**1. Introduction**

For antifouling properties of materials, the surface plays an important role [1–10]. This is because fouling phenomena occur at materials' surfaces as a result of interactions between various materials and environments. The phenomena should be controlled from both the environmental surroundings

and materials' surfaces. The causes of fouling can be grouped into two main categories of animate and inanimate causes [11]. As for the latter, one can mention many types of contamination from organic to inorganic matter. Usually, they are just called contaminants. The former is usually called biofouling [12,13]. Biofouling is a process where organisms generally attach to materials' surfaces and cause their function and/or characteristics to change and deteriorate in many cases. It can be further classified into microfouling (where microbes such as bacteria and microalgae attach to materials' surfaces) and macrofouling like the attachments of barnacles, oysters, and other bigger organisms in marine environments. For the biofouling in marine environments, microfouling is usually followed by macrofouling. Therefore, the microfouling should be controlled to suppress macrofouling [14,15].

In the case of microfouling, microbial attachments are generally followed by biofilm formations [16–19]. Biofilms are matters formed by bacterial activities. Since microfouling is a process involving bacteria and other micro-organisms, biofilms play an important role. This formation process is well known, and it is one of biofouling. Biofilms are formed on materials' surfaces by multiple steps. Firstly, bacteria attach to materials' surfaces to get nutrition (carbon compounds) which generally exists on materials' surfaces as conditioning film. The repeated process of attachment and detachment occurs. If the attachment phenomenon exceeds the detachment process, then the number of bacteria on materials' surfaces begins to increase. When this number reaches a threshold value, a signal transduction phenomenon called quorum sensing occurs. At this time, the attached bacteria simultaneously excrete polysaccharides. As a result, materials' surfaces would be covered with sticky water films. These sticky water films are called biofilms and are actually the product of microfouling. Then, the biofilm with high water content, becomes a very complex matrix that contains microorganisms (extracellular polymeric substances, EPS). In most cases, they are proteins, nucleic acids, ions (organic, inorganic) and molecules, accumulated from the aqueous environment. Since the biofilm formation makes materials' surfaces sticky, various organic and inorganic contaminants could be attached and kept on materials' surfaces. To keep the materials' surfaces free from contaminants, biofilm formation should be controlled.

From the environmental side, chemical agents such as biocides, etc. might be effective countermeasures to control fouling. On the other hand, fouling could be controlled by using materials with appropriate coatings. The coating is important because it can make a new performance (anti-fouling in this case) expressed, while the inherent properties of the material would be kept.

We have investigated the biofilm formation behaviors for many kinds of materials using some unique evaluation methods and applied coating processes to antifouling effects [20–58]. In this experiment, we focused on a polymer brush coating for the anti-fouling effect. There are already many polymer brush coatings which have been investigated and proposed so far [59–61]. All of them have future possibilities from various viewpoints. The polymer brush we selected for this experiment was one made from an ionic liquid. By applying living radical polymerization to graft polymerization with the ionic liquid, some of the authors succeeded in producing concentrated polymer brushes [62–64]. The polymer brush has lots of attractive properties such as good adherence and water repellency. Particularly, this type of polymer brush has a very low friction coefficient, which may be useful to the automobile industry. Several of the authors have tried to apply it to the decoration of solid-state polymer electrolyte.

While such advanced functions and characteristics have been investigated, antifouling properties have not been closely studied yet. However, we are gradually learning that some researchers have proposed polymer brush coatings. A number of researchers believe that most of the fouling on materials' surfaces is a direct or indirect result of biofilms. Biofilms are formed by bacterial activities as previously described. However, they are different from bacteria. Since they are actually sticky water films on materials' surfaces, they may incorporate organic and inorganic components as they grow. This might result in serious fouling. Some researchers think that polymer brush coatings could be used to repel biofilms and related contaminants [65]. However, this strongly depends on the type of brush coating used and the results of the investigations performed. Therefore, we decided to use

biofilms to test the ionic liquid brush coating that we developed. Such an additional characteristic (to make a material's surface free from contaminants due to anti-fouling effects) will improve the overall performance and broaden the field of applications.

#### **2. Materials and Methods**

In this experiment, we issued a polymer brush coating made from ionic liquid. N,N-diethyl-N-methyl-N-(2-methoxyethyl) ammonium bis(trifluoromethanesulfonyl) imide (DEME-TFSI). Some substituents were added to it and N,N-diethyl-N- (2-methancryloylethy)-Nmethylammonium bis(trifluoromethylsulfonyl) imide (DEMM-TFSI) was grafted on glass specimens (10 mm × 15 mm) through surface-initiated living radical polymerization to get a densely grafting polymer brush coating [66,67]. Since the polymer coating was ionic, it was basically composed of a cationic part (DEMM) and an anionic one (TFSI). However, for this paper, it was called DEMM coating. On the other hand, a PMMA was coated as a reference [68]. In this paper, it was referred to as PMMA coating. It was made on the same size glass specimens through polymerization of methyl methacrylate by Activators Regenerated by Electron Transfer Atom Transfer Radical Polymerization (ARGET ATRP) process [69,70]. Glass has always been used for the general and start-up investigations by our research groups, since they are basically inactive and can avoid reactions between the substrates and solutions.

Our polymer coatings had to be swollen so that the surface would be brush-like. The polymer coating glasses were swollen using the following processes. DEMM coating specimens were immersed in acetonitrile solution for about 12 h. Then the specimens were immersed in 50% water-50% acetonitrile mixed solution for an hour. Finally, the specimens were immersed in water for 1 h. As for the PMMA polymer coated specimens, they were immersed in tetrahydrofuran (THF) solution for about 12 h. Then they were immersed in 50% water–50% THF solution for one hour and finally, they were immersed in water for one hour. Through these steps, surface coated polymers became brush-like coating on glass specimens. And since both polymer coatings were hydrophobic, such step-by-step substitution processes were needed. The number of specimens per each measurement was three (*N* = 3). We designed the polymer brush coating according to our previous studies [62–64,66–68]. The glass plate with the concentrated ionic liquid type polymer brush (the brush length: 500 nm in dry state and the graft density: 0.15 polymer chains/nm2) was used in this test. The graft chain length, the graft density and the brush layer thickness were determined by the gel permeation chromatography [64], thermal decomposition analysis [64] and spectroscopic ellipsometry [66], respectively.

Two kinds of bacteria were used for biofilm formation and evaluation. One of them was *Escherichia coli* (*E. coli*, K-12, G6), a Gram-negative bacteria and the other was *Staphylococcus epidermidis* (*S. epidermidis*, ATCC 35984). Both are typical non-pathogenic bacteria as Gram-negative and Gram-positive bacteria, respectively. Therefore, they were the most suitable for the general investigation of biofilm formation. *E. coli* was cultured in LB (1% tryptone–0.5% yeast extract–1% NaCl) liquid broth in advance for 18 h (±2 h). On the other hand, *S. epidermidis* was cultured in Heart Infusion (HI) liquid broth (1% heart extract–1% peptone–0.5% NaCl) for 18 h (±2 h) in advance. Both were cultured at 37 ◦C in a shaking incubator.

A biofilm study is generally composed of two steps. One of them is to artificially produce biofilms. On a laboratory scale, the environment to produce biofilms is called a "laboratory biofilm reactor". In this experiment, we chose a simple screening process using the 12-well plates. It is generally called the microtiter plate method. This method might provide bacteria with too much nutrition and may be a sort of deflection from the real environment. However, it could give us information for a simple, and rapid data screening process. Each specimen was placed in the well of a sterilized 12-Well plate. Each well was filled with liquid broths containing bacteria. In the case of *E. coli*, the plates were kept at 25 ◦C, so that the biofilm formation would be accelerated. On the other hand, the plates for *S. epidermidis* were kept at 37 ◦C. After one day (24 h) passed, the specimens were removed from the wells. Then, they were evaluated by using Raman spectroscopy combined with optical microscopy (NRS-3100, JASCO, Halifax, NS, Canada). Raman spectroscopy is a useful method to detect exopolymeric substances

(EPS), which are excreted from bacteria and exist as one of the biofilm components. We decided to use the Raman method for this study because ionic interactions could take place using the staining technique with crystal violet solution.
