inscribed on a gDR1 thin film. Similarly, Figure 2b is an AFM image of another sample labeled CSRG2, which has nanostructured orthogonal gratings with a pitch value of 448 nm. Figure 2c is a 5 μm × 5 μm 2D AFM image of a metasurface which is inscribed on a gDR1 thin film by superposition of CSRG1 and CSRG2. Both the 306-and 448-nm pitch values can be distinguished on this metasurface. Figure 2d,e,f are the corresponding 3D AFM images of CSRG1, CSRG2 and the metasurface respectively, showing their spiky bioinspired 3D nanostructures similar to the cicada wings presented in Figure 1.
The pitch values of the nanostructured CSRGs and the metasurface in this work were carefully chosen according to the desired surface plasmon resonance (SPR) wavelengths that will be used to obtain enhanced antibacterial properties. Based on theory, the wavelength of a SPR generated by a grating structure along a metal-dielectric interface can be calculated as follows: [43] where Λ is the pitch value of the grating structure, n d is the refractive index of the dielectric medium, ′ r,m is the real part of the permittivity of the metallic film, and i is the light incidence angle.
Previous studies have demonstrated that LED light at wavelengths of 465-and 630-nm can inhibit bacterial growth or inactivate bacterial pathogens effectively at high power levels or close exposure to the pathogens. [44] Therefore, these two wavelengths were chosen as desired SPR emissions to be excited at the interface between the nanostructured surfaces and the bac- terial suspension. Therefore, the pitch values of the nanostructured CSRGs and the metasurface were calculated using Equation 1 in a way that they produce SPR emission peaks at 465 and 630 nm at their interface with the E. coli suspension (n d = 1.3385). The real part of the permittivity ( ′ r,m ) of silver at those specified wavelengths was taken into account for this calculation. Note that other effective antibacterial wavelengths could be similarly selected. Figure 3 illustrates the normalized SPR reflection spectra of the nanostructured silver-coated CSRG1, CSRG2, and the metasurface measured at normal incidence under white light illumination and in water (n d = 1.33). Generally, once the SPR is excited at the interface of a metal-dielectric layer, it will be measured as a dip in reflection and a peak in transmission. [45] Figure 3a shows that the SPR is occurring at 465 nm for CSRG1 and Figure 3b shows that SPR occurs at 630 nm for CSRG2. The metasurface on the other hand has a double reflection dip at 465 and 630 nm according to Figure 3c.
The antibacterial property of the bioinspired nanostructures fabricated in this paper was studied against E. coli bacteria. The experiments were conducted with and without white light illumination to investigate the antibacterial efficacy of the samples via mechanical and optical mechanisms. Epi-fluorescence microscopy images were taken every 5 min for 25 min. At first, the antibacterial property of the nanostructures was studied in darkness, assessing the effect of the nanostructures alone on  bacterial population. Subsequently, the imaging was performed under white light illumination which allowed for the simultaneous study of the effect of both the nanostructures and the SPR generated at their interface as schematically illustrated in Figure  4a. Additionally, the viability of E. coli was also studied on a control sample without any gratings or metasurfaces both under white light illumination and in darkness.
To measure the E. coli viability in contact with the proposed nanostructures in this study, fluorescence images were taken every 5 min for a period of 25 min using GFP and TRITC filter cube sets from the stained E. coli bacteria. Staining was done according to the LIVE/DEAD BacLight Bacterial Viability Kit L7012 which offers an effective two-color fluorescence assay for assessing bacterial viability. This kit utilizes a combination of SYTO 9 green fluorescence, which labels all bacteria, and propidium iodide red fluorescence, which only labels damaged membranes that ultimately result in a decrease of the SYTO 9 green fluorescence once the cells are dead. Therefore, the intensity ratio of greento-red (G/R) fluorescence was measured using ImageJ software, and it was used as a quantitative factor for E. coli viability in this study. For all the nanostructures presented in this work, the viability was calculated by percentage of reduction in G/R ratio every 5 min during a 25-min time span for an average of at least three different samples of each nanostructure, according to the following equation: Figure 4b,c show the percentage of E. coli population reduction for silver-coated nanostructured CSRGs and the metasurface in darkness and under white light illumination, respectively. In general, the mortality rate of E. coli increases with time from 5 to 25 min for all the nanostructures both in darkness and under white light illumination. However, the mortality rates are higher under white light illumination.
According to Figure 4b,c, after 25 min, the flat silver surfaces which were used as control, exhibited almost similar antibacterial behavior in both darkness and under white light illumination, reducing the E. coli population by 58.3% and 64.0%, respectively ( Table 1). Silver has a natural antibacterial property, [46] which may be the cause of bacterial death when in the dark. Under white light illumination, the silver film has a small increase in antibacterial activities which could be attributed to the antibacterial qualities of the light itself. [22] By exposing the silver film to white light, its antibacterial effectiveness could be slightly improved.
In contrast to flat silver film, the antibacterial properties of nanostructured CSRG1, CSRG2, and the metasurface changed significantly depending on lighting conditions, which implies that SPR is effectively contributing to the killing of bacteria. For instance, CSRG1 which has a pitch value of 306 nm, reduced the E. coli population by ≈72.1% in darkness after 25 min of being in contact with the bacterial suspension. That is a 23.7% increase in the antibacterial activity compared to the silver flat control surfaces in darkness. This increase is attributed to the presence of the bioinspired spiky nanostructures having a pitch value of 306 nm (as seen in AFM images in Figure 2a,d) that puncture the E. coli membrane. When white light was shone on the surface of CSRG1, the SPR was excited at the wavelength of 475 nm at the interface between the CSRG1 and the bacterial suspension as illustrated in Figure 3a. The intense blue light from the SPR excitation enhanced the antibacterial activity on the surface of CSRG1 and eradicated 88.0% of the E. coli population after 25 min. This is an increase of ≈22.1% in antibacterial properties of CSRG1 when comparing darkness and white light illumination conditions. Similarly, CSRG2 which has a pitch value of 448 nm was found to neutralize ≈77.3% of E. coli in darkness after 25 min which is 32.6% higher than the flat silver control (Table 1). This could be attributed to the presence of taller spiky nanostructures in CSRG2 compared to CSRG1. Note that in the AFM images of Figure 2, CSRG1 has a maximum undulation height of 71 nm while for CSRG2, it is 194 nm. Therefore, the CSRG2 nanospikes have a higher potential in puncturing E. coli membranes and they have a higher mechano-bactericidal effect under darkness (Table 1). When white light was shone on CSRG2, the SPR was excited at the wavelength of 630 nm at the interface between the nanostructures and the bacterial suspension, as seen in Figure 3b. This SPR-enhanced red light eliminated 86.0% of E. coli bacteria population after 25 min. That is an increase of 11.3% in the antibacterial properties of CSRG2 by illuminating white light compared to dark state and an increase of 34.4% in its performance compared to flat silver film under white light illumination (Table 1).
Blue LED light has been found in previous reports to be effective in eradicating various types of gram-positive and gramnegative bacteria due to the stimulation of endogenous chromophores in microbial cells. [47] At proper power and wavelength, exposure to blue LED light results in the generation of toxic reactive oxygen species (ROS) that can damage components of bacteria, including the cell wall, and even the nucleus; [25] whereas red LED light has been found to have bactericidal effects mostly on gram-negative bacteria such as E. coli through thermal mechanisms, which is less effective compared to ROS. [48,49] For instance, Galo et al. [48] showed that exposure of pathogenic Staphylococcus aureus and Pseudomonas aeruginosa to blue light for 6 h inhibited their growth up to 75% after 24 h while red light was ineffective. Interestingly, in terms of the emitted SPR-enhanced wavelengths at the interface, it can be similarly observed that blue SPR light at 475 nm reduced the viable E. coli population more than the red SPR light at 630 nm (  (Table 1). However, the metasurface structure, which was composed of superimposed CSRG1 and CSRG2 (seen in AFM image of Figure 2c), interestingly eradicated 74.3% of the E. coli population in darkness which is in between the previously reported amounts for CSRG1 and CSRG2 (Table 1). Examining the AFM images in Figure 2, it can be concluded that the mechano-bactericidal activities of the nanostructured surfaces in darkness is directly related to the height of nanospikes' undulations. Meaning that the larger the undulation heights, the more effective their antibacterial activity is in darkness. This is in agreement with literature data that the height of the surface nanostructures directly affects its mechanobactericidal properties. [50] Once the white light was illuminated, the metasurface had an SPR excitation at both blue (475 nm) and red (630 nm) as seen in Figure 3. The reduction in E. coli viability due to this combined SPR emission was remarkable at ≈97% effectiveness within a period of 25 min (Figure 4c and Table 1). This illuminated condition was ≈30.6% more effective at bactericidal activity compared to its dark state, and clearly more effective than both the CSRG1 and CSRG2 nanostructures ( Table 1). The total performance of the nanostructured metasurface compared to the silver flat film is ≈51.6% higher under white light illumination. These results show that the nanostructured surfaces presented in this study not only benefited from the well-established antibacterial properties of silver but also from both the mechano-bactericidal and phototherapy effects generated by light enhancement due to surface plasmon resonance.
A synergistic blue & red or blue & near-infrared light illumination was reported in the literature to be effective at killing some pathogens via phototherapy by simultaneous LED exposures or by using photonic nanoparticles. [51][52][53] Guffey et al. [52] used a combined illumination of a blue 405-nm light and an infra-red 880-nm light to inhibit the growth of gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa. For P. aeruginosa, their proposed treatment reduced a maximum of ≈93.8% of bacterial colonies with an exposure of 90 mW cm −2 . In another study, Vasil'kov and co-workers [54] investigated the antibacterial properties of plasmonic silver nanoparticles illuminated with a blue 470-nm laser. This localized plasmonic effect reduced ≈64% of E. coli population after 5 min of irradiation with a 5-mW 470-nm laser. A synergic localized surface plasmon and propagating surface plasmon resonances have also been shown to be effective in creating photothermal properties in surface relief grating films fabricated from silver nanoparticles. [55] However, the combined bactericidal effect observed in our study is due to a combination of mechanical interactions between nanosized structures and bacterial cell membranes, as well as the generation of propagating surface plasmon resonances of the desired wavelengths which produce photothermal effects. This resulted in a reduction of ≈97% in the population of gram-negative E. coli when exposed to low-intensity white light with an irradiance of 0.64 mW cm −2 . This is achieved for the first time here using a nanoplasmonic metasurface that exhibits mechanobactericidal properties and achieves an SPR at both blue and red wavelengths at the interface between the nanostructure and E. coli suspension. This finding indicates that by exciting plasmonic resonances at the surface of nanostructures, it is possible to significantly improve their antibacterial properties against pathogenic E. coli bacteria and sanitize the surfaces purely by illuminating them with low-intensity white light. This approach can open new research avenues in creating smart antibacterial photonic devices to be used in healthcare facilities, kitchens, or public areas.
To investigate the effect of the metal coating on the antibacterial behavior of the nanoplasmonic structures, other samples were also fabricated and coated with gold this time, instead of silver. A flat gold surface was selected as the control. A nanostructured crossed surface relief grating (CSRG3) was fabricated through the method described in the Experimental section with a pitch value of 434 nm, as seen in AFM images of Figure 5a,b. The pitch value of CSRG3 was calculated using Equation 1 in a way that it would produce a 630-nm red SPR emission at the interface of gold and the E. coli suspension similar to CSRG2 (when comparing Figures 3b and c). Owing to the difference between the real part of permittivity ( ′ r,m ) of gold and silver, the pitch values of CSRG2 and CSRG3 are different but they both excite a SPR at 630 nm. Figure 5d shows that in a time span of 25 min, a flat gold surface eradicated 71.0% of E. coli in darkness while this value was 79.4% under white light illumination ( Table 2). For the gold-coated CSRG3 nanostructure, the reduction of E. coli population was 74.6% in darkness. This value is less than the bactericidal rate reported for the silver-coated CSRG2 in darkness (when comparing values in Tables 1 and 2). Once again looking at their AFM images (Figures 2b and 5a), it can be concluded that the E. coli mortality rate in darkness is dependent primarily on mechanical parameters such as the depth of the undulations, and not the material of the coating layer.
Plasmonic gold nanoparticles were reported to be effective in killing E. coli in short periods of time under high irradiations of white or laser light. For instance, under white light irradiation of 300 mW cm −2 , 77% of E. coli in contact with a gold thin film were eliminated in 15 min. [39] The plasmonic maximum absorption of gold nanoparticles in that study was at 624 nm [39] close to our CSRG2 and CSRG3 absorption. Additionally, an 810-nm CW laser light with irradiation of 6.3 W cm −2 eliminated 90% of E. coli in contact with gold nanoparticles in suspension in less than 10 min. [37] However, in our study, a low-intensity white light (0.64 mW cm −2 ) was used and 84.0% of E. coli in contact with gold-coated CSRG3 were eliminated in 25 min (Figure 5d and Table 2). This represents a 12.6% increase in antibacterial activity of gold-coated CSRG3 under white light illumination which is close to what was obtained for silver-coated CSRG2 that has a similar SPR wavelength of 630 nm. Therefore, under white light illumination phototherapy mechanism is primarily dominant and the antibacterial characteristic of the nanoplasmonic structures depends on the photothermal effects caused by the emitted SPR wavelength at the interface between the nanostructures and E. coli suspension.

Conclusion
Bioinspired 3D nanostructured nanoplasmonic metasurfaces presented in this study can be used as effective antibacterial surfaces with the potential to reduce or, potentially, eliminate the use of antibiotics. The results of this study demonstrate an effective reduction in the number of viable bacteria on these bioinspired 3D nanostructures due to a synergic mechano-bactericidal and photothermal effect. Exposure to white light promotes SPR at the interface between the nanostructures and the bacteria (E. coli suspension in this study) which, due to both texture and surface plasmonic activity, results in a significant decrease in the population of viable bacteria. It is also demonstrated that the bactericidal effect of the surfaces is mainly governed by their texture and the depth of the nanostructured undulations in darkness. However, under white light illumination, the antibacterial properties depend on the wavelength of the surface plasmon resonance being excited at the interface of the nanostructures and the bacterial layer. Exposure to the plasmonic evanescent wave in the blue region of the spectrum proved to be more effective at killing bacteria compared to red wavelengths. However, a combination of blue and red wavelengths had higher antibacterial effect than either one alone. A nanoplasmonic metasurface presented in this work, which produced both blue and red SPR wavelengths, enabled a 97% reduction in E. coli population after irradiation with low-intensity white light for only 25 min.

Experimental Section
Fabrication of Nanostructured Surfaces: The synthesis of the Disperse Red-1 azobenzene molecular glass (gDR1) was carried out according to a method described previously. [56] Solutions of 3 wt% gDR1 in Dichloromethane (CH 2 Cl 2 ) were prepared and spin-coated at 1000 RPM for 30 s onto clean Corning 0215 glass microscope slides (38 × 38 × 1 mm 3 ). The gDR1 films were dried at 75°C for 15 min and their average thicknesses were measured using a Dektak XT surface profiler (Bruker, USA), estimated to be ≈300 nm. A continuous-wave (CW) diode-pumped solid-state laser (Coherent, Verdi V6, = 532 nm) was used to inscribe nanostructured crossed surface relief gratings (CSRG) on gDR1 thin films using a Lloyd mirror setup, by interfering left-and right-handed circularly polarized collimated laser beams. An initial laser exposure resulted in the formation of linear surface relief gratings (SRG). Then, by rotating the sample 90 degrees and performing a secondary exposure for a fraction of the time, CSRGs formed as explained in our previous work. [57] In order to create metasurfaces for this study, two CSRG nanostructures with different pitch values were superimposed consecutively requiring four steps of laser exposures on gDR1 thin films. The laser exposure times for each step were ≈3-5 min and were optimized precisely to increase the circularity of the nanostructures as reported elsewhere. [58] The grating pitch (Λ) was controlled carefully by fine-tuning the angle of incidence of the laser ( ) on the gDR1 thin film via a rotating sample holder assembly (Λ = /2sin ). The Verdi laser irradiance was kept constant at 140 mW cm −2 during all exposures. The surface topography, modulation depths and pitch value of the resulting nanostructured CSRGs and metasurfaces were measured using a Dimension Edge atomic force microscope (AFM, Bruker, USA) and analyzed using the Nanoscope analysis software of the AFM. A 10-mW He-Ne laser with a wavelength of 632.8 nm was also used to confirm the resulting grating pitch by measuring the angles between the 0 and ±1 diffraction order (m) according to the grating equation (Λ = m /sin ). In order to perform the plasmonic and bacterial studies, the fabricated nanostructures on gDR1 thin films were transferred onto UV-curable epoxy transparent films by nanoimprinting and they were then coated with 20 nm of silver using a high vacuum sputter coater (Quorum, UK). The surface plasmon resonance (SPR) spectra of the nanostructured surfaces were recorded in reflection in water using unpolarized white light at normal incidence using an Ocean Insight spectrometer. The reflection from the flat surface of thin films in water was taken as the reference.
Cultures of Escherichia coli BW25113 bacteria cells were prepared as follows: 15 mL of 2xTY medium (16 g L −1 tryptone, 5 g L −1 NaCl, 10 g L −1 yeast extract) was added into a 50 mL falcon tube. No antibiotics were added. The medium was inoculated with 1 μL loopful of E. coli (from frozen glycerol stock), then the bacteria were grown for 16-18 h at 37°C with shaking at 200 RPM. Staining of E. coli was performed in suspension with LIVE/DEAD BacLight Bacterial Viability Kit L7012 by combining and mixing 1.5 μL of SYTO 9 dye (3.34 mm, 300 μL solution in DMSO) and 1.5 μL of Propidium iodide (20 mm, 300 μL solution in DMSO) in a microfuge tube. Then, 1 mL of the E. coli suspension was added to the dye mixture and incubated at room temperature in the darkness for 15 min.
Fluorescence Microscopy: Five μL of the stained E. coli suspension were trapped between the nanostructured thin films and an 18-mm square coverslip. The samples were then observed for 25 min under an Olympus IX83 inverted microscope equipped with an X-Cite 120LED boost high-power LED illumination system and 40X objective lens. Filter cube sets of GFP-4050B-OFF compatible with SYTO9 (Ex: 466/40 nm, Em: 525/50 nm) and TRITC-A-Basic-OFF compatible with Propidium iodide (Ex: 545/25 nm, Em: 620/30 nm) were used for fluorescence microscopy. At least three different samples were tested separately for each nanostructured or flat surface both in darkness and under white light illumination. Images of the bacteria were taken every 5 min both using GFP and TRITC filters. Then, the shutter was closed and the samples remained in darkness. In order to study the effect of SPR excitation on the antibacterial properties of the nanostructured surfaces, the samples were illuminated by white light using bright field mode of the microscope. The irradiance of white light was measured with a photodiode to be ≈0.64 mW cm −2 and it was kept constant throughout all imaging.