artificial cilia as autonomous nanoactuators: design of a gradient … · the basis of this...

R E S EARCH ART I C L E

B IOM IMET I C

1Department of Materials Engineering, School of Engineering, The University of Tokyo,7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. 2Institute of Advanced BiomedicalEngineering and Science, Tokyo Women’s Medical University (TWIns), 8-1 Kawada-cho,Shinjuku-ku, Tokyo 162-8666, Japan.*Corresponding author. Email: [email protected]

Masuda et al. Sci. Adv. 2016; 2 : e1600902 31 August 2016

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10.1126/sciadv.1600902

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

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

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A gradient self-oscillating polymer brush surface with ordered, autonomous, and unidirectional ciliary motionhas been designed. The self-oscillating polymer is a random copolymer composed of N-isopropylacrylamideand 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 gradientby using sacrificial-anode atom transfer radical polymerization. The gradient structure of the polymer brush wasconfirmed 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 uni-directional propagation of the chemical wave from the region with small Ru(bpy)3 amounts to the region with largeRu(bpy)3 amounts. This spatiotemporal control of the ciliary motion would be useful in potential applications tofunctional surface such as autonomous mass transport systems.

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INTRODUCTION
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Spatiotemporally well-ordered mechanical actuation of biomacromol-ecules in the nanometer-order scale driven by chemical reactions, suchas enzymatic reactions, plays an important role in living organisms.For example, motor proteins bind to a polarized cytoskeletal fila-ments and use the energy derived from repeated cycles of adenosine5′-triphosphate hydrolysis to move steadily along them. In addition,many motor proteins carry membrane-enclosed organelles to their ap-propriate locations in the cell. Cytoskeletal motor proteins move uni-directionally along an oriented polymer track. In this process, they usechemical energy to propel themselves along a linear path, and the di-rection of sliding is dependent on the structural polarity of the track.Recently, the construction and design of these biomolecular motorsystems with well-controlled unidirectional motion have become anarea of great focus in advanced sciences (1–3).

On the other hand, inspired by biomolecular motors, a variety ofdynamic material systems based on synthetic polymers have been de-vised. In particular, as bioinspired dynamic materials, many polymersthat can change their physicochemical properties in response to exter-nal stimuli (pH, temperature, electric field, etc.), or stimuli-responsivepolymers, have been extensively studied. As well as the swelling-deswelling motion of a cross-linked polymer (that is, gel), theexpanding-contracting motion of the polymer chains by the on-offswitching of external stimuli is applied to soft actuators, molecularvalves, or switches to control the diffusion or permeation of solutesand the flow direction.

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

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

In contrast to stimuli-responsive polymers and gels, we have sys-tematically investigated “self-oscillating” polymers and gels since thefirst report in 1996 (9). Self-oscillating polymers exhibit autonomousand periodic changes in expansion-contraction or swelling-deswellingbehaviors (in the case of gels) without any on-off switching of externalstimuli (9–11). The basic chemical structure of the self-oscillatingpolymer is a random copolymer composed of N-isopropylacrylamide(NIPAAm) and ruthenium tris(2,2′-bipyridine) [Ru(bpy)3], whichacts as a catalyst for the Belousov-Zhabotinsky (BZ) reaction. TheBZ reaction spontaneously generates redox changes of the catalystmoiety, which creates temporal rhythms or spatiotemporal patternscalled “chemical waves,” referred to as dissipative structures (12, 13).Because the hydrophilicity and the lower critical solution temperatureof 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). Onthe basis of this concept, the self-oscillating polymers and gels haveevolved as autonomous biomimetic material systems, such as bio-mimetic actuators exhibiting a looper-like self-walking motion andtubular gels with an intestine-like peristaltic pumping motion (14, 15).Recently, during the study of AB diblock copolymers containing aself-oscillating polymer segment, un–cross-linked and cross-linkedvesicles (polymersomes) that exhibit self-beating motion, similar tothat of a cardiac muscle cell, were developed (16, 17). Next, extendingthe studies to ABA triblock or ABCBA pentablock copolymers, auto-nomous viscosity oscillation of the polymer solution that mimicsamoeba 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 glasssubstrate, to serve as an artificial model of cilia exhibiting autono-mous wave propagation of polymer chains at the nanometer scale.However, the direction of wave propagation on the polymer brushsurface was observed to be random. In view of the potential applica-tions of self-oscillating polymer brushes (for example, autonomousmass transport systems), controlling the direction of the propagatingchemical wave is an important factor. For bulk gels, both experimen-tal and theoretical studies are reported describing various strategiesto control the dynamic behavior of the self-oscillating gels, such asusing triangle- or pentagon-shaped gel arrays, fabricating the gelswith a gradient in thickness, and designing the composite gel systemswith a nonactive gel and the BZ gel patches having different sizes orcatalyst concentrations (21–23). These studies suggest that introduc-ing heterogeneity into the reaction media is effective in regulatingthe propagating wave during the BZ reaction, and we envision thatthis approach could be applied to the polymer brush systems.

Here, we have prepared a polymer brush surface similar to a livingcilium, exhibiting self-oscillating and unidirectional wave motion ofthe grafted polymer at the nanometer scale. In particular, to achievecontrolled unidirectional motion, we introduced a gradient structureof the polymer brush (Fig. 1) by using the sacrificial-anode atomtransfer radical polymerization (saATRP) method, that is, preparingthe self-oscillating polymer brush with gradient Ru(bpy)3 amount(Fig. 2). saATRP allows the preparation of the target surface with asimple experimental setup and a small volume of the reaction solution(24). saATRP and related methods have been previously used for thefabrication of polymer brush surfaces with gradient structure (24–26);however, this is the first report on the application of saATRP to con-trol the direction of the synchronized nanoactuation of the polymerbrushes. The polymer brush prepared by saATRP was characterizedby attenuated total reflection Fourier transform infrared (ATR/FTIR)spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, and atomicforce microscopy (AFM). The spatial distribution of Ru(bpy)3 andthe chemical wave propagation on the gradient polymer brush duringthe BZ reaction were observed by fluorescence microscopy. This study


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

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RESULTS AND DISCUSSION

Preparation of self-oscillating polymer brushes by saATRPThe self-oscillating polymer [designed to be a random copolymer con-taining 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 graftedonto the glass substrate by saATRP, followed by conjugation ofRu(bpy)3 (Fig. 2). The saATRP reaction is carried out by injectingthe solution containing Cu(II)/tris(2-(N,N-dimethylamino)ethyl)amine[Cu(II)/Me6TREN] into the wedge-shaped gap between the ATRPinitiator–immobilized glass substrate and the sacrificial anode (Zn plate).Cu(II)/Me6TREN is reduced to Cu(I)/Me6TREN on the surface of thesacrificial anode. The generated Cu(I) complex, which acts as an ac-tivator of ATRP, diffuses to the ATRP initiator–immobilized glasssubstrate to promote polymerization (see fig. S1 for the mechanismof saATRP). The gap distance between the two substrates (D) is animportant parameter in controlling the brush thickness, because thepolymerization rate is dependent on the D value (24). A previous studyrevealed that there is an appropriate structure of the self-oscillatingpolymer brush to obtain stable oscillation (20). Thus, the relationshipbetween the amount of grafted poly(NIPAAm-r-NAPMAm) and theD values was investigated. ATR/FTIR spectroscopy was used toestimate the amount of the grafted polymer. The strong Si-O ab-sorption due to the glass substrates appeared at 1000 cm−1, and theabsorption 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 substrateswas determined from the peak intensity ratio I1650/I1000 using a cali-bration curve (20). As the D value increased, the concentration ofCu(I)/Me6TREN catalyst near the ATRP initiator–immobilized glasssubstrate decreased, resulting in a reduced amount of the grafted poly-mer (Fig. 3). Thus, the amount of the grafted polymer is controllableby adjusting the D value.

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

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

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

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


Preparation of a gradient self-oscillating polymer brushOn the basis of these results, a gradient self-oscillating polymer brushwas prepared by saATRP in which the Zn plate was placed with a tiltangle 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 sub-strate every 1 mm along the x axis on the substrate by using x-rayphotoelectron spectroscopy (XPS) (spot size, 400 mm) and summa-rized the results in Table 1. The D value decreases as the position num-ber (0 to 7) increases (Fig. 5A). The C/Si values increased as the positionshifted toward the areas with smaller D value (Fig. 5B), indicating thatthe grafted polymer layer gradually thickened as expected. In addition,the spatial distribution of immobilized Ru(bpy)3 on the polymer brushsurface was evaluated by fluorescence microscopy (585 nm). As shownin Fig. 5C, the gradient of fluorescence intensity, which corresponds tothe gradient of the amount of immobilized Ru(bpy)3, was observed. Theamount of immobilized Ru(bpy)3 was found to increase with theamount of the grafted polymer, which agrees with a previous study(20). The AFM measurement of the gradient self-oscillating polymerbrush height, was also conducted in air. The height of the regionaround the top of the gradient was 77 nm, and that of the regionwith the lower amount of Ru(bpy)3 was 34 nm (see fig. S3). Thus, theobtained polymer brush showed a thickness of several tens of nano-meters in the modified area (5 mm × 10 mm).

Fig. 2. Preparation of the gradient self-oscillating polymer brush. Preparation of the gradient self-oscillating polymer brush grafted on the glasssurfaces by saATRP. (1) Immobilization of the ATRP initiator onto the glass substrate. ClMPETMS, ((Chloromethyl)phenylethyl)trimethoxy silane. (2) Prep-aration 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.

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

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Direction control of chemical wave propagationThe self-oscillating behavior of the self-oscillating polymer brush wasinvestigated by using fluorescence microscopy. The glass substratewith the gradient self-oscillating polymer brush was immersed in anaqueous 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 ob-served for the flat polymer surface prepared by saATRP (see fig.S4A). Next, the dynamic behavior of the gradient surface was inves-tigated. Chemical wave propagation started in the region of the glasssubstrate with larger amounts of Ru(bpy)3. Immediately after that,the starting point of the chemical wave moved toward the regionwith smaller amounts of Ru(bpy)3. Note that the movement of thestarting point of the chemical wave did not occur for the flat surface.Then, the stable wave propagation toward the region with higher Ru(bpy)3amounts was observed (Fig. 6 and movie S1). Figure 6B shows thespatial profile of the chemical wave obtained from Fig. 6A. The

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 reducedstate. 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-oscillatingpolymer 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 gra-dient 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
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waveform was amplified at the site containing larger Ru(bpy)3amounts. The spatiotemporal pattern of the wave propagation (Fig.6C) shows that the velocity of propagation remained almost constantat steady state. At two voluntarily selected positions with small andlarge Ru amounts, stable oscillating profiles with smaller and largeramplitude, respectively, were observed, and a phase difference was de-tected (Fig. 6D). After the stable stage, the derangement of the chem-ical wave started in the Ru(bpy)3 large region and attenuated to thereduced state (the long-duration spatiotemporal analysis is shown infig. S4B).

The behavior observed at the initial stage of the propagation can beexpected as follows. Generally, the BZ reaction has an induction pe-riod before the beginning of the oscillations [the mechanism of the BZreaction using the Field-Körös-Noyes model (27) is shown in the Sup-plementary Materials]. The generation of induction period is due tothe accumulation of bromomalonic acid, and it persists until its crucialconcentration is reached (28). The initial behavior observed in thisstudy suggested that the induction period was shorter in the regionwith a larger amount of Ru(bpy)3. This is consistent with the study


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

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

Fig. 5. Characterization of the gradient polymer brush. (A) Illustrationfor the position on the gradient self-oscillating polymer brush–modifiedglass substrate. (B) The C/Si values analyzed by XPS at each position of thegradient self-oscillating polymer brush. Inset: Illustration of the gradient self-oscillating polymer brush showing the direction of x axis. (C) Cross-sectionalanalysis of the fluorescence intensity of the gradient self-oscillating polymerbrush observed by fluorescence microscopy.

Fig. 6. Chemical wave propagation. (A) Propagation of the chemicalwave on the gradient self-oscillating polymer brush observed by fluores-cence microscopy. Scale bar, 500 mm. The time interval between eachimage 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 ob-tained by image analysis of each image shown in (A). (C) Spatiotemporalpattern of the chemical wave propagation on the gradient self-oscillatingpolymer brush. (D) Oscillating profiles at two different positions with largeand small Ru amounts on the gradient self-oscillating polymer brush. Thedistance between the Ru large and Ru small is 200 mm.

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

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CONCLUSION

In summary, a gradient self-oscillating polymer brush was prepared bysaATRP to control the direction of the chemical wave propagation onthe polymer brush. The amount of grafted polymer can be regulatedby adjusting the distance between the ATRP initiator–immobilizedglass substrate and the sacrificial anode (Zn plate). On the basis of thisfinding, saATRP was performed to prepare the gradient polymerbrush by placing the sacrificial anode with a tilt angle, with respectto the ATRP initiator–immobilized glass substrate. Elemental analysisat different positions and fluorescence observation of immobilizedRu(bpy)3 were conducted by XPS and fluorescence microscopy, respec-tively, revealing the successful formation of the gradient self-oscillatingpolymer brush. During the BZ reaction, the chemical wave propagatedfrom the region with a low Ru(bpy)3 amount to the region with a highRu(bpy)3 amount. The characteristics of the BZ reaction suggestedthat the region with a smaller amount of Ru(bpy)3 acted as thepacemaker of the oscillation. Hence, control of the direction of chem-ical wave on the self-oscillating polymer brush was achieved bydesigning the surface gradient. This functional surface can be appliedto develop spatiotemporally controlled mass transport systems.

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MATERIALS AND METHODS
MaterialsNIPAAm 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 fromTokyo 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 synthe-sized according to the work of Peek et al. (30). ClMPETMS was pur-chased from Gelest. Copper(II) chloride (CuCl2), dehydrated toluene,DMF, DMSO, acetone, methanol, HNO3 aqueous solution, and MAwere purchased from Wako Pure Chemical Industries. Sodium bro-mate (NaBrO3) was purchased from Kanto Chemical. Glass coverslips(24 mm × 50 mm) were obtained from Matsunami Glass Industry.The zinc (Zn) plate was purchased from Junsei Kagaku.

Immobilization of the surface-initiated ATRP initiator ontothe surface of glass substratesThe ATRP initiator (ClMPETMS) was immobilized onto the surfaceof 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 drycleaner (PC-1100) (SAMCO) and placed for 2 hours into a separableflask in which the relative humidity was 60%. A toluene solution


containing ClMPETMS (1.2% v/v) was poured into the separableflask, and the solution was stirred for 18 hours at 25°C. The ATRPinitiator–immobilized glass substrate was washed with toluene and ac-etone and dried in a vacuum oven for 2 hours at 110°C. BeforesaATRP, the ATRP initiator–immobilized glass substrates were cutinto 24-mm × 10-mm rectangles.

Preparation of poly(NIPAAm-r-NAPMAm)-grafted glasssubstrate with gradient structure by saATRPPoly(NIPAAm-r-NAPMAm)-grafted surface with gradient structurewas 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 dis-solved 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 thesolution, and the solution was stirred for 15 min to obtain theATRP catalyst system. The ATRP initiator–immobilized glass sub-strate and the Zn plate were placed face to face, and the distancebetween the two plates was adjusted by using polydimethylsiloxanesheets. The prepared ATRP reaction solution was injected into thegap space between the two plates, and the saATRP reaction wasconducted for 1 hour at 25°C. After completion of the reaction, thepoly(NIPAAm-r-NAPMAm)-grafted glass substrate was washed withacetone, methanol, and water and was then dried in a vacuum ovenfor 3 hours at 100°C.

Conjugation of Ru(bpy)3 to graftedpoly(NIPAAm-r-NAPMAm)The prepared poly(NIPAAm-r-NAPMAm)–grafted surface was con-tacted 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 stepin fig. S1). After completion of the reaction, the substrate was washedwith DMSO and water and dried in a vacuum oven for 3 hours at100°C.

Elemental analysis of the gradient polymer brush by XPSElemental analysis of the gradient self-oscillating polymer brush wasconducted by XPS (Ka) (Thermo Fisher Scientific) using a mono-chromatic Al Ka x-ray source and a take-off angle of 90°. The spotsize of the x-ray was 400 mm, and the distance between the measuredpoints was 1 mm.

Quantitative estimated amount of poly(NIPAAm-r-NAPMAm)grafted on glass substratesThe amount of grafted poly(NIPAAm-r-NAPMAm) was determinedby ATR/FTIR (Nicolet 6700) (Thermo Fisher Scientific) according toa previous study (20). The strong Si-O absorption due to glass substratesappeared at 1000 cm−1, and the absorption of the amide carbonylderived from poly(NIPAAm-r-NAPMAm) on the glass substratesappeared at around 1650 cm−1. The amount of poly(NIPAAm-r-NAPMAm) was determined from the peak intensity ratio I1650/I1000using a calibration curve (20).

Quantitative estimated amount of Ru(bpy)3 immobilized onthe grafted polymerThe amount of Ru(bpy)3 immobilized on the polymer brush surfacewas determined by measuring the absorbance of the substrates at460 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. Theamount of Ru(bpy)3 per area was estimated with the following equa-tion (Lambert-Beer Law)

A ¼ eCL ¼ eC0 ð1Þ

where A is the absorbance, e is the adsorption coefficient, C is the con-centration 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 (micromolesper square centimeter).

Thickness measurement of the grafted polymerThe thickness of the grafted polymer brushes grafted onto the glasssubstrates was analyzed using an AFM (NanoScope, Veeco) in airat 25°C, using tapping mode measurement (cantilever, silicon; scanrate, 0.5 Hz). A part of the polymer brush was removed from the glasssubstrate by scratching with a scalpel blade.

Observation of chemical wave propagation on theself-oscillating polymer brush surfaceThe chemical wave propagation on the self-oscillating polymer brushsurface grafted on glass substrate was observed using a fluorescencemicroscope (DFC 360FX, Leica) (excitation wavelength, 425 nm; flu-orescence wavelength, 585 nm) in a catalyst-free BZ reaction solutioncontaining MA, sodium bromate, and nitric acid at 25°C. Spatiotem-poreal images were analyzed using ImageJ software (NationalInstitutes of Health).

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SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/8/e1600902/DC1fig. 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-Körös-Noyes model)

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AcknowledgmentsFunding: This work was supported in part by a Grant-in-Aid for Scientific Research to R.Y. fromthe 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 YoungScientists (no. 14J09992). Author contributions: T.M. performed all experiments and authored themanuscript. A.M.A., K.N., T.O., and R.Y. conceived and directed the study, as well as authored themanuscript. 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 arepresent in the paper and/or the Supplementary Materials. Additional data related to this papermay be requested from the authors.

Submitted 26 April 2016Accepted 5 August 2016Published 31 August 201610.1126/sciadv.1600902

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

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

Tsukuru Masuda, Aya Mizutani Akimoto, Kenichi Nagase, Teruo Okano and Ryo Yoshida

DOI: 10.1126/sciadv.1600902 (8), e1600902.2Sci Adv

ARTICLE TOOLS http://advances.sciencemag.org/content/2/8/e1600902

MATERIALSSUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2016/08/29/2.8.e1600902.DC1

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