Programmed Transport and Release of Cells by Self-Propelled

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Programmed Transport and Release of Cells by Self-Propelled Micromotors Yoshitaka Yoshizumi, Kyohei Okubo, Masatoshi Yokokawa, and Hiroaki Suzuki Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04206 • Publication Date (Web): 29 Aug 2016 Downloaded from http://pubs.acs.org on September 3, 2016

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Programmed Transport and Release of Cells by SelfPropelled Micromotors Yoshitaka Yoshizumi, Kyohei Okubo, Masatoshi Yokokawa, Hiroaki Suzuki*

Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan

ACS Paragon Plus Environment

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ABSTRACT

Autonomous transport and release of bacterial cells by self-propelled micromotors was achieved. The motors consisted of zinc and platinum hemispheres formed on polystyrene beads and moved as a result of simultaneous redox reactions occurring on both metal ends. The highly negative redox potential of zinc enabled the selection of a wide variety of organic redox compounds as fuels, such as methanol and p-benzoquinone. The movement of motors was observed in solutions of fuels. To realize autonomous capture, transport, and release of cargo, a self-assembled monolayer (SAM) was formed on the platinum part of the motor. This SAM could be desorbed by coupling the reaction with the dissolution of zinc, which could also be controlled by adjusting the concentration of Zn2+ ions. Escherichia coli (E. coli) cells were captured by the motor (due to hydrophobic interactions), transported, and released following SAM desorption at the mixed potential.


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INTRODUCTION The demand for individual cell manipulation and analysis has remarkably increased over the last decade in a wide range of research fields, including in vitro fertilization, single cell transfection, inter-cell interaction, microbiology, stem cells, and tissue engineering.1–3 To this end, micromanipulators,4,5 optical and magnetic tweezers,6,7 electrokinetic systems,8 and microfluidic devices9,10 have been reported and are widely used. However, each method has its own advantages and disadvantages. Micromanipulators in combination with an inverted microscope are a standard way of handling cells. However, they require highly skilled operators, and the time needed for cell transfer limits their throughput. On the other hand, microfluidic devices can potentially realize easy, high-throughput, and low sample volume cell manipulation. However, they require external instruments such as pumps and valves. In this respect, micro/nanomotors are advantageous, since they can move by themselves and allow additional functions to be imparted, depending on the improvement. In this sense, micro/nanomotors may provide a new paradigm for conducting efficient chemical analysis even in still-standing solutions. The development of artificial self-propelled micro/nanomotors is currently a subject of great interest for futuristic smart devices,11,12 since they move spontaneously and can be batchfabricated.

Representative

motors

include

self-powered

nanowires,13–16

tubular-shaped

micromotors,17–22 spherical Janus micromotors,23–26 and a polymer single-crystal micromotor.27 The driving force of these motors is provided by the direct catalytic conversion of chemical energy.28–30 Segmented bimetallic gold/platinum (Au/Pt) nanorods powered by electrochemical decomposition of hydrogen peroxide (H2O2) have been used as common micro/nanomotors. Motors with similar bimetallic structures, such as Pt/Cu,31 Ni/Au, Au/Pd, Au/Ru, and Ru/Pt32 have also been reported. However, the fuels used for these devices are limited to concentrated


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H2O2 (~1 M), strong acids, or other strong redox reagents. Therefore, to expand the range of applications to biology, motors using biologically friendly fuels such as glucose33 and water34 have been proposed. Bubble-propelled microtube motors utilizing H2O2 at low concentrations (< 100 mM) have also been reported.35 Furthermore, in addition to simple movement, selective capture and transport of objects such as drugs, proteins, and cells using specific DNA probes or antibodies have also been reported.21,36–40 These recent advances demonstrate that self-propelled micro/nanomotors are promising tools for cell manipulation. Herein, we propose a novel method to transport targeted living Escherichia coli (E. coli) cells and release them autonomously. We employed a zinc/platinum (Zn/Pt) spherical micromotor with a self-assembled monolayer (SAM) of alkanethiolates to capture cells via non-specific hydrophobic interactions. A key point was the use of zinc, which exhibits a highly negative redox potential, expanding the range of suitable organic redox compounds as fuels and enabling autonomous reductive desorption of the alkanethiolates for cell release. We demonstrated the capture, transport, and release of living E. coli cells in solutions containing organic redox compounds, such as p-benzoquinone and methanol.

EXPERIMENTAL Materials. The reagents and materials used for motor fabrication were obtained from the following commercial sources: a suspension of spherical polystyrene beads (5 µm in diameter) from Spherotech (Lake Forest, IL); cover slips (24 mm × 24 mm and 22 mm × 40 mm, 0.12–0.17 mm thick) from Matsunami Glass Industries (Osaka, Japan); paraffin film (Parafilm, 130 µm thick) from Bemis Company, Inc. (Chicago, IL); methanol, ethanol, p-benzoquinone, 1decanethiol, 1-hexanethiol, and zinc chloride (ZnCl2) from Wako Pure Chemical Industries (Osaka, Japan); and Luria-Bertani (LB) medium from Sigma-Aldrich (Saint Louis, MO). Unless


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otherwise stated, all chemicals were of reagent grade. Deionized Milli-Q water (18.2 MΩ cm, Millipore, Billerica, MA) was used for all solutions, including those of fuels.

Micromotor Fabrication and Motion Principle. Zn/Pt micromotors were fabricated by depositing platinum and zinc on polystyrene beads of 5 µm diameter (Figure 1A,B). The suspension of beads was diluted with pure ethanol to 0.5% (w/v) and dispersed on a cover slip. After drying at room temperature, the beads were coated with platinum (thickness: 50 nm) by RF magnetron sputtering (CFS-4ES, Shibaura Mechatronics, Kanagawa, Japan) in argon gas at a pressure of 3.3 × 10−1 Pa (background pressure: 3.0 × 10−3 Pa) and 100 W RF power, resulting in ca. 70% surface coverage. The beads were suspended in ethanol using an ultrasonic cleaner and collected by centrifugation at 7,740 g for 10 s. The precipitate was re-suspended in pure ethanol, and the beads were dispersed on a cover slip and dried. Since the beads partially coated with platinum were randomly oriented, two more platinum deposition cycles were performed to achieve uniform coverage, followed by deposition of gold and zinc. Gold served as an intermediate layer between platinum and zinc, since the mutual adhesion of these two metals was extremely poor. Gold (50 nm) was deposited at a rate of 0.3 nm/s (background pressure: 1.0 × 10−5 Pa) using an electron beam evaporator (Eiko Engineering, Tokyo, Japan), whereas zinc (300 nm) was deposited at a rate of 0.1 nm/s (background pressure: 1.0 × 10−3 Pa) by a thermal evaporator (Biemtron, Ibaraki, Japan). The covered beads were finally released from the substrate, centrifuged, repeatedly washed with ethanol, and finally dispersed in Milli-Q water. Zn/Ni/Pt micromotors and Au/Pt beads used for comparison (Figure 1C,D) were fabricated in the same manner. To fabricate the Zn/Ni/Pt micromotors, nickel (50 nm) was additionally deposited at 0.1 nm/s (background pressure: 1.0 × 10−5 Pa) using the electron beam evaporator


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before the deposition of gold and zinc layers. Thicknesses of metals other than nickel equaled those of the Zn/Pt micromotors. When a Zn/Pt micromotor is put in a solution of fuel, pH and ionic gradients are formed due to the following redox reactions occurring on the zinc and platinum ends: Zn → Zn2+ + 2e−

E° = − 0.763 V vs. SHE

(1)

CH3OH (aq) + 2H+ + 2e− → CH4 (g) + H2O

E° = 0.588 V vs. SHE

(2)

E° = 0.699 V vs. SHE

(3)

or p-benzoquinone (aq) + 2H+ + 2e− → hydroquinone (aq)

The electric field generated by the concomitant charge separation subsequently generates an electroosmotic flow along the motor surface.32,41,42 Due to the Galilean inverse of the fluid flow, the motor the motor moves with the zinc end at front. Owing to its highly negative redox potential, zinc can reduce a relatively wide range of redox compounds or ions.

Short-Circuit Current Measurement between Zinc and Platinum Electrodes. The progress of redox reactions was investigated by measuring the short-circuit current that flowed at the mixed potential between independent planar zinc and platinum electrodes (4.4 cm2 each) connected with each other. The electrodes were fabricated by depositing chromium and platinum, or chromium, gold, and zinc onto 22 mm × 40 mm cover slips in the above order. Gold and chromium were used as intermediate layers to promote adhesion. The short-circuit current was measured 180 s after immersing the electrodes in aqueous methanol or p-benzoquinone solutions using a potentiostat (HA-151, Hokuto Denko, Tokyo, Japan). This current was also measured in the same manner using independent gold and platinum electrodes connected with each other.


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Micromotor Characterization. The motors were structurally characterized by scanning electron microscopy (SEM) (Helios NanoLab 600i, FEI, Hillsboro, OR) at an acceleration voltage of 10 kV. An energy-dispersive X-ray spectroscopy (EDX) module (Apollo XL, EDAX, Mahwah, NJ) attached to the SEM unit was used to obtain the elemental maps of Zn/Pt beads at 20 kV. Movement of the motors was observed using an optical microscope (BX51, Olympus, Tokyo, Japan) with a 40× objective (NA: 0.9). Video images were recorded using a CMOS camera (EOS 60D, Canon, Tokyo, Japan). An observation chamber was fabricated by inserting two paraffin film spacers (130 µm thick) between a slide glass (76 mm × 26 mm) and a cover slip (24 mm × 24 mm) (Figure 4A). A 20-µL aliquot of the bead suspension containing fuels of various concentrations was injected into the chamber, and the latter was sealed with glue to prevent evaporation. All experiments were carried out at room temperature. Images were processed using the ImageJ software (Wayne Rasband, NIH) to analyze the trajectories of individual beads.

Preparation of E. coli. E. coli K-12 strain MG1655 was grown overnight by shake culturing at 37 °C in 20 mL of the LB medium. After centrifugation for 10 min at 3,000 g and 4 °C, the precipitate of E. coli was re-suspended in ice-cold LB medium. The number of E. coli cells was then determined by optical density measurement (OD600, UV-1800, Shimadzu, Kyoto, Japan). The suspension of E. coli was diluted to an OD of 1.0 with ice-cold phosphate buffered saline (PBS). The solution was then aliquoted and stored as individual standard stocks in a deep freezer until use.

Release of E. coli from a Zn/Pt Composite Electrode with SAM. A Zn/Pt composite electrode (4.4 cm2 of each metal) was fabricated by first depositing chromium and platinum onto


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a cover slip (22 mm × 40 mm) and then covering half of the platinum area with gold and zinc in the order stated. To form a hydrophobic SAM, the platinum area was immersed in a mixture of 10 µM 1-decanethiol (or 10 µM 1-hexanethiol) and 1.0 mM ethanolic ZnCl2 for 30 min. After washing the electrode with ethanol and Milli-Q water, it was immersed in an E. coli suspension (OD600 = 1.0) containing 1.0 mM ZnCl2 for 15 min and washed with a 1.0 mM aqueous ZnCl2 solution. The electrode was then immediately immersed in a 1.0 mM aqueous ZnCl2 solution, and the number of E. coli on the SAM was counted under an optical microscope. Subsequently, the electrode was immersed in Milli-Q water, the released E. coli was washed out with Milli-Q water every 5 min, and the number of residual cells on the SAM was counted. The same experiment was conducted using a 100 mM aqueous methanol solution.

Modification of Zn/Pt Micromotors with SAM and Transport of E. coli. The SAM was formed on the Zn/Pt micromotors as explained earlier. After centrifugation for 10 s at 7,740 g, the precipitate was re-suspended in the same volume of ethanol, and the above steps were repeated twice. The motors were then immersed in an E. coli suspension (OD600 = 1.0) containing 1.0 mM ZnCl2 for 15 min and were centrifuged at 7,740 g for 10 s. The precipitate of the motors was then immediately dispersed in a 100 mM aqueous methanol solution of the same volume, and the solution was injected into the observation chamber.

RESULTS AND DISCUSSION Characterization of Zn/Pt Micromotors. Zn/Pt and Au/Pt structures were fabricated by sputtering and vacuum vapor deposition techniques, as described by Wheat et al. (Figure 1).24 Sputter-deposition of platinum resulted in ~70% surface coverage of the beads. By repeating the sputtering three times, complete coverage of the bead surface with platinum was achieved, based


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on SEM imaging. Exposed polystyrene surfaces are blurred in the images as a result of charging; however, such areas were rarely found. On the other hand, high-vacuum vapor deposition of gold and zinc features a strong shadowing effect43 due to the long mean free path of metal clusters coming from the evaporation source. Thus, the metals were deposited only on the hemisphere facing the evaporation source. Figures 1E–G show a typical Zn/Pt micromotor SEM image, the zinc layer elemental mapping, and the corresponding EDX spectra, respectively. EDX results showed that the zinc layer was formed only on the hemisphere, with the Zn-Pt boundary clearly observed.

Behavior of Zn/Pt Micromotors. The behavior of Zn/Pt micromotors was examined in aqueous methanol solutions of different concentrations. Figure 2A shows the trajectories observed in a 0.3 wt.% aqueous methanol solution. The spontaneous movement of Zn/Pt micromotors could clearly be distinguished from Brownian motion (Figure 2A (left) and Video S1). Similar movement was observed with p-benzoquinone as a fuel. For comparison, the trajectories of Au/Pt beads are also shown in Figure 2A (right). In this case, the beads hardly moved due to the lack of appropriate agents for methanol reduction. The average velocities of Zn/Pt micromotors in 100 mM aqueous methanol and 100 mM aqueous p-benzoquinone solutions were 0.13 ± 0.03 µm/s (n = 50) and 0.15 ± 0.02 µm/s (n = 50), respectively. The force Fp acting on the motor can be estimated using the Stokes equation, Fp = 6πηav, where η is the dynamic viscosity of the solution, a is the radius of the motor, and v is the average velocity of the motors. From this equation, the force acting on a Zn/Pt micromotor in a 100 mM aqueous methanol solution is approximately 4 fN.


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The dependence of Zn/Pt micromotor velocity on fuel concentration is shown in Figure 2B,C. The velocity increased with increasing methanol or p-benzoquinone concentration. When a dissolved oxidant (fuel) is reduced on platinum, the electrons should flow from the zinc part to the platinum part. To confirm this, the short-circuit current flowing between the macroscopic planar zinc and platinum electrodes in aqueous fuel solutions was measured (Figure 2B,C). The current increased with increasing methanol or p-benzoquinone concentrations, in agreement with the trend observed for motor velocity. The motion of Zn/Pt micromotors and Au/Pt beads can be expressed in terms of the mean square displacement (MSD), defined as MSD(t) = < (r(t0 + t) − r(t0))2 >

(4)

where r(t) is the position of the motor or bead at time t, and t0 is the initial time. Figure 3 shows MSD time courses for the Zn/Pt micromotors and Au/Pt beads (Figure 1D). For Brownian and spontaneous directional motion, MSD changes are proportional to t and t2, respectively.44,45 Therefore, the origin of this behavior can be inferred from the double logarithm plots of the above relation. The plot slopes for Zn/Pt micromotors and Au/Pt beads are 1.5 (~2) and 0.6 (~1), respectively (Figure S1). This suggests that spontaneous directional motion is dominant in the case of Zn/Pt micromotors, while the motion of Au/Pt beads is Brownian (Figure 3). A slope of less than 2 in the case of the Zn/Pt micromotors suggests that fluctuation of the motion direction cannot be neglected, as in the case of Brownian motion. The motor running time is a concern for practical use, since zinc is consumed. Under our experimental conditions, the running time was approximately 1 h, with the motor movement being undistinguishable from Brownian motion after this period. In contrast, the running time estimated from the current flow within the motor and the amount of zinc is approximately 180 h.


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Possible reasons of this large discrepancy are inefficient electric field generation around the motor and the formation of zinc oxide that suppresses the dissolution of zinc.

Remote Control of Micromotor Movement. To control the direction of movement, Zn/Ni/Pt micromotors (Figure 1C) were used. A magnet was placed at a distance of 1 cm from one side of the observation chamber for 30 s to make the zinc side of the motors face it. When the magnet was removed, the self-propulsive motion of the motors was observed for 30 s (Figure 4A). The same steps were repeated for the other three sides in a clockwise manner. The motor trajectories in aqueous fuel solutions are shown in Figure 4B and Video S2, with the dashed and solid lines showing trajectories in presence and absence of the magnetic field, respectively. The motors moved in the direction of the magnet even after it was removed. It should be noted that the inertial motion of the motor after removing the magnet is negligible for such a low Reynolds number due to low inertia and high viscous forces. Under our experimental conditions, the relaxation time τ was approximately one microsecond, corresponding to an inertial motion displacement of 1 nm (Supporting Information). This means that the motor requires a continuous and efficient propulsive force to overcome the frictional constraints of viscosity and Brownian motion. Therefore, these results support the hypothesis that the Zn/Pt micromotors self-propel in the direction of the zinc end, i.e., the Zn end is the "head" in solutions of fuels.

Transport and Release of E. coli by Zn/Pt/SAM Micromotors. Autonomous capture, transport, and release of target cells can be realized by adsorbing them onto the alkanethiolate SAM on the platinum surface of the Zn/Pt/SAM micromotor by hydrophobic interactions and subsequently releasing cells by the reductive desorption of SAM thiolates.46–48 The autonomous


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release of E. coli can be explained by a shift of the mixed potential. Figure 5A shows the anodic polarization curve for the oxidation of zinc and the cathodic polarization curve for the reductive desorption of alkanethiolate, Pt–S–R + H+ + e− → Pt + HS–R

(5)

where R is an alkyl chain. The mixed potential is determined as a potential at which the sum of anodic and cathodic currents originating from all oxidation and reduction reactions is zero. When the Zn/Pt/SAM composite electrode fabricated using 1-decanethiol is immersed in Milli-Q water, the mixed potential settles at approximately −0.9 V (vs. Ag/AgCl). SAM thiolates are reductively desorbed at this potential; however, the desorption is inhibited in the presence of Zn2+ ions due to the reduction of the Zn2+ ions and the accompanying positive shift of the mixed potential (Figure 5B). In other words, Zn2+ ions can stop SAM desorption. Figure 5C also shows the case where methanol is used as a fuel. Here, the desorption of thiolates and reduction of methanol to move the Zn/Pt micromotors proceed simultaneously. This indicates that capture, transport, and release of cells can be controlled by appropriately changing the solution components and their concentrations. The capture and release of E. coli cells was preliminarily examined using a macroscopic planar composite electrode consisting of zinc and platinum. The platinum side was modified with the hydrophobic 1-decanethiol to create a SAM that nonspecifically adsorbed E. coli by virtue of hydrophobic interactions49 in an aqueous solution containing Zn2+ ions. Here, 2.3 cells per 39 µm2 SAM area were initially captured on average, translating into two cells per motor (SAM area: 39 µm2). When the composite electrode with the adsorbed E. coli was immersed in Milli-Q water and a Zn2+-free 100 mM aqueous methanol solution, the adsorbed cells were expected to be released from the Zn/Pt/SAM electrode together with the SAM, as depicted in Figure 6A. The


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desorption of E. coli was actually observed in 100 mM aqueous methanol solution (Figure 6B). The decrease in the number of E. coli cells was also confirmed by counting the residual cells on the surface in Milli-Q water and 100 mM aqueous methanol solution every 5 min (Figure 6C). As a control, the release of E. coli was also investigated in 1.0 mM aqueous ZnCl2 solution with and without methanol (Figures S2 and 6C, respectively). However, no release of E. coli was observed, indicating that methanol had no adverse influence on nonspecific adsorption. Zn2+ ions shifted the potential in the positive direction and decreased the apparent rate of zinc oxidation and SAM reductive desorption for the Zn/Pt/SAM micromotors, as shown for the Zn/Pt/SAM electrode. E. coli was captured by the Zn/Pt/SAM micromotors, which were then washed to remove Zn2+ ions and mixed with a Zn2+-free E. coli suspension containing 100 mM methanol as fuel. The images in Figure 7 and Video S3 clearly show that Zn/Pt/SAM micromotors moved in a straight line by approximately 100 µm together with its E. coli cargo after the removal of Zn2+ ions (Figure 7A,B), releasing the cargo after 12 ± 1 min (n = 5) (Figure 7C), as expected from the results obtained for the macroscopic composite electrode (Figure 6C). The above findings indicate that the reductive desorption of thiolates can be coupled with the reduction of methanol to move the motors. To confirm the viability of E. coli in aqueous methanol solution, its growth in the culture medium was investigated by measuring OD600 after immersion in 100 mM aqueous methanol solution for 1 h. Subsequently, these data were compared with those obtained for E. coli grown in the culture medium only. Additionally, the growth of E. coli was also investigated in the culture medium after immersion in 1.0 mM aqueous ZnCl2 solution for 1 h. E. coli immersed in aqueous methanol or Zn2+ solutions grew normally, although the growth rate was slightly decreased (Figure S3).


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When Zn/Pt micromotors are stored in an aqueous solution containing Zn2+ ions, zinc may be deposited on platinum at equilibrium or mixed potential, possibly affecting the velocity of the motors when they are placed in a Zn2+-free aqueous fuel solution. To check this suggestion, macroscopic short-circuited zinc and platinum electrodes separated by approximately 1 mm were immersed in 1.0 mM aqueous ZnCl2 solution. After 1 h, several off-white deposits (< 1 mm) were observed within a 1 cm2 area of the platinum part. Therefore, the re-deposition of zinc is anticipated to similarly occur on the motors. However, this deposition was very slow and exhibited no influence on velocity after the motors were moved from the aqueous Zn2+ solution to an aqueous fuel one. The short-circuit current between the zinc and platinum electrodes in Zn2+free 100 mM aqueous methanol solution recorded after immersing the short-circuited electrodes in 1.0 mM ZnCl2 for 1 h also showed negligible differences compared to the values in Figure 2B. Although there is still room to improve the velocity of the motors and the timing of cell release, this is the first case of using electrochemical SAM desorption for the autonomous transport and release of cargo by self-propelled micromotors. This function can be introduced for other micro/nanomotors by adding the zinc part to the motor structure. Furthermore, the timing of cell release could be adjusted by changing the related redox species (fuel, fuel concentration, and alkanethiolates) on the motor.50 As an example, the release of E. coli from a planar platinum electrode modified with a different alkanethiol, 1-hexanethiol, was also examined in 100 mM aqueous solution. As shown in Figure S4, desorption of E. coli from this electrode was much more rapid than that from the SAM formed using 1-decanethiol (Figure 6C). For practical applications, the capture of E. coli should be made more specific by using biomolecules, such as antibodies and aptamers. Moreover, nonspecific adsorption of molecules such as proteins and polysaccharides in real samples could be avoided by blocking the surface with reagents such as bovine serum albumin (BSA). By combining these techniques with the


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autonomous release function of the motor, more precisely controlled cell processing, including animal cells, could be realized.

CONCLUSIONS The proposed Zn/Pt/SAM micromotors capture, transport, and release bacterial cells spontaneously. The highly negative redox potential of zinc enables the use of a wide variety of organic redox compounds as fuels, such as methanol and p-benzoquinone. The hydrophobic SAM formed on the platinum part of the motor enables the adsorption of E. coli cells via hydrophobic interactions. This SAM can be desorbed to release the cells by changing the mixed potential of Zn/Pt. The above desorption can be stopped by shifting the mixed potential in the positive direction, e.g., by storing the Zn/Pt/SAM micromotors in aqueous Zn2+ solutions of appropriate concentrations. Although issues such as low velocity and the need to use concentrated fuels still remain, the technique used in our motors may be applicable to other micro/nanomotors for future applications including in vitro cell separation and manipulation or drug delivery in biological environments.

ASSOCIATED CONTENT Supporting Information. Video files showing the movement of Zn/Pt micromotors under the conditions of Figure 2A (left), the movement of a Zn/Ni/Pt micromotor in absence and presence of a magnetic field (using the same motor as in Figure 4B), and the transport of E. coli (same motor as in Figure 7A,B). Supplementary explanations for numerical calculations and figures (PDF). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.xxxxxxx.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions Y.Y., M.Y., and H.S. conceived and designed the experiments. Y.Y., K.O., and M.Y. performed the experiments. Y.Y., K.O., M.Y., and H.S. analyzed the data. Y.Y., M.Y., and H.S. wrote the paper. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT We thank Dr. Y. Horiike (University of Tsukuba), Prof. E. T. Carlen (University of Tsukuba), and Dr. M. Aoyagi (National Institute of Advanced Industrial Science and Technology (AIST)) for providing critical advice and discussion. This work was supported by JSPS KAKENHI Grant Numbers 23656501, 26-2405. A part of this work was conducted at the AIST Nano-Processing Facility, supported by “Nanotechnology Platform Program” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. ABBREVIATIONS SAM, self-assembled monolayer; E. coli, Escherichia coli; LB, Luria-Bertani; SHE, standard hydrogen electrode; SEM, scanning electron microscopy; EDX, energy-dispersive X-ray


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diffraction; NA, numerical aperture; CMOS, complementary metal-oxide semiconductor; PBS, phosphate buffered saline; MSD, mean square displacement; BSA, bovine serum albumin.


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REFERENCES (1) (2) (3)

(4) (5)

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Lu, X.; Huang, W. H.; Wang, Z. L.; Cheng, H. K. Recent Developments in Single-Cell Analysis. Anal. Chim. Acta 2004, 510, 127-138. Todd, R.; Margolin, D. H. Challenges of Single-Cell Diagnostics: Analysis of Gene Expression. Trends Mol. Med. 2002, 8, 254-257. Lutolf, M. P.; Hubbell, J. A. Synthetic Biomaterials as Instructive Extracellular Microenvironments for Morphogenesis in Tissue Engineering. Nat. Biotechnol. 2005, 23, 47-55. El-Badry, H. M. Micromanipulators. In Micromanipulators and Micromanipulation, 1 ed.; Springer-Verlag Wien: New York, 2012; pp 22-71. Wakayama, T.; Perry, A. C. F.; Zuccotti, M.; Johnson, K. R.; Yanagimachi, R. Full-Term Development of Mice from Enucleated Oocytes Injected with Cumulus Cell Nuclei. Nature 1998, 394, 369-374. Chiou, P. Y.; Ohta, A. T.; Wu, M. C. Massively Parallel Manipulation of Single Cells and Microparticles Using Optical Images. Nature 2005, 436, 370-372. Ashkin, A.; Dziedzic, J. M. Optical Trapping and Manipulation of Viruses and Bacteria. Science 1987, 235, 1517-1520. Voldman, J. Electrical Forces for Microscale Cell Manipulation. Annu. Rev. Biomed. Eng. 2006, 8, 425-454. Yi, C. Q.; Li, C. W.; Ji, S. L.; Yang, M. S. Microfluidics Technology for Manipulation and Analysis of Biological Cells. Anal. Chim. Acta 2006, 560, 1-23. Nilsson, J.; Evander, M.; Hammarstrom, B.; Laurell, T. Review of Cell and Particle Trapping in Microfluidic Systems. Anal. Chim. Acta 2009, 649, 141-157. Wang, J. Can Man-Made Nanomachines Compete with Nature Biomotors? ACS Nano 2009, 3, 4-9. Wu, Z.; Lin, X.; Si, T.; He, Q. Recent Progress on Bioinspired Self-Propelled Micro/Nanomotors via Controlled Molecular Self-Assembly. Small 2016, 12, 3080-3093. Paxton, W. F.; Kistler, K. C.; Olmeda, C. C.; Sen, A.; St Angelo, S. K.; Cao, Y. Y.; Mallouk, T. E.; Lammert, P. E.; Crespi, V. H. Catalytic Nanomotors: Autonomous Movement of Striped Nanorods. J. Am. Chem. Soc. 2004, 126, 13424-13431. Kline, T. R.; Paxton, W. F.; Mallouk, T. E.; Sen, A. Catalytic Nanomotors: RemoteControlled Autonomous Movement of Striped Metallic Nanorods. Angew. Chem., Int. Ed. 2005, 44, 744-746. Fournier-Bidoz, S.; Arsenault, A. C.; Manners, I.; Ozin, G. A. Synthetic Self-Propelled Nanorotors. Chem. Commun. 2005, 441-443. He, Y. P.; Wu, J. S.; Zhao, Y. P. Designing Catalytic Nanomotors by Dynamic Shadowing Growth. Nano Lett. 2007, 7, 1369-1375. Manesh, K. M.; Cardona, M.; Yuan, R.; Clark, M.; Kagan, D.; Balasubramanian, S.; Wang, J. Template-Assisted Fabrication of Salt-Independent Catalytic Tubular Microengines. ACS Nano 2010, 4, 1799-1804. Mei, Y. F.; Solovev, A. A.; Sanchez, S.; Schmidt, O. G. Rolled-Up Nanotech on Polymers: from Basic Perception to Self-Propelled Catalytic Microengines. Chem. Soc. Rev. 2011, 40, 2109-2119. Orozco, J.; Garcia-Gradilla, V.; D'Agostino, M.; Gao, W.; Cortes, A.; Wang, J. Artificial Enzyme-Powered Microfish for Water-Quality Testing. ACS Nano 2013, 7, 818-824.


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Orozco, J.; Cortes, A.; Cheng, G.; Sattayasamitsathit, S.; Gao, W.; Feng, X.; Shen, Y.; Wang, J. Molecularly Imprinted Polymer-Based Catalytic Micromotors for Selective Protein Transport. J. Am. Chem. Soc. 2013, 135, 5336-5339. Sanchez, S.; Solovev, A. A.; Schulze, S.; Schmidt, O. G. Controlled Manipulation of Multiple Cells Using Catalytic Microbots. Chem. Commun. 2011, 47, 698-700. Wu, Z.; Wu, Y.; He, W.; Lin, X.; Sun, J.; He, Q. Self-Propelled Polymer-Based Multilayer Nanorockets for Transportation and Drug Release. Angew. Chem., Int. Ed. 2013, 52, 7000-7003. Gao, W.; Pei, A.; Feng, X.; Hennessy, C.; Wang, J. Organized Self-Assembly of Janus Micromotors with Hydrophobic Hemispheres. J. Am. Chem. Soc. 2013, 135, 998-1001. Wheat, P. M.; Marine, N. A.; Moran, J. L.; Posner, J. D. Rapid Fabrication of Bimetallic Spherical Motors. Langmuir 2010, 26, 13052-13055. Gao, W.; Pei, A.; Dong, R.; Wang, J. Catalytic Iridium-Based Janus Micromotors Powered by Ultralow Levels of Chemical Fuels. J. Am. Chem. Soc. 2014, 136, 22762279. Wu, Y.; Wu, Z.; Lin, X.; He, Q.; Li, J. Autonomous Movement of Controllable Assembled Janus Capsule Motors. ACS Nano 2012, 6, 10910-10916. Liu, M.; Liu, L.; Gao, W.; Su, M.; Ge, Y.; Shi, L.; Zhang, H.; Dong, B.; Li, C. Y. Nanoparticle Mediated Micromotor Motion. Nanoscale 2015, 7, 4949-4955. Paxton, W. F.; Sundararajan, S.; Mallouk, T. E.; Sen, A. Chemical Locomotion. Angew. Chem., Int. Ed. 2006, 45, 5420-5429. Gao, W.; Sattayasamitsathit, S.; Wang, J. Catalytically Propelled Micro-/Nanomotors: How Fast Can They Move? Chem. Rec. 2012, 12, 224-231. Wang, W.; Chiang, T.-Y.; Velegol, D.; Mallouk, T. E. Understanding the Efficiency of Autonomous Nano- and Microscale Motors. J. Am. Chem. Soc. 2013, 135, 10557-10565. Liu, R.; Sen, A. Autonomous Nanomotor Based on Copper-Platinum Segmented Nanobattery. J. Am. Chem. Soc. 2011, 133, 20064-20067. Wang, Y.; Hernandez, R. M.; Bartlett, D. J.; Bingham, J. M.; Kline, T. R.; Sen, A.; Mallouk, T. E. Bipolar Electrochemical Mechanism for the Propulsion of Catalytic Nanomotors in Hydrogen Peroxide Solutions. Langmuir 2006, 22, 10451-10456. Mano, N.; Heller, A. Bioelectrochemical Propulsion. J. Am. Chem. Soc. 2005, 127, 11574-11575. Gao, W.; Pei, A.; Wang, J. Water-Driven Micromotors. ACS Nano 2012, 6, 8432-8438. Gao, W.; Sattayasamitsathit, S.; Orozco, J.; Wang, J. Highly Efficient Catalytic Microengines: Template Electrosynthesis of Polyaniline/Platinum Microtubes. J. Am. Chem. Soc. 2011, 133, 11862-11864. Campuzano, S.; Orozco, J.; Kagan, D.; Guix, M.; Gao, W.; Sattayasamitsathit, S.; Claussen, J. C.; Merkoci, A.; Wang, J. Bacterial Isolation by Lectin-Modified Microengines. Nano Lett. 2012, 12, 396-401. Orozco, J.; Campuzano, S.; Kagan, D.; Zhou, M.; Gao, W.; Wang, J. Dynamic Isolation and Unloading of Target Proteins by Aptamer-Modified Microtransporters. Anal. Chem. 2011, 83, 7962-7969. Balasubramanian, S.; Kagan, D.; Hu, C.-M. J.; Campuzano, S.; Lobo-Castanon, M. J.; Lim, N.; Kang, D. Y.; Zimmerman, M.; Zhang, L.; Wang, J. Micromachine-Enabled Capture and Isolation of Cancer Cells in Complex Media. Angew. Chem., Int. Ed. 2011, 50, 4161-4164.


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Dong, B.; Zhou, T.; Zhang, H.; Li, C. Y. Directed Self-Assembly of Nanoparticles for Nanomotors. ACS Nano 2013, 7, 5192-5198. Liu, M.; Liu, L.; Gao, W.; Su, M.; Ge, Y.; Shi, L.; Zhang, H.; Dong, B.; Li, C. Y. A Micromotor Based on Polymer Single Crystals and Nanoparticles: toward Functional Versatility. Nanoscale 2014, 6, 8601-8605. Paxton, W. F.; Baker, P. T.; Kline, T. R.; Wang, Y.; Mallouk, T. E.; Sen, A. Catalytically Induced Electrokinetics for Motors and Micropumps. J. Am. Chem. Soc. 2006, 128, 14881-14888. Paxton, W. F.; Kistler, K. C.; Olmeda, C. C.; Sen, A.; St Angelo, S. K.; Cao, Y. Y.; Mallouk, T. E.; Lammert, P. E.; Crespi, V. H. Catalytic Nanomotors: Autonomous Movement of Striped Nanorods. J. Am. Chem. Soc. 2004, 126, 13424-13431. Stowell, W.; Foster, J.; Berner, W.; Wan, C.; Chambers, D.; Hanes, H. Throwing Power and Shadowing Effect in Planar Magnetron Sputtering Process. J. Vac. Sci. Technol., A 1985, 3, 572-575. Einstein, A. The Motion of Elements Suspended in Static Liquids as Claimed in the Molecular Kinetic Theory of Heat. Ann. Phys. 1905, 17, 549-560. Monnier, N.; Guo, S. M.; Mori, M.; He, J.; Lenart, P.; Bathe, M. Bayesian Approach to MSD-Based Analysis of Particle Motion in Live Cells. Biophys. J. 2012, 103, 616-626. Jiang, X. Y.; Ferrigno, R.; Mrksich, M.; Whitesides, G. M. Electrochemical desorption of self-assembled monolayers noninvasively releases patterned cells from geometrical confinements. J. Am. Chem. Soc. 2003, 125, 2366-2367. Inaba, R.; Khademhosseini, A.; Suzuki, H.; Fukuda, J. Electrochemical desorption of selfassembled monolayers for engineering cellular tissues. Biomaterials 2009, 30, 3573-3579. Wildt, B.; Wirtz, D.; Searson, P. C. Programmed subcellular release for studying the dynamics of cell detachment. Nat. Methods 2009, 6, 211-213. Rowan, B.; Wheeler, M. A.; Crooks, R. M. Patterning bacteria within hyperbranched polymer film templates. Langmuir 2002, 18, 9914-9917. Williams, J. A.; Gorman, C. B. Alkanethiol Reductive Desorption from Self-Assembled Monolayers on Gold, Platinum, and Palladium Substrates. J. Phys. Chem. C 2007, 111, 12804-12810.


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FIGURE CAPTIONS Figure 1. Fabrication of micromotors. (A) Fabrication process of the Zn/Pt micromotors. Crosssection of the Zn/Pt micromotor (B), Zn/Ni/Pt micromotor (C), and Au/Pt bead (D). (E) SEM image of the Zn/Pt micromotor. (F) Elemental mapping of zinc on the Zn/Pt micromotor shown in (E). (G) EDX spectra of the areas indicated by arrows in (E). Figure 2. Movement of Zn/Pt micromotors. (A) Trajectories of Zn/Pt micromotors (left) and Au/Pt beads (right) recorded for 5 and 10 min, respectively, in a 100 mM aqueous methanol solution. Red arrows indicate the direction of movement. (B, C) Relation between the velocity of Zn/Pt micromotors (solid red circles) and the steady-state short-circuit current densities measured using macroscopic zinc and platinum electrodes (4.4 cm2) (solid black squares) in aqueous methanol (B) and p-benzoquinone (C) solutions of different concentrations. The current was measured 180 s after short-circuiting the electrodes. Error bars represent the standard deviations of velocities and currents (n = 50 and 10, respectively). Figure 3. Changes in the MSD of Zn/Pt micromotors (solid black squares) and Au/Pt beads (solid red circles) in a 100 mM aqueous methanol solution, with the corresponding averages and standard deviations (n = 50). Figure 4. Magnetic control of the Zn/Ni/Pt micromotor movement direction. (A) Experimental setup. (B) Recorded trajectory of a Zn/Ni/Pt micromotor. The dashed and solid lines represent movement in presence and absence of a magnetic field, respectively. Red arrows indicate the direction of movement. Sections I–IV represent 1-min motor movement. Figure 5. Changes in the mixed potential of a Zn/Pt/SAM micromotor. The mixed potentials are indicated by red dots. (A) Polarization curves for the dissolution of zinc and the reductive SAM


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desorption. (B) Polarization curves after addition of Zn2+ to (A). The mixed potential shifts to a positive direction, terminating SAM desorption within the experimental timescale. (C) Polarization curves in the presence of methanol and in the absence of Zn2+. Currents for zinc dissolution (red), SAM desorption (green), and methanol reduction (orange) add up to a net zero current. Figure 6. Autonomous release of E. coli from a Zn/Pt/SAM composite electrode. (A) Left: adhesion of E. coli to the SAM. Right: reductive desorption of alkanethiolates taking place simultaneously with the dissolution of zinc and releasing E. coli cells from the electrode. (B) Time-lapse microscopy images during the release of E. coli cells. The images were recorded after changing the liquid phase of the E. coli suspension from 1.0 mM aqueous ZnCl2 to a Zn2+-free 100 mM aqueous methanol solution. (C) Ratio of E. coli cells remaining on the Zn/Pt/SAM composite planar electrode. The number of residual E. coli cells on the SAM was counted after changing the phase to 1.0 mM aqueous ZnCl2 solution (solid red circles), 100 mM aqueous methanol solution (solid black squares), and Milli-Q water (open blue triangles). Figure 7. Transport and release of E. coli. (A) Trajectory of the Zn/Pt/SAM micromotor with E. coli captured by the SAM moving in a 100 mM aqueous methanol solution. (B) Time-lapse sequential images of (A) taken at 50 s intervals. (C) Release of E. coli from the motor observed 720 s after removing Zn2+ ions.


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Table of Contents Graphic


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Figure 1. Fabrication of micromotors. (A) Fabrication process of the Zn/Pt micromotors. Cross-section of the Zn/Pt micromotor (B), Zn/Ni/Pt micromotor (C), and Au/Pt bead (D). (E) SEM image of the Zn/Pt micromotor. (F) Elemental mapping of zinc on the Zn/Pt micromotor shown in (E). (G) EDX spectra of the areas indicated by arrows in (E). 75x146mm (150 x 150 DPI)


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Figure 2. Movement of Zn/Pt micromotors. (A) Trajectories of Zn/Pt micromotors (left) and Au/Pt beads (right) recorded for 5 and 10 min, respectively, in a 100 mM methanol solution. Red arrows indicate the direction of movement. (B, C) Relation between the velocity of Zn/Pt micromotors (solid red circles) and the steady-state short-circuit current densities measured using macroscopic zinc and platinum electrodes (4.4 cm2) (solid black squares) in methanol (B) and p-benzoquinone (C) solutions of different concentrations. The current was measured 180 s after short-circuiting the electrodes. Error bars represent the standard deviations of velocities and currents (n = 50 and 10, respectively). 75x145mm (150 x 150 DPI)


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Figure 3. Changes in the MSD of Zn/Pt micromotors (solid black squares) and Au/Pt beads (solid red circles) in a 100 mM methanol solution, with the corresponding averages and standard deviations (n = 50). 75x71mm (150 x 150 DPI)


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Figure 4. Magnetic control of the Zn/Ni/Pt micromotor movement direction. (A) Experimental setup. (B) Recorded trajectory of a Zn/Ni/Pt micromotor. The dashed and solid lines represent movement in presence and absence of a magnetic field, respectively. Red arrows indicate the direction of movement. Sections I–IV represent 1-min motor movement. 75x113mm (150 x 150 DPI)


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Figure 5. Changes in the mixed potential of a Zn/Pt/SAM micromotor. The mixed potentials are indicated by red dots. (A) Polarization curves for the dissolution of zinc and the reductive SAM desorption. (B) Polarization curves after addition of Zn2+ to (A). The mixed potential shifts to a positive direction, terminating SAM desorption within the experimental timescale. (C) Polarization curves in the presence of methanol and in the absence of Zn2+. Currents for zinc dissolution (red), SAM desorption (green), and methanol reduction (orange) add up to a net zero current. 150x67mm (150 x 150 DPI)


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Figure 6. Autonomous release of E. coli from a Zn/Pt/SAM composite electrode. (A) Left: adhesion of E. coli to the SAM. Right: reductive desorption of alkanethiolates taking place simultaneously with the dissolution of zinc and releasing E. coli cells from the electrode. (B) Time-lapse microscopy images during the release of E. coli cells. The images were recorded after changing the liquid phase of the E. coli suspension from 1.0 mM aqueous ZnCl2 to a Zn2+-free 100 mM aqueous methanol solution. (C) Ratio of E. coli cells remaining on the Zn/Pt/SAM composite planar electrode. The number of residual E. coli cells on the SAM was counted after changing the phase to 1.0 mM aqueous ZnCl2 solution (solid red circles), 100 mM aqueous methanol solution (solid black squares), and Milli-Q water (open blue triangles). 75x113mm (150 x 150 DPI)


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Figure 7. Transport and release of E. coli. (A) Trajectory of the Zn/Pt/SAM micromotor with E. coli captured by the SAM moving in a 100 mM methanol solution. (B) Time-lapse sequential images of (A) taken at 50 s intervals. (C) Release of E. coli from the motor observed 720 s after removing Zn2+ ions. 75x112mm (150 x 150 DPI)


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Programmed Transport and Release of Cells by Self-Propelled

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