Electrodeposition of Zwitterionic PEDOT Films for Conducting and

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Electrodeposition of Zwitterionic PEDOT Films for Conducting and Antifouling Surfaces Tatsuro Goda* and Yuji Miyahara Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda, 101-0062 Tokyo, Japan

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S Supporting Information *

ABSTRACT: Conferring antifouling properties can extend the use of conducting polymers in biosensors and bioelectronics under complex biological conditions. On the basis of the antifouling properties of a series of zwitterionic polymers, we synthesized new thiophene-based compounds bearing a phosphorylcholine, carboxybetaine, or sulfobetaine pendant group. The monomers were synthesized by a facile reaction of thiol-functionalized 3,4-ethylenedioxythiophene with zwitterionic methacrylates. Electrochemical copolymerization was performed to deposit zwitterionic poly(3,4ethylenedioxythiophene) (PEDOT) films with tunable conducting and antifouling properties on a conducting substrate. Electrochemical impedance spectroscopy showed that the conductivity and capacitance decreased with increasing zwitterionic content in the films. Protein adsorption and cell adhesion studies showed the effects of the type and content of zwitterions on the antifouling characteristics. Optimization of the electrodeposition conditions enabled development of both conducting and antifouling polymer films. These antifouling conjugated functional polymers have promising applications in biological environments.

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INTRODUCTION Conducting polymers composed of 3,4-ethylenedioxythiophene (EDOT), i.e., PEDOTs, have attracted attention in bioengineering and bioelectronics because of their properties such as electrical and ionic conductivities, chemical stability, low cytotoxicity, mechanical flexibility, and processability.1 Low impedance on an electrode surface underpins the efficacy of electrical recording and stimulation in biological environments.2−4 Many biosensors use PEDOT or its derivatives as selective layers.5−7 The interactions between a conducting polymer and an analyte modulate the physical parameters of the transducer. A target recognition element or bioactive molecule is frequently introduced into either the conjugated polymer or the dopant.8−12 These biocomponents determine the selectivity in biological sensing and signaling. Moreover, organic conducting materials allow ingress of aqueous solutes and ions into their matrixes.13 This is in sharp contrast to silicon-based solid-state electronics, which contain metal oxide surfaces as thin insulating barriers. A structure that is open to charged aqueous species can enable specific applications of PEDOT-based transducers, including organic electrochemical transistors and organic electronic ion pumps.14−17 Although PEDOT use is promising, it has some limitations. When used in biological environments, PEDOT initially undergoes nonspecific interactions with biomolecules. In biomaterials science, protein adsorption onto an implant surface is known to trigger undesired inflammatory and immune responses. The induction of foreign body reactions © XXXX American Chemical Society

limits the lifetimes of implanted electrodes that are used for extended periods. Previous studies have shown that PEDOTbased microelectrodes recruit astrocytes in systematic biological reactions in brain tissue implants.18 Encapsulation by glial cells electrically passivates the PEDOT surface over time. For label-free biosensors, the signal/noise ratio decreases as a result of nonspecific adsorption of biomolecules onto the electrode surface. Fouling is a major hurdle to measurements in real samples in clinical settings and environmental monitoring. Surface modification of PEDOT-based electrodes with fouling-resistant compounds is critical for biological interfaces. Antifouling agents that are covalently bonded with the substrate give coating layers that are more durable and mechanically robust than layers formed by physical attachment.19 Poly(ethylene glycol) (PEG) and poly(methoxyethyl acrylate) [(poly(MEA)] are representative nonionic polymers that show nonthrombogenicity. 19−21 However, PEG is oxidatively degraded over time under physiological conditions.22 Recent studies have also raised concerns regarding the immunogenicity of PEG-conjugated compounds.23 In addition, poly(MEA) is not completely bioinert, and this allows Special Issue: Zwitterionic Interfaces: Concepts and Emerging Applications Received: May 7, 2018 Revised: July 1, 2018 Published: July 13, 2018 A

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Figure 1. Synthesis of antifouling and conducting polymer films. (a) Synthesis of zwitterionic EDOTs via thiol−ene reaction. (b) Electrocopolymerization of zwitterionic EDOTs and EDOT in the presence of NaClO4 dopant. (c) Schematic diagram of antifouling properties of electrodeposited zwitterionic PEDOT films.

attachment of cancer and epithelial cells.24 Zwitterionic polymers such as poly(2-methacryloyloxyethyl phosphorylcholine) [poly(MPC)], poly(carboxybetaine methacrylate) [poly(CBMA)], and poly(sulfobetaine methacrylate) [poly(SBMA)] have high resistance to biofouling.25−33 Their antifouling effectiveness is probably based on the unique hydration mechanisms of zwitterions.32,34 On the basis of these findings, we investigated the development of zwitterionic PEDOT films with antifouling properties. Yu et al. developed PEDOT bearing a phosphorylcholine group in the side chain for efficient neurite recruitment by reducing nonspecific enzyme/cell binding on the surface.35 Polythiophenes bearing sulfobetaine and carboxybetaine moieties were also reported by other group.36,37 Here, we synthesized a series of zwitterionic EDOTs by a simple method via a thiol−ene click reaction (Figure 1).38,39 Electrochemical copolymerization was used to synthesize PEDOT films with various zwitterionic contents on the surfaces of conducting substrates. The electrodeposited films were characterized by X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transfer infrared (ATR-FTIR) spectroscopy, water contact angle measurements, and electrochemical impedance spectroscopy (EIS). The zwitterionic surfaces were also subjected to nonspecific protein adsorption and cell adhesion to evaluate their antifouling effectiveness.

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commercial sources, and used without further purifications. Milli-Q water (EMD Millipore, Billerica, MA) was used throughout the study. Synthesis of Zwitterionic EDOTs. EDOT-SH was synthesized as previously described.39 EDOT-SH (0.28 g, 1.5 mmol) was reacted with MPC (0.30 g, 1.0 mmol), SBMA (0.28 g, 1.0 mmol), or CBMA (0.20 g, 0.86 mmol) by a thiol−ene reaction in the presence of diisopropylamine (DIPA, 23 μL, 20 mol %) in dry chloroform/ methanol (1/1 v/v, 2.0 mL) under a nitrogen atmosphere for 24 h at 50 °C. After the reaction, a brown solid was obtained from the solution containing EDOT-MPC (EDOTPC) by reprecipitation in THF. EDOT-CBMA (EDOTCB) and EDOT-SBMA (EDOTSB) were purified by extraction with dichloromethane and washing three times with brine in a separating funnel. The organic layer was dried over magnesium sulfate and the solvent was removed by evaporation to give EDOTCB and EDOTSB as dark brown oils. EDOT-SH and the zwitterionic EDOTs were characterized by 1H NMR spectroscopy (400 or 500 MHz in CDCl3, Bruker Daltonics, Billerica, MA) (Figure S1). Electropolymerization. A glassy carbon electrode (inner diameter 3 mm, CH Instruments, Austin, TX), which was used as a working electrode, was polished with alumina powder (grain size 0.05 μm, Baikowski International, Charlotte, NC) and washed with water prior to use. The monomers (10 mmol L−1 in total) were added to degassed water (1 mL) containing 100 mmol L−1 NaClO4 in a glass cell. The three-electrode system with a Pt disk counter electrode and an Ag/AgCl wire reference electrode was placed in a glass cell and connected to a potentiostat (PGSTAT302, Metrohm Autolab, Utrecht, The Netherlands). Electropolymerization was achieved by performing consecutive cycles of CV over a potential window from −0.6 to +1.1 to −0.6 V in water, or from −0.6 to +1.4 to −0.6 V in acetonitrile, at a scan rate of 0.1 V s−1 at room temperature. The interface capacitance (C, mF cm−2) at each electropolymerization cycle was determined from the difference between the current densities in the forward and reverse scans (ΔI, mA) at +0.2 V (vs Ag/AgCl):

MATERIALS AND METHODS

Materials. EDOT, MPC, SBMA, potassium ferricyanide, potassium ferrocyanide, and human serum (HS) type AB were purchased from Sigma-Aldrich Japan (Tokyo, Japan). CBMA was purchased from Tokyo Chemical Industry (Tokyo, Japan). NaClO4 was obtained from Wako Pure Chemicals (Tokyo, Japan). 4′,6Diamidino-2-phenylindole (DAPI) was purchased from Dojindo (Tokyo, Japan). Orgacon conducting transparent PET films (thickness 120 μm) were purchased from Japan AGFA-Gevaert (Tokyo, Japan) and used after equilibration in water for at least 4 days. Dulbecco’s phosphate buffered saline (DPBS, pH 7.4), Dulbecco’s modified Eagle’s medium (DMEM), modified Eagle’s medium (MEM), trypsin/EDTA, and Mitotracker CMXRos were obtained from Invitrogen (ThermoFisher Scientific, Waltham, MA). NIH/3T3 mouse embryo fibroblast cell line was provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. Human Caucasian hepatocyte carcinoma (HepG2) cell line was purchased from DS Pharma Biomedical Japan (Osaka, Japan). Fetal bovine serum (FBS) was purchased from MP Biomedicals Japan (Tokyo, Japan). All other reagents were of extra pure grade, purchased from

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C = ΔI /νA −1

2

where v (V s ) and A (cm ) are the scan rate and geometric surface area of the working electrode, respectively. The mean and standard deviation (SD) were obtained from five independent measurements. The proportion of each constituent was expressed as a molar percentage. The thickness of electrodeposited films after 10 cycles of CV scans on a planar gold electrode was measured using a Dektak 150 profilometer (Veeco, Plainview, NY). The mean and SD were obtained from five measurements. Characterization. XPS was performed using an AXIS-HSi165 instrument (Shimadzu-Kratos, Kyoto, Japan) equipped with a 15 kV Mg Kα radiation source at the anode. The takeoff angle of the B

DOI: 10.1021/acs.langmuir.8b01492 Langmuir XXXX, XXX, XXX−XXX

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Figure 2. CV characteristics of zwitterionic PEDOTs. (a) Ten cycles of CV scans for poly(EDOT-co-EDOTPC) at various EDOTPC proportions in feed. (b) Changes in CV capacitance at +0.2 V (vs Ag/AgCl) as a function of CV cycles at different zwitterionic contents in feed. n = 5. (c) Thickness of electrodeposited films. n = 5, *p < 0.05. electrodeposited films on an ORGACON substrate at 1.6 × 104 cells cm−2, and then incubated for 24 h at 37 °C, 5% CO2. Unattached cells were removed by washing twice with DPBS. Adhered cells were treated in 500 nmol L−1 Mitotracker CMXRos for 30 min at 37 °C, 5% CO2, and then washed twice with DPBS. The adhered cells were fixed with 4% paraformaldehyde in DPBS for 15 min and then washed twice with DPBS. The fixed cells were then treated with 10 μg mL−1 DAPI in DPBS for 10 min, and washed twice with DPBS. Fluorescence microscopy images were captured and processed with a Nikon Eclipse Ti inverted microscope with a confocal laser scanning system (Tokyo, Japan). A 20× objective lens (0.75 NA, Nikon) was used. Statistical Analysis. Values were analyzed by ANOVA (oneway), followed by Tukey’s test for multiple comparisons. p < 0.05 was considered statistically significant.

photoelectrons was set at 90°. The composition of the zwitterionic EDOT units was determined by quantitative elemental analysis. The mean and SD were obtained from five measurements at three positions. ATR-FTIR spectra were obtained using a Spectrum 100 FT-IR spectrometer with universal ATR sampling assembly (PerkinElmer, Waltham, MA). The zwitterionic PEDOT films were electrodeposited on a planar gold electrode. Static water contact angle measurements were performed by the sessile drop method using a DM-501 goniometer (Kyowa Interface Science, Saitama, Japan). The mean and SD were obtained from four measurements. EIS was performed using a PGSTAT302 potentiostat (Metrohm Autolab) with a three-electrode cell consisting of a zwitterionicPEDOT-coated glassy carbon working electrode, Pt counter electrode, and Ag/AgCl (in 3.3 mol L−1 KCl aqueous solution) reference electrode in DPBS containing 5 mmol L−1 ferricyanide/ ferrocyanide over the frequency range 0.1 Hz to 10 kHz (10 points per decade), with a 50 mV AC voltage superimposed on a DC bias of +0.2 V. The solution resistance (Rsol), charge-transfer resistance (Rct), film resistance (Rfilm), constant-phase elements of the electrical double layer (CPEdl) and electrodeposited film (CPEf ilm), and Warburg impedance (W) were determined by curve fitting Bode plots to equivalent circuits. The mean and SD were obtained from five measurements. Protein Adsorption. The amounts of protein adsorbed from 10% and 100% HS were determined with a NAPiCOS quartz crystal microbalance (QCM) twin sensor (Nihon Dempa Kogyo, Tokyo, Japan) at a fundamental resonance frequency of 30.8 MHz. After hydration of the electropolymerized films on gold in DPBS, adsorption experiments were performed by dripping a drop of HS solution on the QCM sensors. After incubation for 30 min at 25 °C, the sensor chip was gently rinsed with DPBS and water, and dried in air at 25 °C. Changes in the resonance frequency (ΔF) after protein adsorption were measured in air at 25.00 ± 0.02 °C. The dry mass of adsorbed material (Δm) was determined using the Sauerbrey equation: −Δm/ΔF = 7.23 pg mm−2 Hz−1.40 The mean and SD were obtained from five measurements. Cell Adhesion. NIH/3T3 and HepG2 cells were cultured on a polystyrene tissue culture dish in DMEM supplemented with 10% FBS and 100 μg mL−1 penicillin/streptomycin at 37 °C, 5% CO2. Subconfluent cultures (70−80%) were passaged by treating with 0.25% trypsin/EDTA. Cell suspensions were seeded on the surfaces of

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RESULTS AND DISCUSSION Electropolymerization. Electrochemical random copolymerization was achieved by performing CV cycles with zwitterionic PEDOTs on a working electrode (Figures 2a and S2). Because EDOTCB and EDOTSB are poorly soluble in water, the poly(EDOT-co-EDOTCB) and poly(EDOT-coEDOTSB) films were eletrodeposited in acetonitrile. The peaks corresponding to electropolymerization were observed at +1.1 V and +1.4 V (vs Ag/AgCl) in water and acetonitrile, respectively. The increases in the current with increasing number of CV cycles are characteristic of nucleation and growth of conducting polymers.41 The capacitance C increased with the number of CV cycles for 0, 25, and 50 mol % EDOTPC and 25 mol % EDOTCB (Figure 2b). In contrast, C was unchanged at higher proportions of zwitterionic EDOTs. Good capacitive properties of conducting polymers are derived from fast charge−discharge and doping−undoping processes.42 The lower capacitances at higher zwitterionic contents therefore indicate decreased conductivity as a result of introduction of zwitterionic groups, which caused structural disorder of the conjugated polythiophene main chain. Scanning electron microscopy (SEM) images indicate that the zwitterionic PEDOT surfaces had rough surfaces composed of small particles (Figure S3). Enhanced surface C

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Figure 3. Surface analysis results. (a) High-resolution P 2p and N 1s spectra in XPS and atomic ratios P/(S−P) for films prepared at 25 and 50 mol % EDOTPC. (b) High-resolution N 1s and O 1s spectra and atomic ratios 2N/(O−4N) for films prepared at 25 and 50 mol % EDOTCB. (c) High-resolution N 1s and S 2p spectra and atomic ratios N/(S−2N) for films prepared at 25 and 50 mol % EDOTSB. n = 3. (d) ATR-FTIR spectra of the original and zwitterionic PEDOT films. (e) Static water contact angles of the original and zwitterionic PEDOT films. n = 4. *p < 0.05.

areas correspond to high interfacial capacitances (Figure 2).3 The particle size decreased with increasing zwitterionic proportion from 25 to 50 mol %. The morphology of the conducting polymer film depends on the nucleation and growth processes, and electromigration phenomena.43 Surface Characterization. The O 1s, N 1s, P 2p, and S 2p XPS peaks confirmed incorporation of the zwitterionic EDOTs in the electrodeposited films (Figure 3, Table S1). The proportion of EDOTPC on the surface of the poly(EDOT-coEDOTPC) film was estimated from the atomic ratio P/(S−P) (see the Supporting Information). This ratio was close to stoichiometric at 25 mol % EDOTPC, but was lower than stoichiometric at 50 mol %. Similarly, the estimated EDOTCB [2N/(O−4N)] and EDOTSB [N/(S−2N)] ratios were lower than the stoichiometric value for feeds containing 25 and 50 mol % of the monomer. The results indicate that the reactivities of the zwitterionic EDOTs were lower than that of pristine EDOT during copolymerization because of the presence of bulky side chains. The ATR-FTIR spectra showed common peaks of EDOT− CH2−, −CO, and C−N+ for the zwitterionic films (Figure 3d). The spectral feature at 3400 cm−1 (−OH) implies the hygroscopic nature of the zwitterions. Bands corresponding to O−PO and −SO were observed for the films containing phosphate and sulfate groups, respectively. Static water contact angles on the zwitterionic films were not significantly lower than the original PEDOT films, except for the film containing EDOTPC at 50 mol % in feed (Figure 3e). Because the zwitterions are highly hydrophilic, we attribute the

results to the poor incorporation of the zwitterionic moieties in the electrocopolymerized films as shown by XPS analysis. Electrochemical Impedance Spectroscopy. The introduction of functional groups into the side chains of conjugated polymers can impair the conductivity.39,44 We therefore examined the impedances of the electrodeposited films at various zwitterionic contents (Figure 4a−c). The impedance increased with increasing content of zwitterionic EDOTs. For in-depth analysis, the data were modeled by equivalent circuits (Figure 4d).45 For the electrical double layer, Rct increased with increasing zwitterionic content (Figure 4e). The Rfilm element appeared at high proportions of zwitterionic EDOTs. The increased impedance caused by zwitterionic moieties is consistent with the CV results (Figure 2). CPEdl and CPEfilm decreased at high zwitterionic contents, in agreement with the SEM observations (Figure S3). Among the zwitterionic monomers, EDOTPC had the lowest impedance and highest capacitance. Protein Adsorption. The protein repellency of the electrodeposited zwitterionic PEDOT films was evaluated by measuring the amount of nonspecifically adsorbed proteins in 10% and 100% HS solutions (Figure 5). The amount adsorbed was significantly lower on the zwitterionic PEDOT surface than on pristine PEDOT in both 10% and 100% HS solutions. The antifouling effect was pronounced for 50 mol % zwitterionic EDOTs and was better with EDOTPC and EDOTSB than with EDOTCB. On the basis of the molecular dimensions of major serum proteins and the adsorption amounts, we suggest that the original PEDOT surface D

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Figure 4. EIS analysis of electrodeposited zwitterionic PEDOTs. Bode plots for films prepared at 0, 25, 50, 75, and 100 mol % EDOTPC (a), EDOTCB (b), and EDOTSB (c). Solid lines show curve fitting results. (d) Electrically equivalent circuits used to model data. (e) Summary of curve fitting results for R, CPE, and W. n = 5, N.D.: not determined.

protein adsorption occurs at surface packing defects formed by insufficient zwitterionic contents. For MPC copolymers, full surface coverage by phosphorylcholine groups requires more than 30 mol % of MPC units; this endows nonfouling properties.25 This value is in agreement with our observation that the threshold EDOTPC content for protein resistance was between 25 and 50 mol %. The surfaces containing EDOTCB failed to prevent protein adsorption. Increased adsorption may be caused by uneven coverage or surface vacancies caused by differences between zwitterionic head-groups (Figure 3b,e). However, EIS showed that high contents of zwitterions impair the PEDOT conductivity (Figure 4). The copolymer composition therefore must be carefully tuned to obtain conductivity and antifouling properties. Cell Adhesion. Cell adhesion studies were conducted to further investigate the antifouling properties. Representative fluorescence microscopy images show the nuclei and mitochondria of NIH/3T3 fibroblasts spread on the surface (Figure 6a). Fluorescence from Mitotracker indicates functioning of mitochondria in live cells without any cytotoxicity of the electrodeposited films.59 Loose adhesion of cells with round morphologies (i.e., no migration) was observed at 25 mol % EDOTPC and 50 mol % EDOTSB. Almost no cell adhesion occurred at 50 mol % EDOTPC. The number of adhered cells decreased significantly with increasing proportion of zwitterions (Figure 6b). Protection against cell adhesion was low on the films containing EDOTCB. Similar trends were observed for adhesion of HepG2 cells on these surfaces (Figures S4). Nonspecific adsorption of protein on the surface of a material has been postulated as an initial step in integrinmediated cell adhesion.60 The cell adhesion studies were performed in the condition of 10% FBS, hence nonspecific

undergoes monolayer adsorption and multilayer stacking in 10% and 100% HS, respectively.46,47 Protein adsorption on the zwitterionic PEDOT films is therefore below monolayer coverage in 10% HS and monolayer in 100% HS. In this work, the determining factors in achieving lowfouling surfaces were the zwitterionic type and content. PC consists of an anionic phosphate with a cationic quaternary ammonium end, while SB is a cationic quaternary ammonium with an anionic sulfonate end (Figure 1). A difference between poly(MPC) and poly(SBMA) in the antipolyelectrolyte effect was reported in the literature: poly(MPC) showed completely no change in dimension with an addition of electrolytes, but poly(SBMA) expanded with added salt only at low concentration due to the breakup of intermolecular pairing.48,49 In our study, a major difference between EDOTPC and EDOTSB was the solubility to water. As supported by wettability tests (Figure 3e), EDOTPC was more hydrophilic than EDOTSB. Despite the difference, the films prepared at 50 mol % EDOTPC and EDOTSB commonly gave good surface passivation against nonspecific adsorption in dilute HS solution. In previous reports, zwitterionic polymers and selfassembled monolayers (SAMs) have extremely low fouling properties.19,28−32,50−52 SAMs composed of a 1:1 mixture of alkanethiols with anionic and cationic end groups also show nonfouling properties.53,54 The protein resistance of zwitterions is considered to come from maintenance of the network and number of H-bonds in the surrounding water compared with bulk water.26,55−57 In the molecular design of zwitterions, a low cationic and high anionic charge densities are considered to be essential for antifouling properties.58 The low amounts of protein adsorbed at higher contents of zwitterionic units are attributed to surface coverage by the zwitterions. Nonspecific E

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adsorption (Figure 5), surface passivation against cell adhesion was imperfect on the films containing EDOTCB. The film prepared with 50 mol % EDOTPC prevented cell adhesion, indicating that cell resistance is caused by protein resistance.

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CONCLUSIONS We developed a series of zwitterionic EDOTs as monomers for synthesizing antifouling conjugated polymer films via electrodeposition. Tuning the type and composition of the zwitterionic EDOTs in the electrochemical copolymerization process enables the production of both low-impedance and antifouling surfaces. Among the films investigated, poly(EDOT-co-EDOTPC) gave the lowest impedance and the best resistances to protein adsorption and cell adhesion. This study will help in the molecular design of conducting and antifouling polymers for applications in biosensors and bioelectronics.

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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/acs.langmuir.8b01492. 1

Figure 5. Amount of protein in dry mass adsorbed on electrodeposited films prepared at 0, 25, and 50 mol % EDOTPC, EDOTCB, and EOTSB in 10% HS (a) and 100% HS (b) solutions. n = 7. *p < 0.05.

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H NMR spectra for EDOT-SH, EDOTPC, EDOTCB, and EDOTSB; CV scans for poly(EDOT-co-EDOTCB) and poly(EDOT-co-EDOTSB); SEM images showing surface morphology of zwitterionic PEDOT films; surface characterization by XPS; cell adhesion procedure; Merged confocal fluorescence microscopy images; (PDF)

AUTHOR INFORMATION

Corresponding Author

adsorption of plasma proteins led to cell attachment and migration. In accordance with the results for protein

*E-mail: [email protected]. Tel.: +81-3-5280-8097.

Figure 6. NIH/3T3 cell adhesion results. (a) Merged confocal fluorescence microscopy images (mitochondria in red and nuclei in blue) of cells adhered on electrodeposited films prepared at 0, 25, and 50 mol % EDOTPC, EDOTCB, and EDOTSB. Scale bar: 100 μm. (b) Number of cells adhered on each film. n = 8. *p < 0.05. F

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Langmuir ORCID

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Tatsuro Goda: 0000-0003-2688-8186 Yuji Miyahara: 0000-0003-2703-0958 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.G. is grateful for financial support from the Futaba Electronics Foundation and the Asahi Glass Foundation. Part of the research is based on the Cooperative Research Project of the Research Center for Biomedical Engineering from MEXT Japan. We thank Prof. K. Ishihara at the University of Tokyo for providing access to XPS, Prof. K. Mitsubayashi for offering the use of the surface stylus profiler, and Prof. N. Yui at TMDU for offering the use of the water contact angle goniometer. We thank T. Hatano and M. Toya at TMDU for experimental support and discussion about organic synthesis, respectively.

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DOI: 10.1021/acs.langmuir.8b01492 Langmuir XXXX, XXX, XXX−XXX