Development of a Sperm‐Flagella Driven Micro‐Bio‐Robot

A new biohybrid micro-robot is developed by capturing bovine sperm cells inside magnetic microtubes that use the motile cells as driving force. These micro-bio-robots can be remotely controlled by an external magnetic field. The performance of micro-robots is described in dependence on tube radius, cell penetration, and temperature. The combination of a biological power source and a microdevice is a compelling approach to the development of new microrobotic devices with fascinating future applications. The design of nanomotors and micro-bio-robots is of rising interest in nanomedicine and nanotechnology. There have been many successful approaches to develop artificial micromotors of various architectures which are driven by chemical fuels,1-6 surface tension gradients,7, 8 magnetic9-11 or electric fields.12, 13 The use of micromotors towards biologically related applications has led to interesting advances such as the drilling of tissues14, 15 and fixing cancer cells.2, 16 However, the use of toxic fuels is currently a challenge for any potential biomedical application of such motors and hence, a next generation of self-propelled microdevices is desperately sought after.2 The fascinating biomolecular motors found in nature offer a great source of inspiration for the design of artificial motors. As Steven Boxer mentions, “since we can't beat them (biomolecular systems) we should join them.”17 Motors found in nature are small but powerful and thus a reason to take them as archetypes. The first demonstration of integrating a biomolecular motor in a nanodevice was a combination of ATPase with a metal propeller.18 It has been demonstrated that there is potential in using motile microorganisms for the development of micro-bio-robots when they are integrated into microsystems.19-26 There are several mechanisms towards the directional control of micro-bio-robots such as taxis-based motion (magnetotaxis,22 chemotaxis,22, 23 thermotaxis etc.), electrokinetic control24 or geometrical asymmetry.25, 26 Bacteria-powered microrobots, magnetotactic bacteria, artificial bacterial flagella, flexible magnetic filaments, and our sperm-flagella driven micro-robots have all in common that they use flagella as driving force in low Reynolds conditions. Magnetotactic bacteria require generally a small magnetic field strength (0.4 mT)22 and the speed of a MC-1 cell (marine coccus strain) decreases only 15% when steered by an external magnetic field. Artificial bacterial flagella that are driven by a rotating magnetic field tend to require a slightly higher magnetic field strength for actuation. Magnetotactic, bacteria-powered and sperm-driven microrobots have in common, that they do not need an external power source for actuation, can be controlled by an external signal and their activity range is restricted to physiological conditions (temperature and pH tolerance of bacteria species and spermatozoa, respectively). Artificial bacterial flagella10 and flexible magnetic swimmers27 offer more flexibility in the design of the device and can tolerate a large temperature range. However, the artificial flagella are only actuated and steered if a continuously rotating magnetic field is applied. Flexible magnetic microswimmers require an oscillating transverse field in order to be actuated. Both kinds of artificial swimmers do not have an on-board power source. The artificial flagella presented by Tottori et al.10 moves at 100 μm s−1 at a frequency of magnetic rotation of 50 Hz and a field strength of 4 mT. However, none of these approaches offer the selective encapsulation of a single motile cell and directed control over its motility. Our sperm-tube hybrid system provides more flexibility regarding the type of organism that can be integrated: it can be adjusted to any flagellated organism if needed. One advantage of using sperm cells for the micro-bio-robot actuation is that these cells are readily available, easy to handle, do not need to be cultivated, and are completely harmless. They are adapted to swim in highly viscous media such as serum. The motivation for using sperm cells as driving force is not primarily to develop a more efficient micro-bio-robot (compared to existing hybrid robots) but rather to go towards a medical application that involves the controlled guidance of a single sperm cell to an egg cell. Compared to existing magnetically or bacterial driven systems, this sperm-driven micro-robot does not only offer a biological propulsion method, but comes with a very promising motivation of developing new fertilization methods, where the transport of a single spermatozoon to the egg cell location is required. The incorporation of magnetic nanoparticles is one attempt to provide magnetic control in a moving system. Although magnetic nanoparticles have been successfully applied to sperm cells for drug and gene delivery28 as well as tracking and targeting of cells in vivo,29, 30 their major disadvantage is that they can pass through the cell membrane and affect vital functions of the organism. As a consequence, they have been found to be toxic and are controversially discussed regarding their biocompatibility.31 With the future purpose of using our sperm-driven micro-bio-robot for in-vivo guidance of sperm cells and fertilization, the capture of a sperm cell inside a microtube seems a good approach to a control method without altering the cell. Microtubes are sufficiently large to not be taken up by cells and thus can be combined with sperms without any effect on sperm activity or its ability to undergo acrosome reaction. Compared to other types of microparticles (e.g. spheres), rolled-up tubes are advantageous because they consist of carefully designed thin nanomembranes,6, 32 and thus consist of light-weight structures that could serve as multi-functional carriers or chassis for cell guidance. Furthermore, the physical confinement of the cell inside the tube cavity circumvents the complex biochemical functionalization of the inner tube surface to bind specifically to sperm membrane proteins without limiting the cell motility, in this particular case sperm activity. In addition, the mechanical trapping method inside a magnetic microtube can be applied to any flagellated or ciliated cell. Here we present the first example of combining a single motile cell with a microtube to create a micro-bio-robot which can be guided to defined positions. The sperm cell can enter the microtube and is trapped in the tube cavity. In this work, the performance of the micro-bio-robot is described and the influence of temperature, tube radius and penetration length of cell inside the microtube is evaluated. The incorporation of thin films of magnetic material into the microtube offers the possibility to remotely control the motor by the application of an external magnetic field. We demonstrate the remote-controlled separation of a selected micro-bio-robot on-chip. This micro-bio-robot is created by combining sperm cells with rolled-up magnetic microtubes, which are fabricated by rolling up thin ferromagnetic layers as previously reported.32 We can adjust the parameters regarding desired materials, nanofilm thickness and template size during the fabrication process in a way that the resulting microtubes have a certain diameter, wall thickness, shape, length and advanced functionality.33-35 By doing so, we fabricated tubes with a diameter slightly larger than the sperm head (5–8 μm), as shown in the scanning electron microscope images in Figure 1b,d and the optical image in Figure 1e; consequently, the cells become trapped once they enter the hollow structure of the microtubes (see also Video S1, Supporting Information). Spermatozoa display very powerful movements and are able to interact with rolled-up magnetic microtubes in a way that they are mechanically caught inside the tubes and push them forward. The motile cell that is incorporated as driving force into the hybrid system is a bovine sperm cell. Generally, all mammalian sperm cells display similar composition by head, midpiece and tail. We selected bovine sperm cells because of their size and shape which are similar to human sperm cells. A representative microscopic image of a bull sperm cell used in our experiments is shown in Figure 1a. A bovine sperm head is about 10 μm long, 5 μm in diameter but only about 1 μm thick36 containing a flagella of about 60 μm in length. Spermatozoa are perfectly adapted to swimming in viscous media on the microscale and thus are promising as driving force of micro-robots. Cryopreserved bovine sperm cells are thawed and diluted in SP-TALP (modified Tyrode's Albumin-Lactate-Pyruvate Medium) to a cell density of about 108 cells mL−1. After addition of about 100 magnetic microtubes (50 μm long, 5–8 μm in diameter) to the petri dish containing 2 mL of SP-TALP Medium with suspended cells, the coupling of microtubes with spermatozoa takes place randomly. The coupling process is illustrated in Figure 1 f,g and video S2. Video S3 demonstrates that most tubes (14 out of 21) are coupled with spermatozoa after just 10 minutes incubation. There are several factors influencing the performance of the micro-bio-robot, which are subject of this study. In order to evaluate the performance of this kind of micro-bio-robot, we analyzed penetration, tube radius and speed of over 40 cases of micro-bio-robots depending on the initial swimming speed of the spermatozoon at room temperature. The schematic in Figure 2a displays how the radius and length of the microtube as well as penetration x are defined. The length of the cell (measured from head tip) that is confined inside the tube (0–50 μm) divided by the total length of the microtube (50 μm) and multiplied by 100, results in the relative penetration percentage x. Since the natural variation of swimming speeds of thawed bovine spermatozoa is very high, we eliminate this variation by relating the speed of our micro-bio-robots to the initial cell speed of each case before entering the microtube. That means, that only recorded capturing processes of cells inside the microtubes are considered in this analysis. In general, we can conclude that type A micro-bio-robots display a higher relative speed than type B micro-bio-robots. This is caused by the influence of penetration into the confinement of sperm flagella which in turn reduces the cell speed. The average speed of type A micro-bio-robots is 10 μm s−1 compared to 5 μm s−1 for type B. In both cases the speed reduces significantly from the free cell speed which can be explained by both, the cargo the cell is pushing, and the confined flagella amplitude. The performance of natural and artificial biomotors is influenced by the temperature of the media in which they swim.39 Similarly, the sperm-driven micro-bio-robot is sensitive to those changes. The effect of temperature on the performance of the micro-bio-robot was analyzed by heating and cooling the sample while recording videos of the moving microtubes. The temperature was kept between 5–40 °C in the tolerance range of sperm cells. Trajectories of the same micro-bio-robot at 12 °C and 37 °C over 10 seconds are illustrated in Figure 3a. Generally, for all 73 observed cases an increase of speed with increasing temperature was observed. The reason for the speed increase at higher temperatures is caused by physiological changes in the metabolism of the sperm cell. Sperm thermotaxis is a control mechanism that also takes place in vivo to guide and accelerate the cells towards the oocyte in the fallopian tubes in the female body.40 The response of sperm cells to changing temperature can be used to control the speed of this micro-bio-robot. Therefore, we can perform a “stop and go” control of the micro-bio-robots, as demonstrated in Video S4. Once the coupling between sperm cell and microtube is completed, the micro-bio-robot (coupled sperm with microtube) can be steered through the alignment with an external magnet, as shown in Figure 4a and Video S5, in a similar fashion as presented with nanoparticles, magnetotactic bacteria and self-propelled microjets.41 In all these experiments, we used a permanent neodymium magnet with a magnetic field intensity of 540 mT, which was applied to the sample at a distance of about 2 cm leading to a magnetic field intensity of 22 mT. This field intensity is enough to align and guide the microtubes.42 In order to quantify the directed motion of the cell once it is trapped inside the microtubes, we measured the directionality of a free cell compared to the trapped cell. For the measurement and calculation of directionality we rely on the work by Paxton et al.43 where the directionality factor is defined as cos (θ), θ being the angle between directionality vector and axis of the sperm head when looking at free cells or tube axis when observing the whole micro-bio-robot over one time interval (t = 0.2 s in our work). Thus, an object that moves straight in axial direction has a directionality factor of 1, whereas a 90 degree turn means a directionality factor of 0. Hence, directionality is a measure for how straight a motion is performed. Figure 4b shows that prior to the coupling between the sperm cell and microtube, the directionality of the freely swimming cell is variable and ranges from 0.65 to 1. This is a result of the natural motion of the sperm cell with rotational and wavy movements. Before the coupling, the sperm cell reaches a speed of up to 100 μm s−1. However, when the sperm cell connects with the microtube, the speed decreases to an average of 10 μm s−1. After the coupling is completed, the cell inside the tube moves with a high directionality factor of close to 1. This value becomes even more invariable when an external magnet is applied, since the alignment of the tube is induced by the magnetic field lines. For the proof of concept of separating a desired sperm-microtube robot from a mixture of uncoupled microtubes and free sperm cells by external magnetic control, we used a commercial microfluidic chip from ibidi. A schematic of the separation concept is depicted in Figure 5a. This microchip, as displayed in Figure 5b, consists of two 80 μL chambers that are connected with a narrow 70 μm high channel (1 mm wide and 10 mm long). The red circles in Figure 5b and c mark the position of the micro-bio-robot that is guided from the top 80 μL chamber into the channel over a time length of 5 minutes. Using a permanent magnet that is mounted on a stand underneath the sample, the microtube is aligned parallel to the magnetic field lines and the propulsion by the sperm cell leads to the forward motion of the robot. In the video S6, it can be observed that all microtubes align according to the external magnetic field, but only the microtube containing a trapped sperm cell is able to move to the selection channel. After less than 5 minutes, the selected microtube has reached the channel and is separated from all other microtubes. At this point, the selected micro-bio-robot can be removed from the chip. The micro-bio-robot in this separation process displays an average velocity of 15 μm s−1. This process can be followed in Video S6. The fabrication of ferromagnetic rolled-up microtubes was described previously32, 44 and the concept of magnetization of self-propelled microtubes has been reported recently.45 When microtubes containing a ferromagnetic layer are exposed to a strong magnetic field the result is the re-alignment of their domains according to the external field. The microtubes acquire a defined north and south pole and become magnets themselves. Consequently, the magnetized microtubes can be attracted and repulsed by an external magnetic field depending on its polarity and therefore an acceleration of the hybrid micromotor could be achieved (see Figure S1 in Supporting Information). We conducted a long-term experiment of recording the sperm-driven micro-bio-robot over 90 minutes at room temperature to determine how long the sperm cells can operate as driving force (see Figure S2 in Supporting Information). In vivo, it was reported that sperm cells travel from the uterus to the fertilization site in only a few minutes46 which to some extent is due to vaginal, cervical and uterine contractions. In the long-term experiment, the motor velocity slightly decreases over time from 8 μm s−1 to 6 μm s−1. However, the operation of the micro-bio-robot for over an hour suggests that this micro-bio-robot is suitable for medical applications and can assist in vivo fertilization methods. In conclusion, we have presented the development of a micro-bio-robot comprising a motile sperm cell and a magnetic microtube. This micro-bio-robot moves without any toxic fuel and is solely based on the flagellar propulsion of the cell. The performance of the micro-bio-robot was investigated regarding the influence of tube radius, penetration and temperature on the motor speed. In addition, the magnetic microtube consisting of rolled-up thin films offers the advantage of having a cavity to trap the cell as well as direct the motion of a single sperm cell with an external magnet. We demonstrated the separation of a selected sperm-driven micro-bio-robot by remote magnetic guidance on-chip. Improving the design of the microtubes, e.g. length and geometry, could further improve the motility and performance of the sperm-driven micro-bio-robot in future works. The implementation of a cell release mechanism could be helpful to transport single sperm cells to a desired location. In doing so, the sperm-driven micro-bio-robot can become a promising device for developing an alternative fertilization method that takes place in vivo when the hybrid motors with selected sperm cells are remotely guided to the egg cell. This bio-hybrid micro-robotic approach demonstrates the potential of utilizing microtubes driven by motile sperm cells for various applications in medicine, such as micromanipulation and targeted drug delivery. Fabrication of Microtubes: Magnetic microtubes are fabricated by rolling up thin magnetic layers as follows: A 50 μm long microtube consisting of titanium and iron is fabricated using roll up technology. A glass substrate with 50 × 50 μm squared structures is coated with photoresist AR-P 3510, exposed to UV light for 7 seconds and developed with an AR 300–35:water (1:1) solution. 10 nm layers of titanium and iron are deposited onto the substrate using electron beam evaporation. The substrate is immersed in acetone and the layers roll up immediately because the photoresist layer is removed. Microtubes are formed with a length of 50 μm and a diameter of 5–8 μm. These microtubes are dried using the Critical Point Dryer. Preparation of Sperm-Driven Micro-Bio-Robot: The interaction between bull sperm cells and microtubes was investigated by adding microtubes that were released from their substrate, into a solution of bovine sperm cells in SP-TALP medium (modified Tyrode's Albumin-Lactate-Pyruvate Medium from CaissonLabs). This medium has a composition that comes close to oviduct fluid of mammalians. The sperm cell solution was prepared as follows: The previously cryopreserved bovine semen straws were thawed for 10 minutes in the incubator at 37 °C and diluted in 2 ml SP-TALP medium and incubated for another 10 minutes at 37 °C. After addition of microtubes the sample was observed under the optical microscope. For magnetic guidance a neodymium magnet was used with a magnetic field intensity of 22 mT at a distance of 2 cm from the sample. The temperature experiments were carried out under the same conditions while using a Peltier Element for heating and cooling the sample to the desired temperature. In order to take scanning electron microscope images, sperm cells were fixed on glass substrates by incubating the cell solution with 2% glutaraldehyde solution for 30 minutes. The samples were subsequently washed three times with PBS and a final rinsing step with water was carried out. To create a conducting surface, the glass wafers were coated with 20 nm chromium using a sputter coater. The scanning electron microscope images were taken with an NVision SEM/FIB 40 machine from Zeiss. Speed Measurements: Frozen sperm vials were thawed for 10 min at 37 °C. The sample was diluted in 2 mL SP-TALP and incubated for additional 10 min at 37 °C. The videos were taken with a Phantom Miro eX2 high-speed camera from VisionResearch mounted onto an inverted microscope. For the tracking of cell paths the video analyzing software FIJI was used. The length of trajectories of sperm cells were measured by Fiji software and with the known frame rate the speed of tracked cells can be determined. Separation On Chip: For the separation experiments we used a μ-Slide Chemotaxis2D from ibidi®. At first, SP-TALP medium was filled into one of the 80 μL chambers. The bovine sperm cells are prepared as described in previous paragraphs. A solution containing Ti/Fe microtubes and bovine sperm cells was filled into the second 80 μL chamber. Afterwards, the middle channel was filled with clean SP-TALP medium. The inlets and outlets were closed with plugs and the chip was placed under the microscope with a permanent magnet mounted on a stand underneath the sample holder. The separation process was recorded at room temperature with 5× magnification with 1 frame per second using a Zeiss Axiocam camera. Authors thank the Volkswagen Foundation (# 86 362). The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement n° 311529 (Samuel Sanchez). The authors would also like to thank Masterrind GmbH Meißen, and R. Naumann from MPI-CBG Dresden for providing cell samples, S. Harazim for SEM imaging, ITI GmbH Dresden for SimulationX software, ibidi for free samples of chemotaxis chips and Dr. R. Traeger for the schematic in Figure 5. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.