Scholarly article on topic 'Artificial cilia as autonomous nanoactuators: Design of a gradient self-oscillating polymer brush with controlled unidirectional motion'

Artificial cilia as autonomous nanoactuators: Design of a gradient self-oscillating polymer brush with controlled unidirectional motion Academic research paper on "Chemical sciences"

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Academic research paper on topic "Artificial cilia as autonomous nanoactuators: Design of a gradient self-oscillating polymer brush with controlled unidirectional motion"

BIOMIMETIC

Artificial cilia as autonomous nanoactuators: Design of a gradient self-oscillating polymer brush with controlled unidirectional motion

2016 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). 10.1126/sciadv.1600902

Tsukuru Masuda,1 Aya Mizutani Akimoto,1 Kenichi Nagase,2 Teruo Okano,2 Ryo Yoshida1*

A gradient self-oscillating polymer brush surface with ordered, autonomous, and unidirectional ciliary motion has been designed. The self-oscillating polymer is a random copolymer composed of W-isopropylacrylamide and ruthenium tris(2,2'-bipyridine) [Ru(bpy)3], which acts as a catalyst for an oscillating chemical reaction, the Belousov-Zhabotinsky reaction. The target polymer brush surface was designed to have a thickness gradient by using sacrificial-anode atom transfer radical polymerization. The gradient structure of the polymer brush was confirmed by x-ray photoelectron spectroscopy, atomic force microscopy, and ultraviolet-visible spectroscopy. These analyses revealed that the thickness of the polymer brush was in the range of several tens of nanometers, and the amount of Ru(bpy)3 increased as the thickness increased. The gradient polymer brush induced a unidirectional propagation of the chemical wave from the region with small Ru(bpy)3 amounts to the region with large Ru(bpy)3 amounts. This spatiotemporal control of the ciliary motion would be useful in potential applications to functional surface such as autonomous mass transport systems.

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INTRODUCTION

Spatiotemporally well-ordered mechanical actuation of biomacromol-ecules in the nanometer-order scale driven by chemical reactions, such as enzymatic reactions, plays an important role in living organisms. For example, motor proteins bind to a polarized cytoskeletal filaments and use the energy derived from repeated cycles of adenosine 5'-triphosphate hydrolysis to move steadily along them. In addition, many motor proteins carry membrane-enclosed organelles to their appropriate locations in the cell. Cytoskeletal motor proteins move uni-directionally along an oriented polymer track. In this process, they use chemical energy to propel themselves along a linear path, and the direction of sliding is dependent on the structural polarity of the track. Recently, the construction and design of these biomolecular motor systems with well-controlled unidirectional motion have become an area of great focus in advanced sciences (1-3).

On the other hand, inspired by biomolecular motors, a variety of dynamic material systems based on synthetic polymers have been devised. In particular, as bioinspired dynamic materials, many polymers that can change their physicochemical properties in response to external stimuli (pH, temperature, electric field, etc.), or stimuli-responsive polymers, have been extensively studied. As well as the swelling-deswelling motion of a cross-linked polymer (that is, gel), the expanding-contracting motion of the polymer chains by the on-off switching of external stimuli is applied to soft actuators, molecular valves, or switches to control the diffusion or permeation of solutes and the flow direction.

Advances in precise polymerization techniques promoted the synthesis of well-designed polymers with different architectures, such as multiblock copolymers, branched polymers, and polymer brushes. Among them, polymer brushes are the most attractive for the design

department of Materials Engineering, Schoolof Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Corresponding author. Email: ryo@cross.tu-tokyo.acjp

of biomimetic functional surfaces because they allow for the control of the surface properties such as hydrophilicity and friction. In particular, stimuli-responsive polymer brushes have been used as functional surfaces for various applications, including realized oil-repellent surfaces, controlled movement of objects and friction, high-resolution chromatographic stationary phases, and cell culture dishes, which allow cell sheet recovery (4-8).

In contrast to stimuli-responsive polymers and gels, we have systematically investigated "self-oscillating" polymers and gels since the first report in 1996 (9). Self-oscillating polymers exhibit autonomous and periodic changes in expansion-contraction or swelling-deswelling behaviors (in the case of gels) without any on-off switching of external stimuli (9-11). The basic chemical structure of the self-oscillating polymer is a random copolymer composed of N-isopropylacrylamide (NIPAAm) and ruthenium tris(2,2'-bipyridine) [Ru(bpy)3], which acts as a catalyst for the Belousov-Zhabotinsky (BZ) reaction. The BZ reaction spontaneously generates redox changes of the catalyst moiety, which creates temporal rhythms or spatiotemporal patterns called "chemical waves," referred to as dissipative structures (12, 13). Because the hydrophilicity and the lower critical solution temperature of the polymer depend on the redox state, the polymer undergoes self-oscillation by alternatively expanding (hydrated state) and contracting (dehydrated state) (or swelling and deswelling in the case of a gel). On the basis of this concept, the self-oscillating polymers and gels have evolved as autonomous biomimetic material systems, such as bio-mimetic actuators exhibiting a looper-like self-walking motion and tubular gels with an intestine-like peristaltic pumping motion (14, 15). Recently, during the study of AB diblock copolymers containing a self-oscillating polymer segment, un-cross-linked and cross-linked vesicles (polymersomes) that exhibit self-beating motion, similar to that of a cardiac muscle cell, were developed (16, 17). Next, extending the studies to ABA triblock or ABCBA pentablock copolymers, autonomous viscosity oscillation of the polymer solution that mimics amoeba motion has also been achieved (18).

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In parallel, we have developed self-oscillating polymer brushes (19, 20) by immobilizing the self-oscillating polymer onto a glass substrate, to serve as an artificial model of cilia exhibiting autonomous wave propagation of polymer chains at the nanometer scale. However, the direction of wave propagation on the polymer brush surface was observed to be random. In view of the potential applications of self-oscillating polymer brushes (for example, autonomous mass transport systems), controlling the direction of the propagating chemical wave is an important factor. For bulk gels, both experimental and theoretical studies are reported describing various strategies to control the dynamic behavior of the self-oscillating gels, such as using triangle- or pentagon-shaped gel arrays, fabricating the gels with a gradient in thickness, and designing the composite gel systems with a nonactive gel and the BZ gel patches having different sizes or catalyst concentrations (21-23). These studies suggest that introducing heterogeneity into the reaction media is effective in regulating the propagating wave during the BZ reaction, and we envision that this approach could be applied to the polymer brush systems.

Here, we have prepared a polymer brush surface similar to a living cilium, exhibiting self-oscillating and unidirectional wave motion of the grafted polymer at the nanometer scale. In particular, to achieve controlled unidirectional motion, we introduced a gradient structure of the polymer brush (Fig. 1) by using the sacrificial-anode atom transfer radical polymerization (saATRP) method, that is, preparing the self-oscillating polymer brush with gradient Ru(bpy)3 amount (Fig. 2). saATRP allows the preparation of the target surface with a simple experimental setup and a small volume of the reaction solution (24). saATRP and related methods have been previously used for the fabrication of polymer brush surfaces with gradient structure (24-26); however, this is the first report on the application of saATRP to control the direction of the synchronized nanoactuation of the polymer brushes. The polymer brush prepared by saATRP was characterized by attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, and atomic force microscopy (AFM). The spatial distribution of Ru(bpy)3 and the chemical wave propagation on the gradient polymer brush during the BZ reaction were observed by fluorescence microscopy. This study

Fig. 1. Gradient self-oscillating polymer brush. Illustration of the self-oscillating polymer brush inducing unidirectional propagation of the chemical wave.

provides a new concept to design autonomous polymer brush surfaces effective in the nanometer scale as bioinspired dynamic soft materials.

RESULTS AND DISCUSSION

Preparation of self-oscillating polymer brushes by saATRP

The self-oscillating polymer [designed to be a random copolymer containing NIPAAm, Ru(bpy)3, and N-3-(aminopropyl)methacrylamide (NAPMAm) as a binding site of Ru(bpy)3, that is, poly(NIPAAm-r-NAPMAm-r-Ru(bpy)3 NAPMAm)] brush was successfully grafted onto the glass substrate by saATRP, followed by conjugation of Ru(bpy)3 (Fig. 2). The saATRP reaction is carried out by injecting the solution containing Cu(n)/tris(2-(NN"-dimethylamino)ethyl)amine [Cu(II)/Me6TREN] into the wedge-shaped gap between the ATRP initiator-immobilized glass substrate and the sacrificial anode (Zn plate). Cu(II)/Me6TREN is reduced to Cu(I)/Me6TREN on the surface of the sacrificial anode. The generated Cu(I) complex, which acts as an activator of ATRP, diffuses to the ATRP initiator-immobilized glass substrate to promote polymerization (see fig. S1 for the mechanism of saATRP). The gap distance between the two substrates (D) is an important parameter in controlling the brush thickness, because the polymerization rate is dependent on the D value (24). A previous study revealed that there is an appropriate structure of the self-oscillating polymer brush to obtain stable oscillation (20). Thus, the relationship between the amount of grafted poly(NIPAAm-r-NAPMAm) and the D values was investigated. ATR/FTIR spectroscopy was used to estimate the amount of the grafted polymer. The strong Si-O absorption due to the glass substrates appeared at 1000 cm-1, and the absorption of the amide carbonyl derived from poly(NIPAAm-r-NAPMAm) on the glass substrates appeared at around 1650 cm-1. The amount of poly(NIPAAm-r-NAPMAm) on the glass substrates was determined from the peak intensity ratio 11650/11000 using a calibration curve (20). As the D value increased, the concentration of Cu(I)/Me6TREN catalyst near the ATRP initiator-immobilized glass substrate decreased, resulting in a reduced amount of the grafted polymer (Fig. 3). Thus, the amount of the grafted polymer is controllable by adjusting the D value.

Next, the D value was fixed at 500 mm (the amount of the grafted polymer is 9.79 ± 2.90 mg cm-2), and more detailed characterization was carried out after conjugation of the N-hydroxysuccinimidyl (NHS) esters of Ru(bpy)3 [hereinafter, Ru(bpy)3-NHS] to the amino group of NAPMAm. The amount of immobilized Ru(bpy)3 was determined to be 1.05 ± 0.17 nmol cm-2 by UV-vis spectroscopy, and the thickness of the polymer brush was found to be 59 nm by AFM observation in air (AFM images are shown in fig. S2). These values were comparable with those of the self-oscillating polymer brushes that were investigated in a previous study (20). Thus, these results demonstrate the successful preparation of the target polymer brush surface by saATRP, followed by conjugation of Ru(bpy)3.

To clarify whether the thickness of the polymer changes in response to the redox states of the Ru(bpy)3 moiety, we conducted the AFM measurements in aqueous solution (Fig. 4). The heights of the self-oscillating polymer brush prepared by saATRP in reduced and oxidized states were 50 and 95 nm, respectively. Thus, it was revealed that the height in the oxidized state was actually larger than that in the reduced state. As for the phase images, the phase difference between the polymer brush and the cleaved area was observed in the reduced

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Fig. 2. Preparation of the gradient self-oscillating polymer brush. Preparation of the gradient self-oscillating polymer brush grafted on the glass surfaces by saATRP. (1) Immobilization of the ATRP initiator onto the glass substrate. ClMPETMS, ((Chloromethyl)phenylethyl)trimethoxy silane. (2) Preparation of gradient poly(NIPAAm-r-NAPMAm) by saATRP. DMF, dimethylformamide. (3) Conjugation of Ru(bpy)3 to the amino group of NAPMAm. DMSO, dimethyl sulfoxide.

Preparation of a gradient self-oscillating polymer brush

On the basis of these results, a gradient self-oscillating polymer brush was prepared by saATRP in which the Zn plate was placed with a tilt angle of 10°, with respect to the ATRP initiator-immobilized substrate (see fig. S1). To confirm the formation of the gradient polymer brush, we conducted the elemental analysis at several positions of the substrate every 1 mm along the x axis on the substrate by using x-ray photoelectron spectroscopy (XPS) (spot size, 400 mm) and summarized the results in Table 1. The D value decreases as the position number (0 to 7) increases (Fig. 5A). The C/Si values increased as the position shifted toward the areas with smaller D value (Fig. 5B), indicating that the grafted polymer layer gradually thickened as expected. In addition, the spatial distribution of immobilized Ru(bpy)3 on the polymer brush surface was evaluated by fluorescence microscopy (585 nm). As shown in Fig. 5C, the gradient of fluorescence intensity, which corresponds to the gradient of the amount of immobilized Ru(bpy)3, was observed. The amount of immobilized Ru(bpy)3 was found to increase with the amount of the grafted polymer, which agrees with a previous study (20). The AFM measurement of the gradient self-oscillating polymer brush height, was also conducted in air. The height of the region around the top of the gradient was 77 nm, and that of the region with the lower amount of Ru(bpy)3 was 34 nm (see fig. S3). Thus, the obtained polymer brush showed a thickness of several tens of nanometers in the modified area (5 mm x 10 mm).

Fig. 3. Amount of poly(NIPAAm-r-NAPMAm) grafted on the glass substrate. Dependence of the amount of grafted polymer on the gap distance (D) between the sacrificial anode and the ATRP initiator-immobilized glass substrate.

state, which was not observed in the oxidized state (Fig. 4, B and E). Additionally, in the previous study, quarts crystal microbalance measurements revealed that the polymer brush actually undergoes swelling-deswelling changes in response to temperature change or oxidization of the Ru(bpy)3 moiety (20). In these ways, it was confirmed that the self-oscillating polymer brush is more swollen in the oxidized state than in the reduced state.

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2.5 5.0 7.5 10.0 0 2.5 5.0 7.5

Position (jjm) Position (|jm)

Fig. 4. AFM observation. (A to C) Height image (A), phase image (B), and cross-sectional analysis (C) of the self-oscillating polymer brush in the reduced state. Outer solution: 0.81 M HNO3 and 0.15 M NaCl. (D to F) Height image (D), phase image (E), and cross-sectional analysis (F) of the self-oscillating polymer brush in the oxidized state. Outer solution: 0.81 M HNO3 and 0.15 M NaBrO3.

Table 1. Elemental analyses. Elemental analysis at each position of the gradient self-oscillating polymer brush shown in Fig. 4A. n.d., note detected.

Atom (%)

Position Si Cl Ru C N O C/Si N/C

0 19.7 1.3 n.d. 41.3 1.3 36.4 2.1 0.03

1 23.2 0.7 n.d. 30.3 1.6 44.2 1.3 0.05

2 18.0 0.6 0.1 39.6 4.3 37.4 2.2 0.11

3 6.6 0.6 0.2 63.4 9.8 19.4 9.6 0.15

4 2.0 0.5 0.1 72.3 11.0 14.1 36.4 0.15

5 1.4 0.4 0.2 73.2 11.0 13.8 57.2 0.15

6 21.6 0.6 0.1 31.7 2.7 43.3 1.5 0.08

7 22.1 0.9 n.d. 30.8 2.1 44.1 1.4 0.07

Direction control of chemical wave propagation

The self-oscillating behavior of the self-oscillating polymer brush was investigated by using fluorescence microscopy. The glass substrate with the gradient self-oscillating polymer brush was immersed in an aqueous solution containing the reactants of the BZ reaction [HNO3, NaBrO3, and malonic acid (MA)] except for the catalyst at 25°C. First, it was confirmed that autonomous oscillation was successfully observed for the flat polymer surface prepared by saATRP (see fig. S4A). Next, the dynamic behavior of the gradient surface was investigated. Chemical wave propagation started in the region of the glass substrate with larger amounts of Ru(bpy)3. Immediately after that, the starting point of the chemical wave moved toward the region with smaller amounts of Ru(bpy)3. Note that the movement of the starting point of the chemical wave did not occur for the flat surface. Then, the stable wave propagation toward the region with higher Ru(bpy)3 amounts was observed (Fig. 6 and movie S1). Figure 6B shows the spatial profile of the chemical wave obtained from Fig. 6A. The

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Position

Fig. 5. Characterization of the gradient polymer brush. (A) Illustration for the position on the gradient self-oscillating polymer brush-modified glass substrate. (B) The C/Si values analyzed by XPS at each position of the gradient self-oscillating polymer brush. Inset: Illustration of the gradient self-oscillating polymer brush showing the direction of xaxis. (C) Cross-sectional analysis of the fluorescence intensity of the gradient self-oscillating polymer brush observed by fluorescence microscopy.

waveform was amplified at the site containing larger Ru(bpy)3 amounts. The spatiotemporal pattern of the wave propagation (Fig. 6C) shows that the velocity of propagation remained almost constant at steady state. At two voluntarily selected positions with small and large Ru amounts, stable oscillating profiles with smaller and larger amplitude, respectively, were observed, and a phase difference was detected (Fig. 6D). After the stable stage, the derangement of the chemical wave started in the Ru(bpy)3 large region and attenuated to the reduced state (the long-duration spatiotemporal analysis is shown in fig. S4B).

The behavior observed at the initial stage of the propagation can be expected as follows. Generally, the BZ reaction has an induction period before the beginning of the oscillations [the mechanism of the BZ reaction using the Field-Koros-Noyes model (27) is shown in the Supplementary Materials]. The generation of induction period is due to the accumulation of bromomalonic acid, and it persists until its crucial concentration is reached (28). The initial behavior observed in this study suggested that the induction period was shorter in the region with a larger amount of Ru(bpy)3. This is consistent with the study

Time (s)

Fig. 6. Chemical wave propagation. (A) Propagation of the chemical wave on the gradient self-oscillating polymer brush observed by fluorescence microscopy. Scale bar, 500 mm. The time interval between each image is 10 s, and the temperature is 25°C. Outer solution: [HNO3] = 0.81 M, [NaBrO3] = 0.15 M, [MA] = 0.10 M. (B) Chemical wave profiles obtained by image analysis of each image shown in (A). (C) Spatiotemporal pattern of the chemical wave propagation on the gradient self-oscillating polymer brush. (D) Oscillating profiles at two different positions with large and small Ru amounts on the gradient self-oscillating polymer brush. The distance between the Ru large and Ru small is 200 mm.

by Suzuki and Yoshida (29) on self-oscillating microgels, which demonstrated that the induction period increased as the effective Ru(bpy)3 concentration decreased.

The observed stable wave propagation with respect to the Ru(bpy)3 gradient can also be explained by the characteristics of the BZ reaction. In the study by Yashin et al. (23) on the heterogeneous self-oscillating gels, it was demonstrated theoretically and experimentally that chemical wave propagated from the gel with lower Ru(bpy)3 concentration to the gel with higher Ru(bpy)3 concentration (or the smaller gel to the larger gel), because of the shorter oscillation period

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of the self-oscillating gel with lower Ru(bpy)3 concentration. Chemical waves with higher frequency usually predominate in the BZ reaction medium because chemical waves annihilate when they collide. Thus, the region of the gradient self-oscillating polymer brush with a smaller Ru(bpy)3 amount acts as the pacemaker of the oscillation. As a result, the direction of the chemical wave propagating in the self-oscillating polymer brush can be controlled, which will be helpful for applications such as autonomous mass transport system.

CONCLUSION

In summary, a gradient self-oscillating polymer brush was prepared by saATRP to control the direction of the chemical wave propagation on the polymer brush. The amount of grafted polymer can be regulated by adjusting the distance between the ATRP initiator-immobilized glass substrate and the sacrificial anode (Zn plate). On the basis of this finding, saATRP was performed to prepare the gradient polymer brush by placing the sacrificial anode with a tilt angle, with respect to the ATRP initiator-immobilized glass substrate. Elemental analysis at different positions and fluorescence observation of immobilized Ru(bpy)3 were conducted by XPS and fluorescence microscopy, respectively, revealing the successful formation of the gradient self-oscillating polymer brush. During the BZ reaction, the chemical wave propagated from the region with a low Ru(bpy)3 amount to the region with a high Ru(bpy)3 amount. The characteristics of the BZ reaction suggested that the region with a smaller amount of Ru(bpy)3 acted as the pacemaker of the oscillation. Hence, control of the direction of chemical wave on the self-oscillating polymer brush was achieved by designing the surface gradient. This functional surface can be applied to develop spatiotemporally controlled mass transport systems.

MATERIALS AND METHODS Materials

NIPAAm was provided by KJ Chemicals and purified by recrystalli-zation in toluene/hexane. NAPMAm was purchased from Poly-sciences and used as received. Me6TREN was purchased from Tokyo Chemical Industries. Bis(2,2'-bipyridine) (1-(4'-methyl-2,2'-bipyridine-4-carbonyloxy)-2,5-pyrrolidinedione) ruthenium(II) bis(hexafluorophosphate) (abbreviated as Ru(bpy)3-NHS) was synthesized according to the work of Peek et al. (30). ClMPETMS was purchased from Gelest. Copper(II) chloride (CuCl2), dehydrated toluene, DMF, DMSO, acetone, methanol, HNO3 aqueous solution, and MA were purchased from Wako Pure Chemical Industries. Sodium bromate (NaBrO3) was purchased from Kanto Chemical. Glass coverslips (24 mm x 50 mm) were obtained from Matsunami Glass Industry. The zinc (Zn) plate was purchased from Junsei Kagaku.

Immobilization of the surface-initiated ATRP initiator onto the surface of glass substrates

The ATRP initiator (ClMPETMS) was immobilized onto the surface of the glass substrates by silane coupling reaction (first step in Fig. 2). Glass coverslips were cleaned by oxygen plasma irradiation (intensity, 400 W; oxygen pressure, 0.1 mmHg; duration, 180 s) in a plasma dry cleaner (PC-1100) (SAMCO) and placed for 2 hours into a separable flask in which the relative humidity was 60%. A toluene solution

containing ClMPETMS (1.2% v/v) was poured into the separable flask, and the solution was stirred for 18 hours at 25°C. The ATRP initiator-immobilized glass substrate was washed with toluene and acetone and dried in a vacuum oven for 2 hours at 110°C. Before saATRP, the ATRP initiator-immobilized glass substrates were cut into 24-mm x 10-mm rectangles.

Preparation of poly(NIPAAm-r-NAPMAm)-grafted glass substrate with gradient structure by saATRP

Poly(NIPAAm-r-NAPMAm)-grafted surface with gradient structure was prepared by saATRP (the second step in Fig. 2). NIPAAm (1.53 g, 13.5 mmol) and NAPMAm (0.268 g, 1.5 mmol) were dissolved in a DMF (2.5 ml)/water (2.5 ml) (1:1) mixture. CuCl2 (10.1 mg, 0.075 mmol) and Me6TREN (40.0 ml, 0.15 mmol) were added to the solution, and the solution was stirred for 15 min to obtain the ATRP catalyst system. The ATRP initiator-immobilized glass substrate and the Zn plate were placed face to face, and the distance between the two plates was adjusted by using polydimethylsiloxane sheets. The prepared ATRP reaction solution was injected into the gap space between the two plates, and the saATRP reaction was conducted for 1 hour at 25°C. After completion of the reaction, the poly(NIPAAm-r-NAPMAm)-grafted glass substrate was washed with acetone, methanol, and water and was then dried in a vacuum oven for 3 hours at 100°C.

Conjugation of Ru(bpy)3 to grafted poly(NIPAAm-r-NAPMAm)

The prepared poly(NIPAAm-r-NAPMAm)-grafted surface was contacted with 300 ml of a DMSO solution containing Ru(bpy)3-NHS (21.2 mg) and triethylamine (25 ml) for 4 hours at 25°C (the third step in fig. S1). After completion of the reaction, the substrate was washed with DMSO and water and dried in a vacuum oven for 3 hours at 100°C.

Elemental analysis of the gradient polymer brush by XPS

Elemental analysis of the gradient self-oscillating polymer brush was conducted by XPS (Ka) (Thermo Fisher Scientific) using a monochromatic Al Ka x-ray source and a take-off angle of 90°. The spot size of the x-ray was 400 mm, and the distance between the measured points was 1 mm.

Quantitative estimated amount of poly(NIPAAm-r-NAPMAm) grafted on glass substrates

The amount of grafted poly(NIPAAm-r-NAPMAm) was determined by ATR/FTIR (Nicolet 6700) (Thermo Fisher Scientific) according to a previous study (20). The strong Si-O absorption due to glass substrates appeared at 1000 cm-1, and the absorption of the amide carbonyl derived from poly(NIPAAm-r-NAPMAm) on the glass substrates appeared at around 1650 cm-1. The amount of poly(NIPAAm-r-NAPMAm) was determined from the peak intensity ratio 1165o/i1ooo using a calibration curve (20).

Quantitative estimated amount of Ru(bpy)3 immobilized on the grafted polymer

The amount of Ru(bpy)3 immobilized on the polymer brush surface was determined by measuring the absorbance of the substrates at 460 nm using a UV-vis spectrophotometer (UVPC-2500) (Shimadzu) according to a previous study (20). A calibration curve was prepared

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from a series of Ru(bpy)3 solution with known concentration. The amount of Ru(bpy)3 per area was estimated with the following equation (Lambert-Beer Law)

A = eCL = eC

where A is the absorbance, e is the adsorption coefficient, C is the concentration of Ru(bpy)3 (millimolar), L is the length of the light pass (centimeter), and C' is the amount of Ru(bpy)3 per area (micromoles per square centimeter).

Thickness measurement of the grafted polymer

The thickness of the grafted polymer brushes grafted onto the glass substrates was analyzed using an AFM (NanoScope, Veeco) in air at 25°C, using tapping mode measurement (cantilever, silicon; scan rate, 0.5 Hz). A part of the polymer brush was removed from the glass substrate by scratching with a scalpel blade.

Observation of chemical wave propagation on the self-oscillating polymer brush surface

The chemical wave propagation on the self-oscillating polymer brush surface grafted on glass substrate was observed using a fluorescence microscope (DFC 360FX, Leica) (excitation wavelength, 425 nm; fluorescence wavelength, 585 nm) in a catalyst-free BZ reaction solution containing MA, sodium bromate, and nitric acid at 25°C. Spatiotem-poreal images were analyzed using ImageJ software (National Institutes of Health).

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/

content/full/2/8/e1600902/DC1

fig. S1. Mechanism of saATRP.

fig. S2. AFM observation for the flat surface.

fig. S3. AFM observation for the gradient surface.

fig. S4. Spatiotemporal analysis of the BZ reaction.

movie S1. Chemical wave propagation.

Mechanism for the BZ reaction (Field-Koros-Noyes model)

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Acknowledgments

Funding: This work was supported in part by a Grant-in-Aid for Scientific Research to R.Y. from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant no. 15H02198) and a research fellowship to T.M. from the Japan Society for the Promotion of Science for Young Scientists (no. 14J09992). Author contributions: T.M. performed all experiments and authored the manuscript. A.MA, K.N., T.O., and R.Y. conceived and directed the study, as well as authored the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

Submitted 26 April 2016 Accepted 5 August 2016 Published 31 August 2016 10.1126/sciadv.1600902

Citation: T. Masuda, A. M. Akimoto, K. Nagase, T. Okano, R. Yoshida, Artificial cilia as autonomous nanoactuators: Design of a gradient self-oscillating polymer brush with controlled unidirectional motion. Sci. Adv. 2, e1600902 (2016).

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Artificial cilia as autonomous nanoactuators: Design of a gradient self-oscillating polymer brush with controlled unidirectional motion

Tsukuru Masuda, Aya Mizutani Akimoto, Kenichi Nagase, Teruo Okano and Ryo Yoshida (August 31, 2016) Sci Adv 2016, 2:. doi: 10.1126/sciadv.1600902

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