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Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus
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Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus Takeshi Hatsuzawa a,∗ , Daishin Ito b , Takasi Nisisako a , Yasuko Yanagida a a b
Laboratory for Future Interdisciplinary Research of Science and Technology(FIRST) Tokyo Institute of Technology, Japan Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Japan
a r t i c l e
i n f o
Article history: Received 7 November 2016 Accepted 14 November 2016 Available online xxx Key words: Micro-rotary ratchets Plankton driven disks Phototaxis Mmigratory phytoplankton
a b s t r a c t This study proposes micro-rotary ratchets driven by a migratory phytoplankton–Volvox, exhibiting a positive phototaxis. Two types of micro-discs, i.e., ratchet- and starfish-like ratchets are fabricated using conventional photolithography. The ratchet is floated in the center of a Petri dish filled with Volvox suspension under an optical microscope with halogen lamp illumination and is covered by a mask with a small hole so that the microorganisms are concentrated around the micro-ratchets by the phototaxis. Rotations of the ratchets with the same diameter of 0.567 mm were observed through a biological microscope; a rotation speed of 0.86 rpm for the micro-ratchet and 2.01 rpm for the starfish ratchet were obtained for a Volvox density of 1000–3000/mL under an illumination intensity of 0.18 W/cm2 . As the driving mechanism of the ratchet is based on the microorganisms adhesion to the ratchets surface rather than collision impacts, a gelatin coating on the ratchet was used to enhance the adhered number of Volvox. Although the drag force was increased owing to the larger ratchet diameter, a rotation speed of 0.16 rpm was observed. A particle tracking velocimetry measurement using polystyrene beads was performed to study the fluid flow around the micro-ratchet. A vortex generation by the micro-ratchets was confirmed; this effect may work as a micro-mechanical power booster for microorganisms. This drive system may open the possibility of a solar-power-driven and sustainable micro-mechanism using phytoplankton. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Various driving methods regarding micromechanisms have been studied using a microorganism as a kinetic driving source because they do not depend on electrical or chemical sources just like regular machines. Several mechanisms such as bacterial motors [1], micro-objects driven by Escherichia coli [2], Bacillus subtilis [3], and Serratia marcescens [4], micro-bead transportation by Escherichia coli [5] and Chlamydomonas [6], reciprocating linear actuators [7], and micro-ratchet gears [8] driven by Artemia among others have been reported to date. In these mechanisms, the moving vector of each microorganism is random and should be aligned along an uniform direction. To achieve this, the asymmetric shape of micro-objects such as lead-in entrance [1], ratchet gears [2]. Also, a mass- microorganism drive is important for the application of the rotary actuator power source, although a linear beads
∗ Corresponding author at: FIRST, Tokyo Institute of Technology, 4259−R2−6, Nagatsuta−cho, midori−ku, Yokohama 226−8503, Japan. E-mail address: [email protected] (T. Hatsuzawa).
driven system has been realized for small numbers of migratory phytoplankton(6) so far. To gather microorganisms around microratchets, stimulation to microorganisms [6] are typical solutions. Among various types of stimulation, phototaxis is one of the most convenient because of the simplicity of control outside of the breeding environment. We have reported two types of micro-actuators driven by oceanic zooplankton [7,8]; however, they have some difficulties, such as low growth yield to become adults, short lifetime of larva, and the requirement of salt water for breeding. To solve those difficulties, an alternative approach using migratory phytoplankton with phototaxis was proposed, fabricated and evaluated in order to actuate micro-rotary ratchets floating on the water, as well as a surface treatment for the torque enhancement and an image analysis for the application. Compared to zooplankton, phytoplankton has a long driving possibility because of its photosynthesis and lack of feeding requirement, except for a small amount of fertilizer and solar power. Moreover, existence and reproduction can be maintained sustainably as along as the environmental condition is secured, which may lead to a bio-solar power driven micro mechanical system without parts maintenance.
http://dx.doi.org/10.1016/j.precisioneng.2016.11.010 0141-6359/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010
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Fig. 1. Swimming force of Volvox and principle of micro-ratchet drive by multi-Volvox.
Fig. 2. Design of the micro-ratchets; (a) 8-tooth ratchet gear, (b) starfish-like micro-disk, and (c) principle of roll-in clip action.
In the experiment, two types of micro-rotary ratchets, i.e., ratchet gear and starfish, were fabricated via conventional photolithography on a silicon substrate and Volvox was selected as the driving plankton after testing three species. A gelatin coating on the micro-ratchets was also examined to increase Volvox engagement, resulting in the ratchet rotation for large diameters. Finaly for the application purposes, a particle tracking velocimetry (PTV) measurement using polystyrene beads was performed for investigating fluid movement around the micro-ratchets; the PTV measurement confirmed that the fluid was driven by the micro-rotary ratchets. 2. Driving principle and ratchet design and phytoplankton selection The swimming behavior of Volvox is based on the rising force with rotation by the flagellum movement due to the nature of phototaxis [10], which is shown in Fig. 1. When the Volvox reaches the lower face of the ratchet, a precession is expected by the composition of rising force and revolution, because the top of Volvox is fixed on the back surface, resulting in components of horizontal force. Originally, the direction of precession force is random, however, the asymmetry of the ratchet makes the torque in unique direction according to the teeth orientation illustrated in Fig. 1. One direc-
tional rotation by the ratchet is common in micro-ratchet motors [2,3], and pawl is attributed to the difference of fluid resistance by the ratchet asymmetry. An analysis of rotation drag force around a ratchet is performed in the previous study [8], directional difference of the drag force seems to be very small due to slow rotation speed and small radius. Base on the principle, two types of micro-ratchets designed for phytoplankton actuation are shown in Fig. 2. The first one (a) is based on the 8-teeth ratchet gear configuration used in the previous study for the Artemia actuation [7] to confirm the usefulness of the ratchet shape; however, its diameter is reduced to 1/10 because of the size difference between the two planktons. Three types of migratory phytoplankton, i.e., Chlamydomonas sp., Euglena proxima, and Volvox aureus, with diameters of 10–30 m, 20–50 m, and 200–500 m, respectively, and almost the same swimming velocity of 100 m/s, were considered in a preliminary driving experiment under a microscope. The micro-ratchet can be rotated only in the case of Volvox, while the others have no driving power to rotate the ratchet owing to their small sizes. Thus, the superiority of Volvox as a micro actuator has been confirmed. During the experiments, Volvox tends to roll along the edge of micro-ratchet because its rotation originated from the swim action. Incorporating this movement as a roll-in clip between two carving teeth shown in Fig. 2(c),
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010
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Fig. 3. A ratchet micro-ratchet (a) and a starfish micro-ratchet (b). Ratchets stripped from the substrate by a blade edge (c).
Fig. 4. Experimental setup for micro-rotary ratchet observation. A mask with a pinhole covers a Petri dish for the positive phototaxis of Volvox.
a starfish-like disk (b) was designed. This design is also expected to reduce the fluid resistance via a stream-line-like configuration.
4. Experimental results 4.1. Comparison of the rotation for two micro-ratchets
3. Ratchet fabrication and experimental setups A conventional photolithography process was used to fabricate the micro-ratchets. A PMMA (Poly-methyl methacrylate, Wako Pure Chemical Industries Ltd.) solution was dissolved in toluene (Wako Pure Chemical Industries Ltd.) to a density of 1% w/v; the PMMA solution was spin-coated onto a Si substrate as a sacrificial layer. A negative photoresist SU-8 (Micro Chem Corp,) was then overcoated to a thickness of 50 m. Next, the substrate was exposed by a PET film photomask to simultaneously fabricate a 20 × 20 array of micro-ratchets. After a development and rinse process, microratchets were formed on the Si substrate, as shown in Fig. 3. Finally, the substrate was dipped in acetone (Wako Pure Chemical Industries Ltd.) for the dissolution of the sacrificial layer, and then the ratchets are floated on the water surface after stripping from the substrate by a sharp blade (Fig. 3(c)). A biological microscope (Olympus CKX41), shown in Fig. 4, was used for the micro-ratchet observation. A plastic Petri dish with a diameter of 35 mm was filled with Volvox suspension. The dish was illuminated by the halogen lamp of the microscope, and the micro-rotary disk was floated at the center of the dish. The dish was covered with a black mask with a small hole of diameter of 3 mm in order to gather the Volvox around the micro-ratchet by the positive phototaxis. Illumination wavelength can be selected by RGB filters placed on the mask. Two digital cameras (NIKON COOLPIX P600/P7700) were involved in the experimental setup for high sensitivity imaging and capturing movies.
For each experiment, a 2 mL suspension of Volvox with a density of 1000–3000/mL was filled in the Petri dish under an illumination intensity of 0.18 W/cm2 (∼1200 Lx). Initially, they are swimming randomly; subsequently, the density at the illuminated area increased almost three times from its original in 3 min, demonstrating that the positive phototaxis is working in the setup. Both of the micro-ratchets can be rotated in the experiments for 4 min, but translational motion was also simultaneously observed, as shown in Fig. 5 (upper). As Volvox tends to adhere to the bottom surface of the micro-ratchets, swimming action directly affects the ratchet movement. This process is different from the case of Artemia actuation based on the collision impacts and pushing without adhesion. In the experiments, an average rotation speed of 0.89 rpm was obtained for the ratchet engaged with 7 Volvox, whereas 2.01 rpm was obtained for the starfish ratchet with 9 Volvox (Fig. 5 lower), which were measured by a movie analyzing software. The difference may be attributed to the number of adhered Volvox and fluid resistance, however, the former seems to be dominant because of the small size and rotation speed of ratchets. The results were obtained from an average of cultured Vovlox, though, further experiments are required to clear the rotation speed dependence on the number and size of microorganisms. Thus, micro-ratchets with fairly large diameters in sub-mm can be driven by migratory phytoplankton with phototactic concentration, whereas in the conventional method, microbeads with a
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010
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Fig. 5. Tracking records for the two types of micro-rotary ratchets in 4 min. Upper: locus of disks; lower: accumulated rotation angle dependence on the driving time.
diameter of tens of m can be driven. The results indicate the driving potential for the application of rotary actuators.
4.2. On–off control of the rotation via wavelength change and mechanical impact Rotational speed control is a property for the application of a mechanical actuation source. Because no direct relationship between the illumination intensity and the swimming speed of Volvox was observed in the experiments, the possibility of on–off control of the micro-disk was examined using illumination switching. As Volvox is insensible to red light, a red color filter with a cut-off wavelength of 650 nm was employed for the wavelength switching; the red illumination corresponds to no illumination for Volvox but enables continuous observation using the camera on the microscope. When the filter was slid in on the dish as the illumination blackout, as shown in Fig. 6, free Volvox were dispersed from the filtered area and swam to the bright field by the phototaxis, whereas no apparent change was observed for the ratchet-attached Volvox, because illumination does not affect adhesiveness except phototaxtic concentration. To release the microorganism, a slight mechanical impact to the Petri dish was observed to be most effective. Finally, a combination of red filter insertion and mechanical impact proved to be effective in releasing Volvox and stopping the micro-ratchet rotation, as shown in Fig. 6. Immediately after inser-
tion and the impact, the rotation stops within 0.5s, and a small number of Volvox were swimming around the micro-ratchet. As restarting is realized via spot illumination for 3 min, which takes longer compared to release, though, the possibility of on–off control was thus demonstrated.
4.3. Large ratchet drive via chemical surface modification The experimental results show that the micro-ratchets are driven by the attached Volvox rotation rather than collision impacts against the ratchet side surface. Therefore, a possibility for larger ratchet actuation may be realized by increasing the number of attached Volvox. apart from the on-off control. There are several types of adherence mechanisms between the solid surface and the microorganism, such as ionic, chemical, mechanical, meniscus [9], and swimming force [10]. Among these mechanisms, ionic and chemical are the simplest and least harmful because only the surface of the micro-ratchets is modified by specific chemical materials with affinity for the microorganism. As the outer surface of Volvox is covered with an extracellular matrix (ECM) composed of protein fibers, gelatin coating on the ratchet appears be one of the promising methods, based on both ionic and chemical adhesion. A ratchet with a diameter of 2 mm, which is 3.5 times the size of those in previous experiments, was dip-coated in a 2% gelatin solution (Sigma Aldrich Japan, G1393) stained using a red food coloring.
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010
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Fig. 6. Principle of on–off control of micro-ratchet rotation via red-filter switching.
Fig. 7. Adherence enhancement by the gelatin coating on the ratchets surface in 6 min.
Fig. 8. Large ratchet (diameter: 2 mm) driven by adhesion enhancement. Arrows denote the rotation of the tooth plate.
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Fig. 9. A captured image of a starfish disk with Volvox in the bead dispersed solution (a) and its binarization (b).
Fig. 10. A comparison of the particle velocity dependence on the radius of the Volvox and the starfish ratchet.
After 5 min of dipping without a drying process, the ratchet is set in the experimental setup. As shown in Fig. 7, more than 80 Volvox are adhered on the backside of the ratchet within 6 min. An average rotation speed of 0.17 rpm was obtained from an image analysis of Fig. 8; however, in cases without gelatin coating, no rotation occurs, and only translation is observed. Although the drag force generated by the rotation is proportional to the 3rd power of ratchet radius[8], i.e., 43 times in this case, the power sum of Volvox was sufficient to overcome the drag. Therefore, an appropriate surface coating on the ratchet improves the adherence of Volvox, thereby increasing the amount of microorganism-enhanced rotational torque. 5. Fluid flow analysis via PTV In view of application as a fluid mechanical component, it is important to investigate how the micro-ratchet works on the adjacent fluid flow in unconstrained movement. In order to compare the flow difference between free swimming Volvox and Volvox driven starfish disk, an analysis based on PTV was performed as a direct
measurement of the microorganisms [11]. We mixed microbeads (nominal diameter 6 m, Polybead, Polysciences) with Volvox suspension (2000/mL) at a density of 10−4 % v/v. Next, the suspension observed through the biological microscope with an image capture camera having a resolution of 500 × 500 pixels and a capture time of 10 s. As shown in Fig. 9, the image was digitized using ImageJ − open-source software developed by the NIH and the locus was determined using plugin software TrackMate. Finally, the locus was numerically analyzed using Matlab (Math Works). Fig. 10 is a comparison of the particle velocity dependence on the radius of free Volvox and a starfish disk. In the experiment, the size of the Volvox is chosen to be approximately the same, so that the effect of the starfish configuration can be evaluated, and fluid speed generated by rotation was measured to confirm stirring effect without translational component. An apparent velocity distribution difference on the distance from the rotation center is observed in the figure, i.e., the velocity of free Volvox is dispersed randomly, whereas that of the starfish disk is in inverse proportion to radius. This phenomenon may be attributed to the smooth
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swimming characteristics of Volvox without disturbing peripheral fluid flow. On the other hand, Volvox driven micro-ratchet works as a vortex generator due to drag force generated by the fins. 6. Discussion The driving principle of the micro-ratchets with Volvox is based on the continuous swimming power supplied by the microorganisms, in contrast to our previous expectation associated with the Artemia impact drive mechanism. Although the size of phytoplankton is smaller than that of zooplankton, i.e., less power can be obtained from the former, an appropriate surface modification helps the number of adhered phytoplankton with ECM. A ring-type ratchet with a larger radius and surface modification may contribute better torque generation because of the increased number of adhered microorganisms and longer length of the moment arm. The starfish micro-ratchet was proved to function as a vortex generator for an adjacent fluid; although its rotation speed is low, it can be regarded as a mechanical power booster for microorganisms. Combined with an outer casing, which surrounds the micro-disk, a centrifugal micropump with small discharge is one possible application of this mechanism, as a self-sustainable and solar-power-driven bio-mechanism. 7. Conclusions Two types of micro-ratchets driven by Volvox exhibiting a positive phototaxis were designed and successfully actuated. The ratchets were fabricated using a conventional photolithography process. The starfish micro-ratchet with a diameter of 0.537 mm resulted in a better rotational speed of 2.01 rpm than the 8teeth ratchet, which resulted in a speed of 0.86 rpm. An on–off drive possibility was demonstrated using wavelength switching combined with mechanical impact for disengaging the microorganisms from the rotary ratchet. For improved torque generation and larger ratchet rotation, a gelatin coating was found to successfully enhance the number of adhered microorganisms on the ratchets surface, resulting in rotational speed of 0.15 rpm for a ratchet with
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a diameter of 2 mm. A PTV result shows that the fluid around the disk can be swirled, indicating that the disk functions as a stirrer or mechanical booster driven by microorganisms. A solar-powerdriven and sustainable-microorganism-powered mechanism is expected to be developed after further research on this and other related matters. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.precisioneng. 2016.11.010. References [1] Hiratsuka Y, Miyata M, Tada T, Uyeda TQP. A microrotary motor powered by bacteria. PNAS 2006;39:13618–23. [2] Di Leonardoa R, Angelania L, Dell’Arcipreteb D, Ruoccob G, Iebbac V, Schippac S, et al. Bacterial ratchet motors. PNAS 2010;21:9541–5. [3] Sokolova A, Apodacac MM, Grzybowskic BA, Aransona IS. Swimming bacteria power microscopic gears. PNAS 2010;3:969–74. [4] Wong Denise, Beattie Elizabeth E, Steager Edward B, Kumar Vijay. Effect of surface interactions and geometry on the motion of micro bio robots. Appl Phys Lett 2013;103:153707. [5] Koumakis N, Lepore A, Maggi C, Di Leonardo R. Targeted delivery of colloids by swimming bacteria. Nat Commun 2013;4:2588, http://dx.doi.org/10.1038/ncomms3588. [6] Weibel Douglas B, Garstecki Piotr, Ryan Declan, DiLuzio Willow R, Mayer Michael, Seto Jennifer E, et al. Microoxen: microorganisms to move microscale loads. PNAS 2005;34:11963–7. [7] Hatsuzawaa T, Michishita K, Yanagidaa Y. A reciprocating linear actuator driven by anti-phototaxis of plankton. Sens Actuators A 2013;201:316–20. [8] Hatsuzawa T, Yamazakib A, Nisisakoa T, Yanagida Y. Design and evaluation of Artemia-driven micro-ratchet gears. Sens Actuators A 2015;235:182–6. [9] An Yuehuei H, Friedman Richard J. Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. Biomed Mater Res 1998;43:338–48. [10] Drescher Knut, Leptos Kyriacos C, Tuval Idan, Ishikawa Takuji, Pedley Timothy J, Goldstein Raymond E. Dancing volvox: hydrodynamic bound states of swimming algae. Phys Rev Lett 2009:102, http://dx.doi.org/10.1103/PhysRevLett.102.168101. [11] Drescher Knut, Goldstein Raymond E, Michel Nicolas, Polin Marco, Tuval Idan. Direct measurement of the flow field around swimming microorganisms. Phys Rev Lett 2012:105, http://dx.doi.org/10.1103/PhysRevLett.105.168101.
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010
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Contents lists available at ScienceDirect
Precision Engineering journal homepage: www.elsevier.com/locate/precision
Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus Takeshi Hatsuzawa a,∗ , Daishin Ito b , Takasi Nisisako a , Yasuko Yanagida a a b
Laboratory for Future Interdisciplinary Research of Science and Technology(FIRST) Tokyo Institute of Technology, Japan Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Japan
a r t i c l e
i n f o
Article history: Received 7 November 2016 Accepted 14 November 2016 Available online xxx Key words: Micro-rotary ratchets Plankton driven disks Phototaxis Mmigratory phytoplankton
a b s t r a c t This study proposes micro-rotary ratchets driven by a migratory phytoplankton–Volvox, exhibiting a positive phototaxis. Two types of micro-discs, i.e., ratchet- and starfish-like ratchets are fabricated using conventional photolithography. The ratchet is floated in the center of a Petri dish filled with Volvox suspension under an optical microscope with halogen lamp illumination and is covered by a mask with a small hole so that the microorganisms are concentrated around the micro-ratchets by the phototaxis. Rotations of the ratchets with the same diameter of 0.567 mm were observed through a biological microscope; a rotation speed of 0.86 rpm for the micro-ratchet and 2.01 rpm for the starfish ratchet were obtained for a Volvox density of 1000–3000/mL under an illumination intensity of 0.18 W/cm2 . As the driving mechanism of the ratchet is based on the microorganisms adhesion to the ratchets surface rather than collision impacts, a gelatin coating on the ratchet was used to enhance the adhered number of Volvox. Although the drag force was increased owing to the larger ratchet diameter, a rotation speed of 0.16 rpm was observed. A particle tracking velocimetry measurement using polystyrene beads was performed to study the fluid flow around the micro-ratchet. A vortex generation by the micro-ratchets was confirmed; this effect may work as a micro-mechanical power booster for microorganisms. This drive system may open the possibility of a solar-power-driven and sustainable micro-mechanism using phytoplankton. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Various driving methods regarding micromechanisms have been studied using a microorganism as a kinetic driving source because they do not depend on electrical or chemical sources just like regular machines. Several mechanisms such as bacterial motors [1], micro-objects driven by Escherichia coli [2], Bacillus subtilis [3], and Serratia marcescens [4], micro-bead transportation by Escherichia coli [5] and Chlamydomonas [6], reciprocating linear actuators [7], and micro-ratchet gears [8] driven by Artemia among others have been reported to date. In these mechanisms, the moving vector of each microorganism is random and should be aligned along an uniform direction. To achieve this, the asymmetric shape of micro-objects such as lead-in entrance [1], ratchet gears [2]. Also, a mass- microorganism drive is important for the application of the rotary actuator power source, although a linear beads
∗ Corresponding author at: FIRST, Tokyo Institute of Technology, 4259−R2−6, Nagatsuta−cho, midori−ku, Yokohama 226−8503, Japan. E-mail address: [email protected] (T. Hatsuzawa).
driven system has been realized for small numbers of migratory phytoplankton(6) so far. To gather microorganisms around microratchets, stimulation to microorganisms [6] are typical solutions. Among various types of stimulation, phototaxis is one of the most convenient because of the simplicity of control outside of the breeding environment. We have reported two types of micro-actuators driven by oceanic zooplankton [7,8]; however, they have some difficulties, such as low growth yield to become adults, short lifetime of larva, and the requirement of salt water for breeding. To solve those difficulties, an alternative approach using migratory phytoplankton with phototaxis was proposed, fabricated and evaluated in order to actuate micro-rotary ratchets floating on the water, as well as a surface treatment for the torque enhancement and an image analysis for the application. Compared to zooplankton, phytoplankton has a long driving possibility because of its photosynthesis and lack of feeding requirement, except for a small amount of fertilizer and solar power. Moreover, existence and reproduction can be maintained sustainably as along as the environmental condition is secured, which may lead to a bio-solar power driven micro mechanical system without parts maintenance.
http://dx.doi.org/10.1016/j.precisioneng.2016.11.010 0141-6359/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010
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Fig. 1. Swimming force of Volvox and principle of micro-ratchet drive by multi-Volvox.
Fig. 2. Design of the micro-ratchets; (a) 8-tooth ratchet gear, (b) starfish-like micro-disk, and (c) principle of roll-in clip action.
In the experiment, two types of micro-rotary ratchets, i.e., ratchet gear and starfish, were fabricated via conventional photolithography on a silicon substrate and Volvox was selected as the driving plankton after testing three species. A gelatin coating on the micro-ratchets was also examined to increase Volvox engagement, resulting in the ratchet rotation for large diameters. Finaly for the application purposes, a particle tracking velocimetry (PTV) measurement using polystyrene beads was performed for investigating fluid movement around the micro-ratchets; the PTV measurement confirmed that the fluid was driven by the micro-rotary ratchets. 2. Driving principle and ratchet design and phytoplankton selection The swimming behavior of Volvox is based on the rising force with rotation by the flagellum movement due to the nature of phototaxis [10], which is shown in Fig. 1. When the Volvox reaches the lower face of the ratchet, a precession is expected by the composition of rising force and revolution, because the top of Volvox is fixed on the back surface, resulting in components of horizontal force. Originally, the direction of precession force is random, however, the asymmetry of the ratchet makes the torque in unique direction according to the teeth orientation illustrated in Fig. 1. One direc-
tional rotation by the ratchet is common in micro-ratchet motors [2,3], and pawl is attributed to the difference of fluid resistance by the ratchet asymmetry. An analysis of rotation drag force around a ratchet is performed in the previous study [8], directional difference of the drag force seems to be very small due to slow rotation speed and small radius. Base on the principle, two types of micro-ratchets designed for phytoplankton actuation are shown in Fig. 2. The first one (a) is based on the 8-teeth ratchet gear configuration used in the previous study for the Artemia actuation [7] to confirm the usefulness of the ratchet shape; however, its diameter is reduced to 1/10 because of the size difference between the two planktons. Three types of migratory phytoplankton, i.e., Chlamydomonas sp., Euglena proxima, and Volvox aureus, with diameters of 10–30 m, 20–50 m, and 200–500 m, respectively, and almost the same swimming velocity of 100 m/s, were considered in a preliminary driving experiment under a microscope. The micro-ratchet can be rotated only in the case of Volvox, while the others have no driving power to rotate the ratchet owing to their small sizes. Thus, the superiority of Volvox as a micro actuator has been confirmed. During the experiments, Volvox tends to roll along the edge of micro-ratchet because its rotation originated from the swim action. Incorporating this movement as a roll-in clip between two carving teeth shown in Fig. 2(c),
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010
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Fig. 3. A ratchet micro-ratchet (a) and a starfish micro-ratchet (b). Ratchets stripped from the substrate by a blade edge (c).
Fig. 4. Experimental setup for micro-rotary ratchet observation. A mask with a pinhole covers a Petri dish for the positive phototaxis of Volvox.
a starfish-like disk (b) was designed. This design is also expected to reduce the fluid resistance via a stream-line-like configuration.
4. Experimental results 4.1. Comparison of the rotation for two micro-ratchets
3. Ratchet fabrication and experimental setups A conventional photolithography process was used to fabricate the micro-ratchets. A PMMA (Poly-methyl methacrylate, Wako Pure Chemical Industries Ltd.) solution was dissolved in toluene (Wako Pure Chemical Industries Ltd.) to a density of 1% w/v; the PMMA solution was spin-coated onto a Si substrate as a sacrificial layer. A negative photoresist SU-8 (Micro Chem Corp,) was then overcoated to a thickness of 50 m. Next, the substrate was exposed by a PET film photomask to simultaneously fabricate a 20 × 20 array of micro-ratchets. After a development and rinse process, microratchets were formed on the Si substrate, as shown in Fig. 3. Finally, the substrate was dipped in acetone (Wako Pure Chemical Industries Ltd.) for the dissolution of the sacrificial layer, and then the ratchets are floated on the water surface after stripping from the substrate by a sharp blade (Fig. 3(c)). A biological microscope (Olympus CKX41), shown in Fig. 4, was used for the micro-ratchet observation. A plastic Petri dish with a diameter of 35 mm was filled with Volvox suspension. The dish was illuminated by the halogen lamp of the microscope, and the micro-rotary disk was floated at the center of the dish. The dish was covered with a black mask with a small hole of diameter of 3 mm in order to gather the Volvox around the micro-ratchet by the positive phototaxis. Illumination wavelength can be selected by RGB filters placed on the mask. Two digital cameras (NIKON COOLPIX P600/P7700) were involved in the experimental setup for high sensitivity imaging and capturing movies.
For each experiment, a 2 mL suspension of Volvox with a density of 1000–3000/mL was filled in the Petri dish under an illumination intensity of 0.18 W/cm2 (∼1200 Lx). Initially, they are swimming randomly; subsequently, the density at the illuminated area increased almost three times from its original in 3 min, demonstrating that the positive phototaxis is working in the setup. Both of the micro-ratchets can be rotated in the experiments for 4 min, but translational motion was also simultaneously observed, as shown in Fig. 5 (upper). As Volvox tends to adhere to the bottom surface of the micro-ratchets, swimming action directly affects the ratchet movement. This process is different from the case of Artemia actuation based on the collision impacts and pushing without adhesion. In the experiments, an average rotation speed of 0.89 rpm was obtained for the ratchet engaged with 7 Volvox, whereas 2.01 rpm was obtained for the starfish ratchet with 9 Volvox (Fig. 5 lower), which were measured by a movie analyzing software. The difference may be attributed to the number of adhered Volvox and fluid resistance, however, the former seems to be dominant because of the small size and rotation speed of ratchets. The results were obtained from an average of cultured Vovlox, though, further experiments are required to clear the rotation speed dependence on the number and size of microorganisms. Thus, micro-ratchets with fairly large diameters in sub-mm can be driven by migratory phytoplankton with phototactic concentration, whereas in the conventional method, microbeads with a
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Fig. 5. Tracking records for the two types of micro-rotary ratchets in 4 min. Upper: locus of disks; lower: accumulated rotation angle dependence on the driving time.
diameter of tens of m can be driven. The results indicate the driving potential for the application of rotary actuators.
4.2. On–off control of the rotation via wavelength change and mechanical impact Rotational speed control is a property for the application of a mechanical actuation source. Because no direct relationship between the illumination intensity and the swimming speed of Volvox was observed in the experiments, the possibility of on–off control of the micro-disk was examined using illumination switching. As Volvox is insensible to red light, a red color filter with a cut-off wavelength of 650 nm was employed for the wavelength switching; the red illumination corresponds to no illumination for Volvox but enables continuous observation using the camera on the microscope. When the filter was slid in on the dish as the illumination blackout, as shown in Fig. 6, free Volvox were dispersed from the filtered area and swam to the bright field by the phototaxis, whereas no apparent change was observed for the ratchet-attached Volvox, because illumination does not affect adhesiveness except phototaxtic concentration. To release the microorganism, a slight mechanical impact to the Petri dish was observed to be most effective. Finally, a combination of red filter insertion and mechanical impact proved to be effective in releasing Volvox and stopping the micro-ratchet rotation, as shown in Fig. 6. Immediately after inser-
tion and the impact, the rotation stops within 0.5s, and a small number of Volvox were swimming around the micro-ratchet. As restarting is realized via spot illumination for 3 min, which takes longer compared to release, though, the possibility of on–off control was thus demonstrated.
4.3. Large ratchet drive via chemical surface modification The experimental results show that the micro-ratchets are driven by the attached Volvox rotation rather than collision impacts against the ratchet side surface. Therefore, a possibility for larger ratchet actuation may be realized by increasing the number of attached Volvox. apart from the on-off control. There are several types of adherence mechanisms between the solid surface and the microorganism, such as ionic, chemical, mechanical, meniscus [9], and swimming force [10]. Among these mechanisms, ionic and chemical are the simplest and least harmful because only the surface of the micro-ratchets is modified by specific chemical materials with affinity for the microorganism. As the outer surface of Volvox is covered with an extracellular matrix (ECM) composed of protein fibers, gelatin coating on the ratchet appears be one of the promising methods, based on both ionic and chemical adhesion. A ratchet with a diameter of 2 mm, which is 3.5 times the size of those in previous experiments, was dip-coated in a 2% gelatin solution (Sigma Aldrich Japan, G1393) stained using a red food coloring.
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Fig. 6. Principle of on–off control of micro-ratchet rotation via red-filter switching.
Fig. 7. Adherence enhancement by the gelatin coating on the ratchets surface in 6 min.
Fig. 8. Large ratchet (diameter: 2 mm) driven by adhesion enhancement. Arrows denote the rotation of the tooth plate.
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Fig. 9. A captured image of a starfish disk with Volvox in the bead dispersed solution (a) and its binarization (b).
Fig. 10. A comparison of the particle velocity dependence on the radius of the Volvox and the starfish ratchet.
After 5 min of dipping without a drying process, the ratchet is set in the experimental setup. As shown in Fig. 7, more than 80 Volvox are adhered on the backside of the ratchet within 6 min. An average rotation speed of 0.17 rpm was obtained from an image analysis of Fig. 8; however, in cases without gelatin coating, no rotation occurs, and only translation is observed. Although the drag force generated by the rotation is proportional to the 3rd power of ratchet radius[8], i.e., 43 times in this case, the power sum of Volvox was sufficient to overcome the drag. Therefore, an appropriate surface coating on the ratchet improves the adherence of Volvox, thereby increasing the amount of microorganism-enhanced rotational torque. 5. Fluid flow analysis via PTV In view of application as a fluid mechanical component, it is important to investigate how the micro-ratchet works on the adjacent fluid flow in unconstrained movement. In order to compare the flow difference between free swimming Volvox and Volvox driven starfish disk, an analysis based on PTV was performed as a direct
measurement of the microorganisms [11]. We mixed microbeads (nominal diameter 6 m, Polybead, Polysciences) with Volvox suspension (2000/mL) at a density of 10−4 % v/v. Next, the suspension observed through the biological microscope with an image capture camera having a resolution of 500 × 500 pixels and a capture time of 10 s. As shown in Fig. 9, the image was digitized using ImageJ − open-source software developed by the NIH and the locus was determined using plugin software TrackMate. Finally, the locus was numerically analyzed using Matlab (Math Works). Fig. 10 is a comparison of the particle velocity dependence on the radius of free Volvox and a starfish disk. In the experiment, the size of the Volvox is chosen to be approximately the same, so that the effect of the starfish configuration can be evaluated, and fluid speed generated by rotation was measured to confirm stirring effect without translational component. An apparent velocity distribution difference on the distance from the rotation center is observed in the figure, i.e., the velocity of free Volvox is dispersed randomly, whereas that of the starfish disk is in inverse proportion to radius. This phenomenon may be attributed to the smooth
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swimming characteristics of Volvox without disturbing peripheral fluid flow. On the other hand, Volvox driven micro-ratchet works as a vortex generator due to drag force generated by the fins. 6. Discussion The driving principle of the micro-ratchets with Volvox is based on the continuous swimming power supplied by the microorganisms, in contrast to our previous expectation associated with the Artemia impact drive mechanism. Although the size of phytoplankton is smaller than that of zooplankton, i.e., less power can be obtained from the former, an appropriate surface modification helps the number of adhered phytoplankton with ECM. A ring-type ratchet with a larger radius and surface modification may contribute better torque generation because of the increased number of adhered microorganisms and longer length of the moment arm. The starfish micro-ratchet was proved to function as a vortex generator for an adjacent fluid; although its rotation speed is low, it can be regarded as a mechanical power booster for microorganisms. Combined with an outer casing, which surrounds the micro-disk, a centrifugal micropump with small discharge is one possible application of this mechanism, as a self-sustainable and solar-power-driven bio-mechanism. 7. Conclusions Two types of micro-ratchets driven by Volvox exhibiting a positive phototaxis were designed and successfully actuated. The ratchets were fabricated using a conventional photolithography process. The starfish micro-ratchet with a diameter of 0.537 mm resulted in a better rotational speed of 2.01 rpm than the 8teeth ratchet, which resulted in a speed of 0.86 rpm. An on–off drive possibility was demonstrated using wavelength switching combined with mechanical impact for disengaging the microorganisms from the rotary ratchet. For improved torque generation and larger ratchet rotation, a gelatin coating was found to successfully enhance the number of adhered microorganisms on the ratchets surface, resulting in rotational speed of 0.15 rpm for a ratchet with
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a diameter of 2 mm. A PTV result shows that the fluid around the disk can be swirled, indicating that the disk functions as a stirrer or mechanical booster driven by microorganisms. A solar-powerdriven and sustainable-microorganism-powered mechanism is expected to be developed after further research on this and other related matters. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.precisioneng. 2016.11.010. References [1] Hiratsuka Y, Miyata M, Tada T, Uyeda TQP. A microrotary motor powered by bacteria. PNAS 2006;39:13618–23. [2] Di Leonardoa R, Angelania L, Dell’Arcipreteb D, Ruoccob G, Iebbac V, Schippac S, et al. Bacterial ratchet motors. PNAS 2010;21:9541–5. [3] Sokolova A, Apodacac MM, Grzybowskic BA, Aransona IS. Swimming bacteria power microscopic gears. PNAS 2010;3:969–74. [4] Wong Denise, Beattie Elizabeth E, Steager Edward B, Kumar Vijay. Effect of surface interactions and geometry on the motion of micro bio robots. Appl Phys Lett 2013;103:153707. [5] Koumakis N, Lepore A, Maggi C, Di Leonardo R. Targeted delivery of colloids by swimming bacteria. Nat Commun 2013;4:2588, http://dx.doi.org/10.1038/ncomms3588. [6] Weibel Douglas B, Garstecki Piotr, Ryan Declan, DiLuzio Willow R, Mayer Michael, Seto Jennifer E, et al. Microoxen: microorganisms to move microscale loads. PNAS 2005;34:11963–7. [7] Hatsuzawaa T, Michishita K, Yanagidaa Y. A reciprocating linear actuator driven by anti-phototaxis of plankton. Sens Actuators A 2013;201:316–20. [8] Hatsuzawa T, Yamazakib A, Nisisakoa T, Yanagida Y. Design and evaluation of Artemia-driven micro-ratchet gears. Sens Actuators A 2015;235:182–6. [9] An Yuehuei H, Friedman Richard J. Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. Biomed Mater Res 1998;43:338–48. [10] Drescher Knut, Leptos Kyriacos C, Tuval Idan, Ishikawa Takuji, Pedley Timothy J, Goldstein Raymond E. Dancing volvox: hydrodynamic bound states of swimming algae. Phys Rev Lett 2009:102, http://dx.doi.org/10.1103/PhysRevLett.102.168101. [11] Drescher Knut, Goldstein Raymond E, Michel Nicolas, Polin Marco, Tuval Idan. Direct measurement of the flow field around swimming microorganisms. Phys Rev Lett 2012:105, http://dx.doi.org/10.1103/PhysRevLett.105.168101.
Please cite this article in press as: Hatsuzawa T, et al. Micro-rotary ratchets driven by migratory phytoplankton with phototactic stimulus. Precis Eng (2016), http://dx.doi.org/10.1016/j.precisioneng.2016.11.010