Hybrid Hairy Janus Particles as Building Blocks for Antibiofouling

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Hybrid Hairy Janus Particles as Building Blocks for Antibiofouling Surfaces Alina Kirillova,†,‡ Claudia Marschelke,†,‡ Jens Friedrichs,†,‡ Carsten Werner,†,‡ and Alla Synytska*,†,‡ †

Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Strasse 6, 01069 Dresden, Germany Technische Universität Dresden, Fakultät Mathematik und Naturwissenschaften, 01062 Dresden, Germany

‡

S Supporting Information *

ABSTRACT: Herein, we report a new strategy for the design of antifouling surfaces by using hybrid hairy Janus particles. The amphiphilic Janus particles possess either a spherical or a plateletlike shape and have core−shell structures with an inorganic core and hydrophilic/hydrophobic polymeric shells. Subsequently, these bifunctional Janus particles enable the fabrication of surfaces with modularity in chemical composition and final surface topography, which possess antifouling properties. The antifouling and fouling-release capability of the composite Janus particle-based surfaces is investigated using the marine biofilm-forming bacteria Cobetia marina. The Janus particlebased coatings are robust and significantly reduce bacterial retention under both static and dynamic conditions independent of the particle geometry. The plateletlike (kaolinite-based) Janus particles represent a scalable system for the rational design of antifouling coatings as well as their large-scale production and application in the future. KEYWORDS: Janus particles, hybrid Janus particles, antifouling surfaces, Cobetia marina, marine biofouling

1. INTRODUCTION Marine biofouling, the accumulation of microorganisms, plants, algae, or animals on seawater contacting equipment (ship hulls, cooling and filtration systems, fishing nets, etc.) has severe consequences leading to annual worldwide costs running into billions of dollars. Thus, there is substantial demand for the development of effective and economical control measures.1 Biofouling is a complex process that in most cases can be described by an initial formation of a conditioning film due to the adsorption of proteins and polysaccharides on the surface, followed by the attachment of microbes, the formation of mature colonies, and partial cell detachment to spread the biofilm to distant locations. In contrast to the strongly adhering mature biofilms, the initial stages of biofilm formation are generally reversible. Hence, many proposed antifouling strategies rely on the prevention/intervention of the initial bacterial adhesion rather than on the removal of the mature biofilms.2 Initial bacterial adhesion can be greatly influenced by interfacial properties such as the surface chemistry (functional groups, electrostatic charge), surface energy, mechanical properties (elastic modulus, shear forces), environmental conditions (pH, temperature, nutrient levels, competing organisms), and surface topography. Consequently, a plethora of different antifouling approaches based on surfaces with diverse properties has been proposed, most of which rely on the use of homogeneous (such as hydrophobic or hydrophilic),3−14 heterogeneous (such as amphiphilic, patterned, or mixed),15−29 or 3D surfaces (such as microtopographicpatterned surfaces).30−40 © XXXX American Chemical Society

Hydrophobic fouling-release coatings do not inhibit the settlement of biofoulers per se, but allow their easy removal by minimizing the adhesion strength between the organisms and the surface.1,4 Presently, polydimethylsiloxane (PDMS) and fluoropolymers are the commonly used fouling-release materials.8 In particular, PDMS is attractive due to its low surface energy, low glass transition temperature, and low elastic modulus.3 However, the efficiency of the current PDMS coatings is limited by their insufficient mechanical stability, requiring improvement and reinforcement of their properties by adding other moieties or fillers, such as carbon nanotubes, nanoclay, and others.41 Hydrophilic antifouling coatings resist protein adsorption and cell adhesion due to the low polymer−water interfacial energy levels.5,42 In particular, polyethylene glycol (PEG) is a widely used nontoxic, biocompatible antifouling polymer coating.6,7,9−12 The efficacy of PEG is determined by its chain length, grafting density, and type of branching architecture.43,44 However, the majority of the currently applied PEG-based systems employ covalent grafting or adsorption of PEG onto a surface, model self-assembled monolayers (SAMs), or PEG-containing block copolymers, all of which typically result in coatings with limited robustness, a problem to be overcome for industrial applications. Additionally, it was Received: August 23, 2016 Accepted: November 4, 2016 Published: November 4, 2016 A

DOI: 10.1021/acsami.6b10588 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Representative SEM images of coatings based on spherical (a) and plateletlike (b) particles at different magnifications.

recently demonstrated that superoleophobic coatings can also be designed to possess antifouling properties.45 A recent trend in the design of experimental surfaces for antifouling purposes is to create surfaces with compositional (chemical) heterogeneity in order to combine the advantageous properties of hydrophilic antifouling and hydrophobic foulingrelease surfaces. Several methods to create heterogeneous surfaces have been reported, such as surface-initiated polymerization, adsorption of SAMs, or layer-by-layer deposition of alternating hydrophilic and hydrophobic polymers.15−17,46,47 Examples of heterogeneous surfaces with lateral scales in the nanometer range displaying antifouling properties include amphiphilic hyperbranched fluoropolymer (HBFP)−PEG as well as HBFP−PDMS−PEG networks18,48 and amphiphilic triblock surface-active block copolymers with PEG, PDMS, or fluorinated side chains.5,20,21,49−51 Furthermore, the domain size of the heterogeneities can be tuned to larger dimensions (tens of micrometers) using photolithography for the design of antifouling surfaces.52,53 Although the aforementioned surfaces perform well in various antifouling approaches, their potential large-scale application may be flawed by restricted scalability in terms of covering very large surface areas. Moreover, the robustness of the designed surfaces has not been evaluated. Finally, surface topography, the size and spacing of topographic features with regards to the dimension of fouling organisms, may be critical for the surface protection against fouling.30−32,54 Janus particles, i.e., particles having different functionalities at their opposite sides,55−58 are promising heterogeneous building blocks that can be used to construct functional surfaces.59 Therefore, we propose a scalable approach for the design of amphiphilic antifouling surfaces based on hybrid hairy Janus particles as solid amphiphilic block copolymer prototypes. The synthesized micrometer-sized core−shell Janus particles are composed of either an inorganic spherical silica or a plateletlike kaolinite core, as well as hydrophilic poly(poly(ethylene glycol) methyl ether methacrylate) (P(PEGMA)) and hydrophobic polydimethylsiloxane (PDMS) or poly(monomethacryloxypropyl-terminated polydimethylsiloxane) (P(PDMSMA)) polymeric shells grafted at the opposite sides of the core. We pursued a systematic study to test the antifouling capability of a series of bifunctional Janus particlebased surfaces and compared it to the performances of monofunctional and unmodified particle-based as well as those of modified and unmodified flat surfaces by investigating the impact of Cobetia marina bacteria. The advantages of the developed Janus particle-based surfaces over the conventional approaches mentioned above are that (1) they combine both chemical and topographical heterogeneity on the micrometer scale, (2) they are mechanically robust, and (3) they can be

easily prepared by simple solvent casting or spraying and can thereby cover large areas of substrates.

2. RESULTS AND DISCUSSION 2.1. Design and Characterization of Coatings. 2.1.1. Synthesis of Hybrid Hairy Janus Particles and Preparation of Surfaces. Spherical and plateletlike hybrid hairy Janus particles (JPs) with hydrophilic P(PEGMA) and hydrophobic PDMS or P(PDMSMA) polymers grafted on the opposite sides of the particles were fabricated and used for the preparation of antifouling surfaces (Figure 1). The spherical Janus particles (hereinafter referred as SiO2−P(PEGMA)/ PDMS−JP, Table 1) were synthesized via grafting of polymers Table 1. List of the Prepared Samples for the Antifouling Experiments sample ID native wafer native SiO2 native kaolinite P(PEGMA) flat P(PDMSMA) flat K−P(PEGMA) K−P(PDMSMA) SiO2−P(PEGMA) SiO2−P(PDMSMA) K−P(PEGMA)/P(PDMSMA)−JP SiO2−P(PEGMA)/PDMS−JP

description unmodified Si wafer unmodified 1 μm large silica particles unmodified kaolinite particles P(PEGMA) polymer brush on a Si wafer P(PDMSMA) polymer brush on a Si wafer monofunctionalized kaolinite particles with a P(PEGMA) shell monofunctionalized kaolinite particles with a P(PDMSMA) shell monofunctionalized 1 μm large SiO2 particles with a P(PEGMA) shell monofunctionalized 1 μm large SiO2 particles with a P(PDMSMA) shell plateletlike kaolinite-based Janus particles with two polymer shells spherical SiO2-based Janus particles with two polymer shells

on silica particles using a combination of “grafting from” and “grafting to” approaches, as described in the Experimental Section as well as in refs 60−62. As observed in the scanning electron microscopy (SEM) images, the Janus ratio of the obtained particles is 1:2 (PDMS/P(PEGMA), Figure 2). The Janus ratio is evaluated based on the depth of the particle penetration into the wax phase during the colloidosome preparation process.63 The plateletlike Janus particles (hereinafter referred as K−P(PEGMA)/P(PDMSMA)−JP) were synthesized via one-step simultaneous grafting of both polymers from inorganic kaolinite particles, as described in the Experimental Section and in ref 64. The Janus ratio of the plateletlike Janus particles is 1:1. The Janus nature of such particles was demonstrated in our previous work on a Janus B


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particles, as well as flat surfaces functionalized either with P(PEGMA), or P(PDMSMA), were prepared. In this way, all the surfaces used for this study were treated in the same way, unless stated otherwise. The particles were held together on the surface via purely physical interactions based on van der Waals forces with PGMA acting as a kind of glue on the surface. The negative control was an unmodified silicon wafer. A full list of the prepared samples is summarized in Table 1. 2.1.2. Surface Topography and Roughness. The rootmean-square (RMS) roughness of the polymer-modified particle-based coatings was determined by atomic force microscopy (AFM) topography imaging under water (Figure S1). Compared to the SiO2 particle-based surfaces, kaolinite particle-based surfaces were found to be slightly rougher: 180 ± 20 nm versus 130 ± 14 nm. Despite the fact that kaolinite particles are flatter compared to the spherical SiO2 particles, the latter are organized in a regular monolayer on the surface, whereas kaolinite particles form multilayers (present even after sonication), thus increasing the RMS roughness values. Modification of both particle types with polymers did not significantly affect the RMS roughness of the resulting coatings. 2.1.3. Distribution of Particles in the Coating Films. The orientation of spherical Janus particles in the coatings is random, with some particles exposing either their P(PEGMA) or PDMS side or lying on their side, thus exposing both polymers to the surrounding (Figure 2). This is observed owing to the clear Janus boundaries visible in many SEM images (such as in Figure 2a). The false color SEM image in Figure 2b is intended only to show an approximate orientation of the JPs on the surface. Unfortunately, EDX elemental mapping could not be performed on these systems due to the elemental similarity of P(PEGMA) and PDMS (or P(PDMSMA)) polymers (Scheme 1) and the fact that Si in PDMS cannot be distinguished from the strong Si signal coming from the inorganic core of the particles. Based on the SEM image analysis, it was concluded that the Janus particles tend to assemble into heterogeneous structures, where the size of the hydrophilic and hydrophobic domains is between several hundreds of nanometers to several micrometers (Figure 2). To estimate the amount of hydrophobic and hydrophilic moieties exposed to the surrounding (and thereby accessible to the fouling organisms), AFM colloidal probe force spectroscopic measurements were performed on the Janus particlebased surfaces under water. Surfaces prepared from monofunctionalized particles served as a reference (Figure 3). The distance between individual measurement points within the force maps was chosen to be 1 μm. This step size corresponds to the average size of most spherical bacteria and should therefore provide a realistic picture of the interactions of bacteria when approaching the examined coatings under water.

Figure 2. Spherical Janus particle layer: (a) original and (b) false color SEM image of the SiO2−P(PEGMA)/PDMS−JP layer (insets: SEM image of a single JP), the distribution of two polymers is visible in b; orange − P(PEGMA), green − PDMS.

particle lamella (cut with a focused ion beam) using transmission electron microscopy (TEM) and energy filtered TEM methods.64 The grafting density of the polymer chains on the spherical and plateletlike particle surfaces, obtained from thermogravimetric analysis (TGA) and gel permeation chromatography (GPC), is 0.2−0.4 chains nm−2. Subsequently, JPs were immobilized onto silicon wafers premodified with poly(glycidyl methacrylate) (PGMA), which serves as a noncovalent adhesion promoter (see Experimental Section for details). In this way, the particles were physically bound to the substrates via van der Waals interactions and mechanical entrapment and not involving any chemical reactions. Such a preparation technique that does not involve any chemical binding of the particles to the substrate was intentionally chosen over a more time- and resource-consuming chemical immobilization of particles, which typically involves additional steps of chemical reactions. The simple physical adsorption of the particles, their “gluing” to the surface via PGMA, allows the particle-based surfaces to be prepared on large areas via solvent casting. Ultimately, surfaces with different morphologies were formed (Figures 1 and S1). Likewise, reference surfaces formed from mono- and nonfunctionalized spherical and plateletlike Scheme 1. Chemical Formulas of the Grafted Polymersa

a

Poly(poly(ethylene glycol) methyl ether methacrylate) (P(PEGMA), left), poly(monomethacryloxypropyl terminated polydimethylsiloxane) (P(PDMSMA), middle) used for the “grafting from” approach, and carboxy terminated polydimethylsiloxane (PDMS, right) used for the “grafting to” approach. C


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Figure 3. Average AFM retracting force−distance curves acquired from the force maps measured under water for spherical (a and b) and plateletlike (c and d) particle layers: (a) SiO2−P(PEGMA) and SiO2−P(PDMSMA); (b) SiO2−P(PEGMA)/PDMS−JP; (c) K−P(PEGMA) and K− P(PDMSMA); (d) K−P(PEGMA)/P(PDMSMA)−JP. All curves were cut off in the y-direction.

Under these conditions, the hydrophilic P(PEGMA) polymer grafted on the surface of the spherical SiO2 as well as plateletlike kaolinite particles is highly hydrated, swollen, and thus completely nonadhesive to the 5 μm large SiO2 colloidal probe, demonstrating an elastic repulsion (Figure 3a,c). In contrast, the collapsed hydrophobic P(PDMSMA) polymer is significantly more adhesive to the colloidal probe (Figure 3a,c). The detachment force acquired on the plateletlike P(PDMSMA)-modified particles was found to be significantly higher than that on the spherical P(PDMSMA)-modified particles due to the different contact areas between the particles of different geometry and the colloidal probe. In the case of the JP-based layers, both types of force−distance curves could be acquired, indicating on the presence of both polymers (Figure 3b,d). Therefore, due to the significantly different adhesion properties of the polymers, the individual force−distance curves within the acquired force maps for the Janus particles could be classified as typical for either P(PEGMA) or for PDMS. Correspondingly, curves with a considerable adhesion were categorized as PDMS, whereas curves with repulsive forces were classified as P(PEGMA). Statistical analysis of the force− distance curves recorded on the surfaces prepared from spherical JPs revealed a 52%/48% distribution of force− distance curves with characteristic features for hydrophobic or hydrophilic polymers, respectively. It must be noted that this ratio represents the effective statistical distribution of hydrophobic to hydrophilic features of the surface detected with the colloidal probe on several samples and not the particle orientation number distribution. The detachment forces recorded for PDMS domains on the JP-based surfaces are lower compared to those recorded for the monofunctionalized P(PDMSMA) particle surfaces, which is due to the swelling of the neighboring P(PEGMA) areas that minimize the contact area between the PDMS and the colloidal probe. Due to their geometry, the plateletlike JPs randomly lie with either their

P(PEGMA) side or their P(PDMSMA) side facing upward (Figure S1b). The distribution of force−distance curves with features characteristic for hydrophobic versus hydrophilic polymers was found to be 44%/56%, respectively. Therefore, it can be concluded that statistically both spherical and plateletlike JP-based surfaces expose the hydrophobic and hydrophilic moieties in similar amounts to the surroundings. Similar to spherical JPs, plateletlike JPs also form heterogeneous domains but with slightly bigger sizes from 1 to 10 μm, which was concluded from the force maps (Figure S2). 2.1.4. Electrokinetic and Wetting Behavior. In the next step, the electrokinetic behavior of the designed surfaces was evaluated using streaming potential measurements (Figure S3a,b, spherical and plateletlike, respectively). The value of the zeta potential in the representative zeta potential versus pH plots changes linearly with pH as a result of charge formation via unsymmetrical ion adsorption.65 Due to the fact that both P(PEGMA) and P(PDMSMA) polymers do not possess dissociating groups, the change in the zeta potential value with increased pH is caused by the preferential adsorption of hydroxyl (OH−) ions at pH > isoelectric point (IEP) and hydronium (H3O+) ions at pH < IEP from the electrolyte solutions, respectively.66 The IEP of all the examined surfaces is around pH 4, which is typically observed for the chargeformation processes driven by ion adsorption. Representative curves recorded for SiO2−P(PEGMA) and K−P(PEGMA) surfaces have a less pronounced slope due to the strong swelling of the hydrophilic P(PEGMA) polymer on the particle surface. In contrast, representative curves recorded for SiO2− P(PDMSMA) and K−P(PDMSMA) surfaces have a more pronounced slope due to the hydrophobic P(PDMSMA). Representative curves recorded for both spherical and plateletlike Janus particle layers occupy an intermediate position.62 Therefore, it can be concluded that both polymers, hydrophilic swollen P(PEGMA) and hydrophobic nonswollen PD


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Figure 4. Representative light microscopy images at low magnifications and SEM images at high magnifications of the particle-based surfaces before (left) and after (right) 4 days of continuous shaking in DI water at 100 rpm: (a) K−P(PEGMA), (b) K−P(PDMSMA), (c) K−P(PEGMA)/ P(PDMSMA)−JP, (d) SiO2−P(PEGMA), (e) SiO2−P(PDMSMA), and (f) SiO2−P(PEGMA)/PDMS−JP.

measurements and compared it to unmodified, modified flat surfaces, and surfaces made from monofunctionalized particles (Figure S4 and Table S1). Freshly cleaned, unmodified silicon wafers are very hydrophilic, demonstrating a complete wetting

(PDMSMA), are exposed on the surface of the JP-based coatings. Furthermore, we investigated the wetting behavior of the Janus particle-based surfaces using water contact angle E


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Figure 5. Representative false color SEM images (bacteria are marked in red) of the (a) reference flat samples and surfaces with native particles and (b) of the respective modified particle-based (monofunctionalized and Janus) surfaces after the biofilm formation assay under static or dynamic conditions. Original SEM images are displayed in Figure S8. SEM images at lower magnifications are presented in Figures S9 and S10.

for the respective flat surfaces due to the roughness effects. The advancing and receding water contact angles on the surfaces formed from Janus particles (spherical: ΘA = 134°, ΘR = 60°; plateletlike: ΘA = 123°, ΘR = 44°) are in between the values determined for the surfaces formed from monofunctionalized particles (Figure S4). Hence, the advancing/receding contact angle measurements also confirm the presence and accessibility of both polymers on the bicomponent Janus particle-based surfaces. 2.1.5. Coating Robustness. In order to be considered potentially relevant for industrial applications, the particlebased surfaces must be robust under application-relevant conditions. Therefore, we performed two kinds of experiments on both spherical and plateletlike particle-based surfaces in

behavior, whereas unmodified SiO2 as well as kaolinite particle surfaces demonstrate slightly higher advancing contact angles (ΘA = 50 and 41°, respectively), while the receding contact angles are less than 10° (complete wetting). Flat P(PEGMA)and P(PDMSMA)-modified surfaces are hydrophilic (ΘA = 40°, ΘR = 23°), and hydrophobic (ΘA = 105°, ΘR = 82°), respectively (Figure S4). The advancing and receding contact angles on the surfaces formed from P(PDMSMA)-modified particles (SiO2−P(PDMSMA): ΘA = 148°, ΘR = 90°; K− P(PDMSMA): ΘA = 127°, ΘR = 65°) are higher compared to those of the respective flat surfaces, while the contact angles on the surfaces formed from P(PEGMA)-modified particles (SiO2−P(PEGMA): ΘA = 38°, ΘR = 3°; K−P(PEGMA): ΘA = 40°, ΘR = 3°) are slightly lower than the values determined F


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Figure 6. Antifouling and fouling-release performance of the polymer-functionalized flat model and (Janus) particle-based surfaces. Marine, biofilmforming Cobetia marina bacterial cells were incubated for 24 h with the samples under (a) static (45 rpm) or (b) dynamic (90 rpm) conditions. The amount of attached cells was quantified. Due to significant variations between the independent experiments, all values were normalized against the negative control (native wafer, static conditions).

order to test their mechanical robustness under water: (1) AFM imaging in contact mode with sequentially increased contact forces exerted by the AFM tip on the samples (Figures S5 and S6) and (2) incubation of the surfaces for 34 days in DI water on a shaking device (Figure 4). Notably, all surfaces were prepared through physical adsorption of particles onto premodified substrates via solvent casting. In the first set of experiments, AFM topography images were recorded on the particle-based surfaces under water with increasing contact forces (25, 50, and 250 nN), revealing different robustness levels. Surfaces made from P(PEGMA)modified plateletlike particles were found to be rather unstable under water (Figure S5a). Scanning of the surface with a contact force of 25 nN revealed many particle-free areas due to the adhesion of the weakly attached particles to the tip. At higher forces even more particles were removed, eventually adhering to the tip, making further scanning impossible and thus indicating on the limited robustness of the layer. On the contrary, both K−P(PDMSMA) and K−P(PEGMA)/P(PDMSMA)−JP layers were very robust, and almost no particles were dislodged for contact forces up to 250 nN (Figure S5b,c). For all the applied contact forces, no particles were dislodged from the spherical particle-based surfaces, demonstrating their high level of robustness (Figure S6). In the second approach, the surfaces were incubated for 4 days in DI water with continuous shaking. Representative light microscopy and SEM images of the particle-based surfaces before and after incubation are summarized in Figure 4. Notably, under these conditions the P(PEGMA)-modified particle surfaces, both spherical and plateletlike, were almost completely decomposed (Figure 4a,d). This observation can probably be explained by the hydration and swelling of the P(PEGMA)-shell that decreases the interparticle adhesion as well as the adhesion of the particles to the substrate. In contrast, surfaces with P(PDMSMA)-decorated hydrophobic particles and P(PEGMA)/P(PDMSMA) amphiphilic Janus particles remained unaffected after the experiment both in the case of plateletlike (Figure 4b,c) as well as spherical particles (Figure 4e,f). This indicates that the hydrophobic attraction forces between the particles as well as to the substrate were

strong enough to prevent the decomposition of the coating (even in the case of Janus particles, which are amphiphilic). Additionally, the robustness of particle-based surfaces was evaluated by using sonication in water in an ultrasonic bath for different time intervals (up to 5 min) on the example of SiO2based surfaces (Figure S7). Very high shear forces are generated during sonication that are typically used to break glued particles apart in dispersions. It was found that the SiO2−P(PEGMA) surface was rather unstable compared to the SiO 2 − P(PDMSMA) and the Janus particle one (Figure S7). It was almost completely decomposed after 2 min of sonication, while the other two kinds of surfaces remained unaffected. Particles started detaching from the SiO2−P(PDMSMA) and SiO2− P(PEGMA)/PDMS surfaces only after 5 min of sonication, which indicates on the high level of surface robustness. All experiments demonstrate that the surfaces composed of particles monofunctionalized with P(PDMSMA) as well as Janus particles are stable under application-relevant conditions. Since it emerged that the hydrophilic P(PEGMA) coatings are not stable under the tested conditions, we modified the protocols for their preparation to obtain stable coatings for the preceding antifouling experiments. Briefly, the P(PEGMA)decorated particles were immobilized onto the substrate using several steps. First, 3-aminopropyltriethoxysilane (APTES)covered particles (either SiO2 or kaolinite) were attached to the PGMA layer on the silicon wafer via reaction between amino groups on the particle surface and glycidyl groups in PGMA. Second, after modification of the particle layer with the initiator for atom transfer radical polymerization (ATRP), surfaceinitiated polymerization of PEGMA was performed directly on the particle layer. The fact that particles in the surfaces prepared in this way were not detached after the antifouling experiments indicates their successful attachment to the surface. Hence, the P(PEGMA)-modified particle layers presented in this work only serve as reference layers due to their tedious preparation procedure, which disallows their further preparation/application on the large scale. 2.2. Antifouling Performance. Surfaces formed from spherical and plateletlike Janus particles, as well as reference surfaces formed from mono- or nonfunctionalized spherical and plateletlike particles and flat surfaces functionalized with the G


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bacteria mainly attached to the surface of the particle layer, and typically did not adsorb in the voids between the particles (Figures S11 and 5). Even in the case of irregular kaolinitebased coatings, bacteria were mostly located on top of the topographical irregularities (Figure S11 a,b). On these exposed positions, presumably less force has to be applied to remove the undesirable accumulation of bacteria on the surface during cleaning cycles in potential future applications. 2.2.2. Dynamic Conditions. Compared to the static culture conditions, significantly less bacteria attached to the samples under high-shear-stress dynamic conditions (compare the unmodified silicon wafer negative control in Figure 6a,b). Nevertheless, the negative control as well as the particle-based surfaces without polymer coatings still performed significantly worse compared to the polymer-modified flat and particlebased surfaces (Figure 6b). Interestingly, monofunctionalized flat and particle-based surfaces modified with P(PEGMA) performed at similar levels as compared to those under the static conditions, indicating that the hydrophilic coatings already reached their optimal performance under the lowshear-stress conditions. In contrast, monofunctionalized flat and particle-based surfaces modified with P(PDMSMA) as well as the Janus particle-based surfaces performed significantly better (as compared to the static conditions) at levels comparable to those of the P(PEGMA)-based surfaces. The observed tendencies were additionally supported by the SEM analysis (Figures 5, S9, and S10). Notably, the P(PEGMA)-modified surfaces based on physical adsorption were proven to have limited robustness (Figures 4, S5, and S7). Furthermore, the technique based on chemical immobilization by which they were prepared requires additional steps of modification, thus disallowing their real application as antifouling surfaces. Both P(PDMSMA)modified and Janus particle-based surfaces have prevented bacterial adhesion to a great extent compared to the control unmodified silicon wafer under static and dynamic conditions (Figure 6). However, while under dynamic conditions both surface types performed almost equally well; under static conditions Janus particle-based surfaces exhibited a better antifouling performance (Figure 6; −56 to −58% vs −43 to −45%). When compared directly to the P(PDMSMA) particle layers (using them as the control and assuming that the normalized luminescence units corresponding to P(PDMSMA) particle layers equal 1), there are ca. 24% less bacteria observed on the Janus particle-based surfaces in the case of both SiO2− P(PEGMA)/PDMS−JP and K−P(PEGMA)/P(PDMSMA)− JP. Eventually, this could lead to a universal application of such Janus particle-based surfaces under both low shear stress and high shear stress conditions. The Janus particles presented in this study are solid block copolymer prototypes serving as bifunctional single blocks with modularity in chemical composition, geometry, and final surface topography. The designed Janus particle-based surfaces provide interplay between chemical and topographical heterogeneity on the micrometer scale. It was found that the topographical aspect of the surfaces, namely, the geometry of the Janus particle core, had just a minor effect on the antifouling performance of the coatings: The spherical JPs performed slightly better under static conditions, while the plateletlike JPs performed better under dynamic conditions. The difference in the RMS roughness parameters (50 nm) of the spherical and the plateletlike particle surfaces was found to be insufficient for the C. marina bacteria to distinguish or

same polymers were tested for their antifouling performance. Unmodified native silicon wafers were used as a negative control. The marine biofilm-forming bacterial strain C. marina was seeded in growth medium onto different substrates and incubated for 24 h under either static conditions with low shear forces (representing standing water) or dynamic conditions with high shear forces (representing a moving ship hull). Subsequently, the number of the attached bacteria was quantified by a chemoluminescence assay that quantifies the adhering viable bacteria by determining the amount of adenosine triphosphate (ATP) present (see the Experimental Section for details). The SEM images of the samples after incubation and the results of the biofilm formation assay are displayed in Figures 5 and 6, respectively. Due to significant differences in the absolute values of the ATP-based assay between independent experiments, the results were normalized against the negative control (unmodified native silicon wafer) under static conditions. Additionally, we investigated all sample types after their incubation in the bacterial solution by means of SEM in order to visualize the adherent bacteria, and to gain insight into their distribution on the surfaces (Figures 5 and S8−S10). In this way, the qualitative distribution of bacteria on the surfaces is displayed in Figure 5, whereas the quantification of the adhered bacteria via a chemoluminescence assay is displayed in Figure 6. 2.2.1. Static Conditions. Under static conditions surfaces containing particles (spherical and kaolinite) monofunctionalized with hydrophilic P(PEGMA) performed equally well compared to the benchmark flat control surfaces covered with the same polymer (Figures 5 and 6). Interestingly, the performance was independent of the particle geometry, and almost similar amounts of bacteria were found on kaolinite- and SiO2-based surfaces. Hydrophobic, fouling-release coatings are not expected to prevent the adhesion of microorganisms per se but rather to facilitate the detachment/removal of the attached foulers when sufficiently high shear forces are applied. Accordingly, significantly more bacteria attached to flat as well as particle-based P(PDMSMA) surfaces (as compared to the P(PEGMA)-based surfaces) under static conditions (Figures 5 and 6). However, these surfaces still performed significantly better than the negative control (unmodified silicon wafer) and the particle-based surfaces without polymer coatings. These results prove that the hydrophobic polymer coating has a significant effect on the bacterial retention even under these low shear stress conditions. Interestingly, the particle-based surfaces without polymer coatings performed significantly better than the negative control, demonstrating that the topography of the particle-based surfaces positively influences their antifouling properties. As observed on the SEM images, bacteria tend to organize themselves in large colonies on the monofunctionalized P(PDMSMA)-based particle surfaces as compared to the rather uniform distribution of isolated bacteria on the monofunctionaizedl P(PEGMA)-based surfaces (Figures 5 and S10). Notably, the fraction of hydrophilic P(PEGMA) on the JPbased surfaces significantly reduced the attachment of bacteria compared to the monofunctional P(PDMSMA)-based particle surfaces. The distribution of bacteria observed in SEM was found to be intermediate between that observed on P(PEGMA)- and P(PDMSMA)-based surfaces. Some of the bacteria were found to be organized in moderately large colonies, while isolated bacteria were also present on the surface (Figures 5 and S10). Additionally, it was found that H


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hydrophobic as well as control native surfaces. (2) Independent from the geometry of the Janus particles (spherical or plateletlike), the developed surfaces significantly reduced bacterial adhesion under dynamic conditions to the levels similar to homogeneous P(PEGMA)- and P(PDMSMA)-based surfaces. (3) The adhered bacteria were mainly located on top of the topographical irregularities of the particle-based surfaces, which would presumably allow an easier removal of the undesirable bacteria during cleaning cycles. (4) The Janus particle-based surfaces were proven to be robust under application-relevant conditions. Furthermore, kaolinite-based Janus particles, which can be prepared on a large scale through a reduced number of steps, offer feasible and scalable systems for a large-scale application and a rational design of antifouling coatings in the future.

respond. However, introducing chemical heterogeneity to the individual particles (JPs) led to their assembly into networks and thus the formation of heterogeneous hydrophilic and hydrophobic domains on the (sub)micrometer scale from several hundreds of nanometers to several micrometers. This resulted in the pronounced lowering of the bacterial adhesion under static, low-shear-stress conditions, compared to those of pure hydrophobic P(PDMSMA)-modified as well as reference unmodified surfaces. Although surfaces prepared with P(PEGMA)-modified particles demonstrated better antifouling properties, these surfaces were mechanically unstable in all the robustness experiments. Presumably, such easy detachment of the particles occurs due to the swelling of the P(PEGMA) shell that decreases the interparticle adhesion as well as the adhesion of the particles to the substrate. The Janus particle-based surfaces demonstrate the best combination of mechanical stability and antifouling properties. The stability of the surfaces during all the experiments indicates that the hydrophobic attraction forces between the particles as well as to the substrate are strong enough to prevent the detachment of particles. To the best of our knowledge, control of both chemical and topographical heterogeneity in designed antifouling surfaces has not been addressed so far in the previous contributions. Moreover, we have pursued a systematic approach in our study and compared the antifouling performance of the bifunctional Janus particle-based surfaces with the corresponding monofunctional surfaces. Typically, analogous heterogeneous surfaces are only being compared to glass or pure PDMS elastomers but not to their homogeneous counterparts. To fully explore the potential of the Janus particle-based surfaces for real-world antifouling applications, additional experiments are envisioned, such as testing the long-term stability of the surfaces over several months, their effectiveness after multiple cycles of use, and hardness of the final coatings. Here also the aspect of particle size might be considered. C. marina is only one example of bacteria present in seawater and contributing to the process of fouling, whereas there are plenty of other fouling organisms (other bacteria, diatoms, algae, etc.), the impact of which must be tested in the future. The present study is a proof-of-principle systematic investigation that has revealed the antifouling potential of the Janus particle-based surfaces among others. Therefore, we believe that this is the first work that demonstrates such systematic analysis of different heterogeneous Janus-based surfaces as well as compares them to the homogeneous and the unmodified ones. Furthermore, the robust Janus particle-based surfaces developed in this work are potentially a completely new type of antifouling coating, which can easily be prepared on a large scale.

4. EXPERIMENTAL SECTION 4.1. Materials and Methods. 4.1.1. Materials. Kaolinite (SigmaAldrich, natural), ethylenediaminetetraacetic acid (EDTA, SigmaAldrich, +99%), sodium citrate dihydrate (Aldrich, 99%), sodium bicarbonate (Sigma-Aldrich, 99.7%), sodium dithionite (SigmaAldrich, 85%), tetraethylorthosilicate (TEOS, Fluka, 99%), ammonium hydroxide (NH4OH, Acros Organics, 28−30% solution), hydrogen peroxide (H2O2, VWR, 30%), ethanol absolute (EtOH, VWR, 99.9%), 3-aminopropyltriethoxysilane (APTES, ABCR GmbH, 97%), αbromoisobutyryl bromide (Aldrich, 98%), α-bromoisobutyric acid (Aldrich, 98%), anhydrous dichloromethane (Acros Organics, 99.8%), triethylamine (Sigma-Aldrich, 99%), copper(II) bromide (CuBr2, Aldrich, 99.999%), tin(II) 2-ethylhexanoate (Aldrich, 95%), L-ascorbic acid (Sigma, 99%), tris(2-pyridylmethyl)amine (TPMA, Aldrich, 98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDTA, Aldrich, 99%), ethyl α-bromoisobutyrate (EBiB, Aldrich, 98%), toluene (Sigma-Aldrich, 99.8%), chloroform (Sigma-Aldrich, 99.5%), dichloromethane (Acros Organics, 99.99%), tetrahydrofuran (Acros Organics, 99.99%), anisole (Sigma-Aldrich, 99.9%), paraffin wax (mp 53−57 °C, Aldrich), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, Sigma-Aldrich, BioXtra), N-hydroxysuccinimide (NHS, Aldrich, 98%), and hexane (Sigma-Aldrich, 95%) were used as received. Carboxy terminated polydimethylsiloxane (PDMS, Mn: 10 000 g/mol) and poly(glycidyl methacrylate) (PGMA, Mn: 42 000 g/mol) were purchased from Polymer Source and used without further purification. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn: 475 g/mol, Aldrich), and monomethacryloxypropylterminated polydimethylsiloxane (PDMSMA, asymmetric, 6−9 cSt., ABCR) were passed through basic, neutral, and acidic aluminum oxides prior to polymerization. Millipore water was obtained from Milli-Q (Millipore, conductivity: 0.055 μS cm−1). 4.1.2. Scanning Electron Microscopy (SEM). Scanning electron microscopy (SEM) images were acquired on a NEON 40 EsB CrossBeam scanning electron microscope (Carl Zeiss NTS GmbH, Germany), operating at 3 keV in the secondary electron mode. In order to enhance electron density contrast, samples were coated with platinum (3.5 nm) using a Leica EM SCD500 sputter coater. 4.1.3. Atomic Force Microscopy (AFM). AFM measurements were performed using a Bruker Dimension Icon AFM (Bruker, USA) on the particle-based samples immersed in DI water using an open fluid cell within a sealed AFM chamber at room temperature. The samples were left to equilibrate in DI water for 20 min before the measurements. Topography images were acquired in contact mode using triangular sharp Si3N4 cantilevers (DNP-10, Bruker, USA) with a spring constant of 0.06 N m−1. The RMS roughness of topography images was evaluated using the WSxM software (WSxM solutions). Force maps were acquired using tipless Si3N4 cantilevers (NP-O10, Bruker, USA) having a nominal spring constant of 0.35 N m−1. The spring constants were calibrated before each measurement using the equipartition theorem.67 Cantilevers were modified with 5 μm large SiO2 colloidal probes. The colloidal probe (CP) technique was used because it provides a realistic overview of the surface adhesion on a larger scale

3. CONCLUSIONS We demonstrated the first attempt to apply hybrid hairy Janus particles as novel building blocks for the rational design of antifouling surfaces. The amphiphilic Janus particles comprise an inorganic core and are grafted with the current antifouling “gold standards”, P(PEGMA) and PDMS, or P(PDMSMA), polymers, at the opposite sides of the core. JPs combine both chemical and topographical heterogeneity on the micrometer scale. We investigated the antifouling capability of the Janus particle-based surfaces under application-relevant conditions using C. marina. Ultimately, we found the following: (1) Introducing chemical heterogeneity to the Janus particles and the resulting JP-based surfaces significantly lowered bacterial adhesion under static conditions, if compared to pure I


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the exposed particle surface.62 Subsequently, wax was dissolved in hexane, and the initiator-covered particles were used for polymerization. 4.2.3. Grafting of P(PEGMA) Using Surface-Initiated ATRP (“Grafting from” Approach). Poly(poly(ethylene glycol) methacrylate) (P(PEGMA), Scheme 1, left) was grafted from the initiatormodified particle surface as follows: Ethanol (3 mL), PEGMA (3 mL), PMDTA (60 μL, 0.5 M solution in DMF), CuBr2 (60 μL, 0.1 M solution in DMF), and EBiB (0.15 μL) were added to the particles. The mixture was sonicated and purged with Ar, followed by the injection of ascorbic acid (200 μL, 1 M solution in DMF). Polymerization was performed under continuous stirring at 60 °C in a water bath for 3 h. Subsequently, particles with the grafted polymer were washed eight times by centrifugation in ethanol and dried under vacuum at 60 °C. 4.2.4. Grafting of Carboxy Terminated PDMS (“Grafting to” Approach). The “grafting to” approach was utilized to graft the second polymer onto the P(PEGMA)-modified JPs prepared in the previous step. For this purpose, the JPs were dispersed in a 1 wt % carboxyterminated PDMS (Scheme 1, right) solution (20 mL) and stirred for 2 h. Next, the solvent was evaporated, and the particles were annealed at 150 °C overnight. Ungrafted polymer chains were removed by repeatedly dispersing the particles in chloroform or toluene and subsequent centrifugation. As a result, bicomponent spherical P(PEGMA)/PDMS−JP were obtained. 4.3. Synthesis of Plateletlike Kaolinite-Based Janus Particles. 4.3.1. Separation, Purification, and Modification of Kaolinite Particles. Kaolinite particles (Fluka) were first separated according to their size using grain size separation (Atterberg method), as described elsewhere.64 Briefly, 0.1−2 μm large kaolinite particles were obtained by sedimentation of the initial particles in DI water for 2 days and subsequent centrifugation of the supernatant suspension. The acquired kaolinite particles were then purified by EDTA to remove calcium and magnesium carbonates, followed by deferration conducted via the dithionite-citrate-bicarbonate (DCB) method.64 Then the kaolinite particles were modified with APTES by stirring them for 12 h in a 5% APTES solution in ethanol. APTES-modified particles were purified by repeated washing and centrifugation cycles in ethanol and dried under vacuum at 60 °C. Next, the ATRP-initiator (αbromoisobutyryl bromide) was immobilized onto the particle surface by incubation in dry dichloromethane (0.7 mL of α-bromoisobutyryl bromide in 35 mL of dichloromethane) in the presence of triethylamine (1.4 mL) at room temperature under constant stirring for 2 h. Initiator-modified particles were purified by centrifugation/ redispersion in dichloromethane, water, and ethanol and dried under vacuum. 4.3.2. Simultaneous Grafting of Two Polymers Using SurfaceInitiated ATRP. The grafting of two different polymers onto the opposite sides of the kaolinite particles was simultaneously conducted in an emulsion formed by oil in the water phase. For the water phase, water-soluble PEGMA (5 g), PMDTA (1 mL, 0.5 M solution in DMF), CuBr2 (1 mL, 0.1 M solution in DMF), α-bromoisobutyric acid (15 mg), and ascorbic acid (1 mL, 1 M solution in DMF) were dissolved in DI water (15 mL). A two-necked round-bottomed flask with initiator-modified kaolinite particles and the water-phase ATRP mixture was placed in a water bath, heated to 70 °C, and continuously purged with Ar during the whole polymerization process. The oilphase ATRP mixture was prepared from monomethacryloxypropyl terminated polydimethylsiloxane (PDMSMA, 3 g) as a water-insoluble monomer, anisole as a solvent (2 mL), CuBr2 (200 μL, 0.1 M solution in DMF), TPMA (28 mg), EBiB (0.15 μL), and tin 2-ethylhexanoate (200 μL). This mixture was introduced to the water phase and mixed by a mechanical stirrer. The simultaneous ATRPs were conducted for 2 h at a 1200−1400 rpm mixing speed. After polymerization, the bicomponent kaolinite P(PEGMA)/P(PDMSMA)−JP (formulas of the polymers are shown in Scheme 1) were collected by centrifugation and repeatedly washed in appropriate solvents (toluene, chloroform, ethanol, and water). 4.4. Preparation of Coatings with Spherical SiO 2 or Plateletlike Kaolinite Janus Particles. Polished single-crystal

than a sharp tip. The contact area between the bacteria and the surface is also much larger than a sharp AFM tip (several nm) and therefore closer in dimensions to the contact area between the CP and the surface (several hundreds of nm). Individual force−distance curves were acquired in the closed loop, constant height mode using 20 nN contact force and 824 nm s−1 approach/retract velocity. For each force map (16 μm × 16 μm), 256 force−distance curves were acquired and analyzed with a step size of 1 μm between the successive force− distance curves. Despite the fact that the CP itself has a 5 μm diameter, the contact area between the CP and the surface is much smaller (several hundred nm), allowing it to perform 1 μm steps, which correspond to the average size of bacteria used in this study. Average force−distance curves corresponding to each particle layer were calculated from 10 representative curves. 4.1.4. Contact Angle Measurements. Advancing and receding water contact angles were measured by the sessile drop method using a conventional drop shape analysis technique (OCA 35; DataPhysics Instruments GmbH, Germany). Fresh DI water was used for the measurements. For advancing contact angle measurements, a 10 μL water droplet was approached gradually onto the sample surface at a flow rate of 0.25 μL sec−1. For receding contact angle measurements, the pump was reversed, and the droplet volume was removed. Both advancing and receding contact angle values were recorded at five different locations of the sample. All measurements were carried out at 24 °C and a relative humidity of 40 ± 3%. 4.1.5. Electrokinetic Measurements. Streaming potential measurements were carried out using the Electrokinetic Analyzer (EKA; Anton Paar GmbH, Graz, Austria) in order to reveal the surface potential of the prepared coatings. Two identical particle-coated silicon wafers (10 × 20 mm) were placed parallel to one another, face-to-face, building a streaming channel inside a rectangular sample cell. An electrolyte solution (10−3 M KCl solution) was circulated through this channel. The streaming potential versus pressure loss was measured by Ag/ AgCl electrodes. 4.1.6. Ellipsometry. The thickness of flat polymer layers on silicon wafers in dry state was measured at λ= 632.8 nm and a 70° angle of incidence with a null-ellipsometer (Multiscope, Optrel Berlin, Germany) in a polarizer−compensator−sample-analyzer configuration as described elsewhere.68,69 4.1.7. Thermogravimetric Analysis (TGA) and Gel Permeation Chromatography (GPC). Thermogravimetric analysis was performed to measure the polymer layer thickness on the particle surface. All measurements were conducted in air atmosphere on a TGA Q 5000IR analyzer (TA Instruments, USA). The molecular weight of bulk polymers obtained after precipitation was determined using GPC (Gradient HPLC HP Series 1100, Agilent Technologies Inc., USA). The thickness of the grafted layer on spherical SiO2 and plateletlike kaolinite particles as well as the grafting density of the attached polymer chains were determined using the equations described elsewhere.60,64 4.2. Synthesis of Spherical SiO2-Based Janus Particles. 4.2.1. Synthesis and Modification of Monodisperse SiO2 Particles. Large silica particles (1 μm) were synthesized using a multistep hydrolysis−condensation procedure of TEOS in ammonia hydroxide− ethanol solution based on the Stöber approach70 as described in ref 61. In brief, TEOS was added sequentially into a mixture of ethanol and ammonia solution. The particles produced within one step of the synthesis were used as seeds for the next step. Each reaction was carried out by stirring the mixture at 500 rpm overnight at room temperature (starting from the last addition of TEOS). Subsequently, the dispersion with particles of the desired size was separated from the solvent by centrifugation, yielding monodisperse silica spheres that were dried in a vacuum oven at 60 °C. Next, the particles were stirred for 12 h in a 5% APTES solution in ethanol to introduce amino groups onto the surface. The APTES-modified particles were then purified by repeated washing and centrifugation cycles in ethanol and dried. 4.2.2. Preparation of Colloidosomes. Colloidosomes with 1 μm large APTES-modified silica spheres were prepared using the wax− water Pickering emulsion approach described elsewhere.60,61 The ATRP-initiator (α-bromoisobutyric acid) was then immobilized onto J


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ACS Applied Materials & Interfaces silicon wafers of orientation (Si-Mat Silicon Materials, Landsberg, Germany) were used as substrates for the coatings. First, silicon wafers (10 × 20 mm) were cleaned in a mixture of H2O2/ NH4OH/H2O (1:1:1) resulting in a uniform SiO2 layer with silanol groups. The thickness of the SiO2 layer measured by null-ellipsometry was (1.5 ± 0.2) nm. Next, PGMA (1% solution in CHCl3) was spincoated onto the wafers. Afterward, the wafers were annealed for 20 min at 150 °C in a vacuum oven in order to chemically graft PGMA. PGMA was attached to the wafer via reaction between silanol groups on the wafer surface and glycidyl groups in the polymer. The PGMA layer thickness was (80 ± 3) nm, as measured by null-ellipsometry. Janus particles (either spherical or plateletlike) covered with two different polymers on their opposite sides were immobilized onto a PGMA-coated silicon wafer by a solvent-casting method.66 Briefly, a 10 wt % dispersion of JPs, prepared in a mixture of chloroform/ toluene, was deposited dropwise onto a PGMA-coated wafer and dried. The wafer was annealed at 150 °C for 2 h in a vacuum oven to melt the PGMA and let the particles partially sink into the PGMA layer. After cooling and solidification of PGMA, the particles were stuck in the polymer layer that acted as a glue holding them on the surface. Multilayers were removed by sonication in chloroform. The quality of the prepared coatings was assessed by optical microscopy, SEM, and AFM. 4.5. Synthesis of Monofunctionalized SiO2 or Kaolinite Particles and Coatings. Particles homogeneously functionalized with just one polymer, P(PEGMA) or P(PDMSMA) (Scheme 1), were prepared using either silica or kaolinite particles fully covered with the ATRP-initiator α-bromoisobutyryl bromide (the procedure of APTES and ATRP-initiator immobilization is described above). The grafting of P(PEGMA) or P(PDMSMA) polymer chains from the initiator-modified particles was carried out using surface-initiated ATRP (“grafting from” approach), as described above. The polymercovered particles were washed after polymerization in appropriate solvents and dried under vacuum. Coatings with monofunctionalized SiO2 or kaolinite particles were prepared in the same manner as described for the Janus particles. 4.6. Preparation of Reference “Flat” Substrates. Silicon wafers were used as substrates for the grafting of polymer brushes. First, the wafers were cleaned as described above. Next, APTES and the ATRPinitiator (α-bromoisobutyryl bromide) were immobilized onto the wafer surface in the same manner as for the particles. The thicknesses of the APTES and ATRP-initiator layers, measured by nullellipsometry, were (0.8 ± 0.2) nm and (0.4 ± 0.1) nm, respectively. Polymer brushes were then grafted from the wafer surface using the surface-initiated ATRP “grafting from” approach as described above. The thicknesses of polymer layers were (25 ± 5) nm for both P(PEGMA) and P(PDMSMA). All the prepared samples for the antifouling experiments are summarized in Table 1. 4.7. Antifouling Experiments. 4.7.1. Bacteria Culture. C. marina (DSM 4741), an aerobic Gram-negative bacterium, was obtained from DSMZ (Braunschweig, Germany) and stored frozen in stock aliquots in a nutrient medium (40 g L−1 sea salts + 10 g L−1 tryptone [both Sigma-Aldrich, Munich, Germany]; pH 7.8) containing 20% glycerol at −80 °C. Experimental stock preparations were maintained on agar slants (nutrient medium + 5 g L−1 peptone + 1 g L−1 yeast extract + 15 g L−1 agar [all Sigma-Aldrich]) and were stored at 4 °C for up to 4 weeks. 4.7.2. Biofilm Formation Assay. For the experiments, a single colony from an agar slant was inoculated in nutrient medium (50 mL) and grown overnight with shaking at 28 °C. The next day, the overnight culture was diluted 1:200 in fresh nutrient medium and grown until an optical density (measured at a 600 nm wavelength, OD600) of 0.2 was reached. Flat or particle-containing substrates were glued to sterile segmented glass Petri dishes using double-sided tape to prevent movement of the samples and adhesion of bacteria to the unmodified backside of the samples during the experiment. Subsequently, the diluted bacteria suspension (OD600 = 0.2) was added to each segment of the Petri dish, and the samples were incubated at 28 °C at 90 rpm (dynamic culture) or 45 rpm (static culture) on a shaker for 24 h. The bacterial solution was discarded,

unbound cells removed by two washes with nutrient medium, and the samples transferred to centrifuge tubes (15 mL) containing sterile 0.9% NaCl (5 mL). Bacteria were removed from the sample surfaces by vortexing for 15 s followed by sonication for 5 min on ice to prevent heating of the sample. An initial test had verified that close to 100% of the attached bacteria were removed from the samples using this procedure [data not shown]). Samples were removed from the centrifuge tubes; bacteria were pelleted by centrifugation at 3000g for 5 min and resuspended in 1 mL of fresh 0.9% NaCl. Subsequently, the amount of viable microbial cells in culture was determined using the BacTiter-Glo Assay (Promega, Madison, USA) following the manufacturer’s instructions. Briefly, each bacterial suspension (100 μL) was pipetted in triplicate into a 96-well plate followed by an ATP reagent (100 μL). Samples were briefly mixed in a plate shaker, incubated for 5 min at room temperature, and finally chemiluminescence measured in a plate reader (Tecan GENios, Maennedorf, Switzerland). Within a single experiment, four individual samples were analyzed for each substrate type (see Table 1). After incubation in the bacterial solution, one of the samples was withdrawn for SEM imaging, and the other three were used for the quantification of viable microbial cells. Five independent experiments were conducted.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10588. AFM topography images of the particle-based surfaces; AFM-based force mapping of the kaolinite JP-based surface; streaming potential and water contact angle measurements of the prepared surfaces; AFM- and sonication-based evaluation of the coating robustness; low-magnification SEM images of the samples after incubation with bacteria (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.S.). Tel.: +49 (0351) 4658 475. Fax: +49 (0351) 4658 474. ORCID

Alla Synytska: 0000-0002-0643-7524 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.S. gratefully acknowledges the Deutsche Forschungsgemeinschaft (Grant SY 125/4-1) for generous financial support. REFERENCES

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