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Laser-Induced Graphene Biofilm Inhibition: Texture Does Matter Swatantra P. Singh, Sanjayani Ramanan, Yair Kaufman, and Christopher J. Arnusch ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00175 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 14, 2018

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ACS Applied Nano Materials

Laser-Induced Graphene Biofilm Inhibition: Texture Does Matter Swatantra P. Singh, Sanjayani Ramanan, Yair Kaufman, and Christopher J. Arnusch1* Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus 84990, Israel

*Corresponding author: Dr. C. J. Arnusch, [email protected]

Keywords: laser-induced graphene, nano-materials, nano-structured surfaces, biofouling, biofilm, bacterial adhesion

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Abstract: Biofilm formation on surfaces in technology and the environment is a problem that can lead to high costs and can endanger human lives. Namely, infrastructure, medical implants, and food processing units, as well as oil refineries, ship hulls, and membrane technology for water treatment are affected, and underlines the importance of identifying low fouling surfaces. Recently, surfaces coated with laser-induced graphene (LIG) were shown to strongly resist biofilm formation. Here we investigated the role of LIG texture and surface chemistry on biofilm formation, and showed that the rough LIG surface texture correlated to enhanced biofilm inhibition. Fabrication conditions of LIG lead to rough surfaces containing carbon nano-fibers (250-750 nm diameter), and micro-pores (1-25 µm), which were shown to inhibit the attachment and proliferation of bacterial cells. In contrast, LIG surfaces with crushed nano-fibers and covered micro-pores resisted biofilm formation less effectively. By oxidizing the LIG surface using oxygen plasma, we rendered the LIG more hydrophilic (contact angle was zero), but this chemical modification had negligible effects on the biofilm formation. After biofilm growth experiments, SEM images revealed that the morphology of P. aeruginosa cells adhered to polyethersulfone substrate surfaces were rod-like in contrast to more spherical shaped cells on the LIG surfaces, which suggested distress. This study gives evidence of the importance of surface texture in antifouling applications and desirable design features such as nano-fibers for effective antifouling LIG surfaces in energy, environmental and biomedical fields.


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Introduction: Bacterial cells on different surfaces in natural and anthropogenic environments usually exist within biofilm.1,2 The biofilm consists of microbial communities supported at the solid-liquid interface in a highly hydrated matrix of extracellular polymeric substances (EPS).3 Examples of infrastructure and systems that are negatively affected by biofilm formation and biofouling include membranes systems for water treatment, ship hulls, plumbing devices, oil refineries, medical implants, and food processing units.2,4–7 Especially in membrane-based water treatment units, biofouling leads to higher energy demands and increased costs due to increased maintenance and shorter module lifespans.4,6 On other liquid-solid interfaces, such as ship hulls, biofouling leads to increased costs due to increased fuel consumption.8 In addition, biofilm in medical settings have resulted in ~100,000 humans deaths annually, which underlines the need for deeper understanding of bacterial growth on surfaces and materials that prevent or delay biofilm formation.2,9,10 The inactivation of bacterial cells inside the biofilm is challenging since the biofilm matrix can protect the cells from different environmental stresses, biocides and oxidants.10 For example, Staphylococcus aureus cells within the biofilm are 600 times more resistant to chlorine as compared to planktonic cells.11 Similarly, biofilms in pipes persisted even after multiple biocide applications,12 and biofilms have been reported to survive in iodine solution for 15 months.13 Thus prevention of biofilm, especially at initial stages is an exciting topic with far reaching consequences.2,7,10,14 Studies have been performed on the different aspects of novel materials including biocidal effects,15,16 nanoparticles modified surfaces17–19 and different bioinspired textured surfaces7,14,20 for biofilm control. Graphene is an sp2-hybridized carbon allotrope with extraordinary physical, chemical, electrical and mechanical properties.21,22 Thus graphene and graphene derivatives are ideal candidates for electronic, biotechnology, energy and environmental applications.23–25 Many



graphene and graphene-based nanomaterials have been shown to be non-toxic, whereas other types have shown potential for antibacterial properties, where aspects such as one dimensional sharp edges of graphene can induce oxidative stress and lead to bacterial inactivation.26–29 These various graphene-based materials and surfaces are demonstrated as antibacterial and antibiofilm.22,26–29 However, the preparation of graphene-based surfaces usually consists of tedious and expensive multistep fabrication processes. In contrast, laserinduced graphene (LIG) can be fabricated in a single step without the use of chemical reagents on polymer surfaces.30 The possible application of LIG in energy and environmental technology has been demonstrated in recent studies,22,25,31,32 and recently, we have shown the anti-biofilm properties of LIG derived from polyimide.22 Moreover, we have shown that LIG can be fabricated on other polymers such as polyethersulfone (PES),33 which is a highly used polymer in membrane filtration,34,35 fuel cells36–38 and as biomaterials39 because of the excellent chemical, thermal and mechanical properties of PES.40 Previously, we have observed limited biofilm growth on LIG using a pure bacterial culture or a mixed bacterial culture obtained from secondary treated wastewater and suggested that the phenomenon was due to a combination of factors including surface charge, wettability and relatively minor antimicrobial activity.22 However, it has been shown that the nano-structure of surfaces can play a significant role in antibacterial activity and the prevention of biofilm growth.41,42 Thus, in the present study, we have performed a series of perturbations on LIG that was fabricated on PES substrates in order to understand especially how the surface structure of the LIG, as well as the chemical composition can affect the biofilm inhibition properties using a model bacteria Pseudomonas aeruginosa. These perturbations included mechanically crushing the PES-LIG surface and comparing with an uncrushed surface, and subsequent treatment of these surfaces with He/O2 atmospheric pressure plasma. These four different PES-LIG surfaces were tested for susceptibility to biofilm formation and especially


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the surface morphology parameter was identified to play an important role in biofilm inhibition. This information could be applied toward the design of LIG surfaces or other materials that can be used to control the biofilm formation in various energy, environmental and biomedical fields.

Results and Discussion: PES-LIG synthesis and characterization: The LIG was fabricated on the visibly transparent PES polymer film substrates using a CO2 laser (10.6 µm) in an ambient atmosphere (Fig. 1a). Despite the flexibility of the LIG coated substrates, surface structural damage can occur with direct mechanical perturbation. The obtained LIG (PES-LIG) had a porous foam-like morphology that was observed with low and high-resolution SEM images (Fig. 1b and 1c). The Raman spectra (Fig. 1d) showed characteristic peaks for the graphene at ~1350 cm-1 (D peak), ~1580 cm-1 (G peak) and ~2700 cm-1 (2D peak). The presence of the 2D peak confirmed the presence of single layer graphene sheets in the PES-LIG.43 The D peak supported the presence of sp2 carbon bond defects in the graphene, whereas the G peak is the first order allowed peak for graphite and confirms the presence of the sp2 carbon bond.43,44 The 2D peak for PES-LIG was fitted to only one Lorentzian peak (centered at 2700 cm-1) with a larger full width at half maximum of ~87 cm1

. This is also one of the characteristics of the single layer graphene sheets.43,44 The D/G

(0.72) and 2D/G (0.53) intensity ratio also supported the high content of graphene in the material.43,44 The full scan of X-ray photoelectron spectra showed the carbon (C) 1s, oxygen (O) 1s and sulfur (S) 2p peaks (Fig. 1d) and the calculated atomic percentages for C, O and S were 89.1%, 9.1% and 1.8 %, respectively. The above characterization shows successful LIG formation similar to previous reports of LIG on the PES polymer with different laser settings parameters, which can be used to generate LIG with variable composition and properties.33



Fig. 1: Fabrication and characterization of PES-LIG; (a) LIG printing on PES polymer; (b) SEM image of PES-LIG at low resolution; (c) SEM image of PES-LIG at high resolution; (d) Raman spectra of PES-LIG; (e) X ray photoelectron spectra of PES-LIG.

Preparation of different LIG surfaces: As illustrated in Fig. 2a, the present LIG has a rough surface morphology composed of rows of vertically aligned flakes that are transformed into a network of nano-fibers as the distance from the substrate increases. This morphology can vary according to the fabrication conditions and laser settings.33,45 In general, in all these reported examples, we have observed that smaller and finer features are present near the surface, while larger features are seen underneath, similar to the present case. To understand the effect of surface texture on biofilm formation in the context of other surface parameters, such as the chemical composition and the surface wetting property of LIG, we prepared four different samples (see methods and Fig. 2a-d): a pristine LIG surface was used as is, a crushed LIG surface, obtained by pressing a glass slide on top of the pristine LIG at ~7840 N m-2 pressure, a pristine LIG surface treated


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with oxygen plasma, and a crushed LIG surface treated with oxygen plasma. The chemical composition of the surface was measured with XPS (Table 1), and an increase in the oxygen content corresponded with changes in the LIG surface wettability, as measured by the contact angle of deionized water. The surfaces of LIG and crushed LIG (C-LIG) became extremely hydrophilic after plasma treatment: the contact angle was found to be 0° for both LIG plasma (LIG-P) and crushed LIG plasma (C-LIG-P) samples (Table 1). In comparison, the contact angles on LIG and on C-LIG (no plasma treatment) were 72° and 132°, respectively. The relatively large wetting angle of the C-LIG (132°) was similar to a previously reported LIG surface (~120°) in which a hydrophilic LIG sample was scraped from the substrate and a film was obtained by filtration.31 Suggested reasons for the changes from hydrophilic to hydrophobic included changes in the orientation of hydrophilic oxidized edges and the more hydrophobic graphene basal planes, and differences in hydrophobicity between LIG near the surface and LIG underneath toward the substrate. SEM, Raman and XPS were performed to characterize the modified surfaces. The C-LIG surface morphology was seen to be significantly different (Fig. 2i), and especially the cross-section SEM images showed that the fine nano-fibers vertical structures were absent in the C-LIG samples. The high resolution SEM image of C-LIG (Fig. 2j) showed a completely different morphology than the LIG (Fig. 1c), and crushed samples had flatter surfaces (Fig. 2c and 2d). No morphology changes were seen after the plasma treatment (Fig. S1), but changes in oxygen content and the wetting properties of the surfaces were observed (Table 1). The XPS C 1s deconvolution of all four surfaces (Fig. 2e-h) showed differences in carbon bonding and especially the percentage of carbon-oxygen bonding in the LIG changed after plasma treatment (Fig. 2f, Fig. 2h). The combined contribution of -C-OH and –O-C-O bonding increased from 22.3% for LIG to 26.8% for LIG-P and 27.2% for C-LIG to 31.6% for C-LIG-P. The Raman spectrum of C-



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LIG showed characteristic peaks of graphene similar to LIG (Fig. S2), and indicated that graphene content remained after destruction of fine features. Table 1. Surface chemical composition as measured by XPS, and water contact angle of LIG surfaces. LIG Type LIG Crushed-LIG (C-LIG) LIG-Plasma (LIG-P) Crushed-LIG-Plasma (C-LIG-P)

Carbon (%) Oxygen (%) 89.1 90.7 87.7 87.3

9.1 7.5 10.9 11.5

Sulfur (%) 1.8 1.8 1.4 1.2

Contact Angle (°) 72 134 0 0

Fig. 2: Schematic of four different PES-LIG prepared for the biofilm study; (a) LIG; (b) Crushed LIG (C-LIG); (c) LIG plasma (LIG-P); (d) C-LIG plasma (C-LIG-P). XPS C 1s deconvolution of (e) LIG; (f) LIG-P; (g) C-LIG; (h) C-LIG-P. (i) SEM images of LIG and CLIG with cross-section in the insets; (j) C-LIG high resolution SEM image


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Biofilm growth on different LIG surfaces: The different LIG surfaces along with samples comprising only of the substrate PES polymer were tested in a flow cell under biofilm growth conditions suitable for P. aeruginosa.22 The biofilm was analyzed using confocal laser scanning microscopy (CLSM) and the largest amount of biofilm growth (biovolume and average thickness) was seen on PES polymer substrate films (Fig. 3). In comparison, all samples containing LIG had significantly less biofilm growth and were found to be significantly different from each other: a one way ANOVA indicated P>bacterium length allows growth and division that follows the curvature of the fiber without surface detachment.7 On the other hand, when D≤bacterium length, it is less likely for the bacterium to remain attached to the fiber during bacterial cell division.7 On the other hand, if the bacteria cell adheres along (in parallel) the nano-fiber, Fig. 6c2, the cell will be able to divide along the nano-fiber and allow the biofilm to grow. On average, this mechanism is expected to slow down the growth of the biofilm. Note, however, that if the distance between two nano-fibers, L, is roughly the size of a single bacterium, the bacteria


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might be able to bridge to neighboring fibers, and this mechanism might be less effective. Hence, nano-fibers can only slow down the biofilm growth when D is about the size of a single cell, or smaller, and L is larger than the size of the bacterium. Taken together, the nano-fibers and the micro-porous structures on the LIG present an unfavorable environment for bacterial attachment and proliferation, which might inhibit biofilm formation. Whereas, these features are removed on C-LIG samples and the remaining flatter surfaces result in higher biofilm formation.

Fig. 6: Schematic illustration of two mechanisms that can qualitatively explain how nanofibers inhibit biofilm growth on the surface. (a) Mechanism 1: when bacteria adhere to nanofibers, and (b) the nano-fiber diameter, D, is smaller than the bacteria size, the limited bacteria—fiber surface area, A, reduces the adhesion energy between the bacteria and the fiber, which slows down the bacteria attachment to the surface, and allows bacteria to be removed by the flow. Mechanism 2: The limited directions available for bacteria cell division on nano-fibers are expected to slow down the average growth rate of biofilm. For instance, (c1) bacteria that attach perpendicularly to the fiber cannot divide and remain the fiber surface; however, (c2) bacteria that adhere in parallel to the fiber can divide and remain on the fiber. (d) Illustration of a crushed LIG surface, where the nano-fibers are mechanically crushed on the surface, results in increased biofilm growth.

In order to compare the fouling propensity of the LIG and crushed LIG surfaces using other bacterial cultures we tested both a pure culture of E. coli and a mixed bacterial culture obtained from a sample of secondary treated wastewater.51 Similarly to experiments with P.



aeruginosa, we quantitatively observed almost no biofilm growth on the LIG compared with the polymer substrate using a mixed culture of bacteria. When crushed, the LIG surface became more susceptible to biofilm growth (Fig S7). Less adhered bacteria were qualitatively observed using SEM, although due to the many types of bacteria present, the effect on the bacterial aspect ratio could not be observed. Therefore, the biofilm growth experiments were performed with a culture of E. coli, and similarly to P. aeruginosa, we observed that the adhered bacteria also showed a reduction of the aspect ratio: 2.78 ± 0.52, 2.13 ± 0.54, and 1.98 ± 0.49 were observed for the PES polymer substrate, the crushed LIG and the LIG, respectively (Fig. S8).

Concluding Remark: Laser-induced graphene is a facile and scalable approach to produce conformal 3D porous graphene structures in any pattern on surfaces in a single step, without the use of solvents or chemical reagents for electrically conductive, functional coatings. The reduced biofilm growth on LIG, as reported here and in previous publications, warranted study into the identification of the important parameters involved. We identified that the texture of LIG has a dominant effect on the formation of biofilm. Especially the nano-fibers on the LIG are features that could be responsible for inhibiting the attachment and proliferation of bacterial cells to form biofilm. Oxidation and wetting property of LIG had lesser effects on the biofilm formation. These observations support the idea that the antibiofilm effect is specific to LIG, in a sense that other materials with similar chemical composition such as the crushed LIG surfaces or other materials such as graphite have inferior antifouling properties. The complete understanding of how LIG surfaces foul will accelerate its use as antifouling surfaces in energy, environmental and biomedical fields.


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Materials and Methods: Materials: PES polymer (E 6020P, 75kD) pellets were obtained from BASF, Germany. Sodium chloride (NaCl, 99%), sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O, >99%), and monobasic potassium phosphate (KH2PO4, 99%) were procured from Merck, Israel. Dichloromethane (DCM) was procured from Sigma-Aldrich, Israel. LIVE/DEAD BacLight bacterial viability kit, containing propidium iodide (PI) and SYTO 9, and a hydrogen peroxide/peroxidase assay kit (Amplex® Red) were obtained from Thermo Fisher Scientific (Molecular Probes, USA). Deionized (DI) water was obtained from a Milli-Q ultrapure water purification system (Millipore, Billerica, MA), and used unless otherwise specified. A 10.6 µm CO2 pulse laser, 50 W, 2.0 inch lens kit (VLS 3.50) from Universal Laser Systems was used. PES-LIG fabrication and characterization: A thin film PES polymer substrate (~100 µm thick) was prepared by dissolving the polymer pellets (2.5 g) in DCM (20 mL) and the solution was poured into a glass Petri dish (inner diameter 11.5 cm). To slow the evaporation of the DCM, an inverted funnel was placed on top of the Petri dish.33 The thickness of the obtained PES sheet was measured to be 102 ± 4 µm. The substrates were taped on the laser cutting platform, which was set at a distance to the laser such that the laser spot was focused on the surface. LIG samples of size 1×1 cm were fabricated using the following laser settings: 2% duty cycle with 70 PPI (pulse per inch) image density, and 25% scan rate in the ambient environment (23 ± 2°C, 1 atm). Crushed LIG: The LIG surfaces were placed under a glass microscope slide and crushed under a weight of 1.5 kg. The SEM images of the nano-fibers and pores on the LIG surfaces were analyzed using Image J (National Institutes of Health, USA) software. Plasma Treatment of LIG: An LIG sample of size 1×1 cm was subjected to He/O2 atmospheric pressure plasma jet (Atomflo, Surfx, USA). The He and O2 flow rates were 30 L min-1 and 0.15 L min-1 respectively, and distance between the LIG and plasma



head was fixed to 2 cm. The samples were treated for 5 min. using 100 W power, and a scan speed of ~10 mm min-1. Surface characterization: Raman spectroscopy was performed with a Renishaw Raman RE01 scope with 633 nm laser. Scanning electron microscopy was performed using JSM7400F, JEOL. X-ray photoelectron spectroscopy was performed with PHI Quantera SXM scanning X-ray microprobe with 200 µm beam size and 45° takeoff angle. The contact angle was measured on an OCA-20 contact angle analyzer (Data Physics, Filterstadt, Germany) using a sessile drop of DI water. Dried LIG samples were fixed on a glass slide with doublesided tape and an average of 6 measurements with 0.3 µL of DI water was reported for each LIG sample. Biofilm growth: P. aeruginosa (PAO1) wild type, E. coli (MG1655) wild type, and a mixed bacterial culture (from secondary treated wastewater as described earlier22,51) were grown in Luria-Bertani (LB) broth at 30 °C, and harvested in mid exponential phase, verified by measuring the optical density at 600 nm. Cultures were centrifuged at 4000 rpm for 15 min and cells were washed three times with phosphate buffer saline and further diluted to 0.1 OD at 600 nm in LB broth. Biofilm growth experiments were performed in a custom made flow cell as reported earlier.22,46 Briefly, the PES polymer substrate, LIG, LIG-P, C-LIG and CLIG-P (1 cm x 1 cm) were attached with double sided tape to a glass slide and placed in the flow cell vertically. Inoculation of P. aeruginosa or other bacteria culture in the flow cell was done with the bacterial suspension (0.1 OD600 nm in LB, 50 mL) at a flow rate of 2.5 mL min-1. Thereafter, a sterile nutrient media (10 % LB) was flowed at 2.0 mL min-1 (cross flow rate of 0.44 cm min-1) through the flow cell for 36 hours. Confocal laser scanning microscopy (CLSM) analysis of biofilm: The samples were stained and analyzed with CLSM (Zeiss LSM 510, META), with Zeiss dry objective plan-


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NeoFluar as previously described.22 SYTO 9 (live), propidium iodide (dead), and concanavalin A (Con A) conjugated to Alexa Fluor 633 dyes (extracellular polymeric substances (EPS)) were used. The excitation wavelength of 488 nm was used for both the SYTO 9 and the PI, and 633 nm was used for the Alexa Fluor 633. The Imaris 3D imaging software (Bitplane, Zurich, Switzerland) was used for biofilm images preparation, whereas COMSTAT on Matlab 2015b was used for quantitative analysis.52 The average biofilm volume and thickness including standard deviation from 4 images are reported. SEM analysis of biofilm: immediately after the biofilm experiment, the samples were washed with 0.9% sterile saline solution and fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M Sorenson’s buffer (pH 7.2) and stored for 3 h. Afterward, the samples were carefully dehydrated by immersion in a series of water/ethanol solutions (50, 70, 80, 90, and 100% ethanol), and samples were stored in a desiccator until SEM analysis. The high-resolution SEM (JSM-7400F, JEOL) images were recorded after gold sputter coating. The bacterial size distribution in the SEM images was evaluated with Image J (National Institutes of Health, USA) software by measuring 250 bacterial cells for each sample (P. aeruginosa), or 40 bacterial cells for each sample (E. coli). Conflicts of Interest Ben Gurion University hold patent rights to this technology. These rights have been licensed by a company in which none of the authors are employees, officers or directors. Acknowledgements We are grateful to the United States-Israel Binational Science Foundation (BSF Grant No. 2014233) for financial support. C.J.A. wishes to thank the Canadian Associates of Ben Gurion University (CABGU) Quebec region for support.



Supporting Information Available: Surface antimicrobial activity methods. SEM images of LIG and LIG-P. Raman spectrum of C-LIG. Cross-section SEM images of LIG. SEM images of LIG and C-LIG with biofilm. Biofilm growth inhibition on LIG and C-LIG using mixed bacterial culture. SEM images of E. coli on sample surfaces. XPS deconvolution of S 2p for PES and LIG and bacterial surface P. aeruginosa inhibition of LIG.

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