In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.
Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.
Biological actuators and biorobots are being actively studied to provide an alternative to conventional robotics for numerous applications. Biorobots that walk5,6,7,8, swim1,2,3,4, pump9,10, or grip11,12,13 have already been developed. Similarly, muscle cells can be incorporated into a 3D rolled PDMS structure14. Often, the biorobot backbones are fabricated using soft lithography techniques with materials such as hydrogels and PDMS (polydimethylsiloxane). These are attractive choices because of their flexibility, biocompatibility, and easily tunable stiffness. Living muscle cells are usually incorporated with these materials to provide force generation through contraction. Mammalian heart muscle cells (cardiomyocytes) and skeletal muscle cells have dominantly been used for actuation. Besides these two, insect muscle tissues have been used to operate biorobots at room temperature3. In this two-part study, cardiomyocytes were chosen because of their spontaneous contraction6.
Much of earlier research on biorobots was focused on developing the biological actuators while optimization of the biorobot architecture and the development of essential functionalities for the biorobots were largely neglected. Recently, a few reports demonstrated the implementation of different swimming modes which were inspired by propulsion modes found in nature. These methods incorporate PDMS films and muscle cells to mimic various natural propulsion methods. For instance, flagella-based propulsion1, biomimetic jellyfish propulsion2, bio-hybrid ray4, and thin film PDMS swimming devices13 have been reported.
In this paper, we present the fabrication process of self-stabilizing swimming biorobots which can maintain immersion depth as well as pitch and roll. The biorobot has a solid base or body, which is propelled by a single cantilever with cardiomyocytes attached to its surface. The cardiomyocytes cause the cantilever to bend in a longitudinal direction when they contract. This form of swimming is classified as ostraciiform swimming. The ability to add additional functionalities on the base is a unique advantage of ostraciiform swimming. For instance, the base can be utilized to provide excess buoyancy to carry additional cargos or control circuitry for cardiomyocyte contraction.
Stability of the biorobot was often overlooked in previous studies of biorobots. In this study, we implemented self-stabilization by designing the base with different composite PDMS materials of varying mass densities. The biorobot thus exhibits resistance to external disturbances and maintains its submersion depth, pitch and roll, unaided. The first layer is microballoon PDMS (MB-PDMS), i.e PDMS mixed with microballoons, which lowers the density of the biorobot, enabling it to float in media. The second layer is the PDMS cantilever, and its thickness is tailored such that force generated by the cardiomyocytes can dramatically bend the cantilever from 45° to 90°. The bottom layer is nickel-PDMS (Ni-PDMS), i.e. PDMS mixed with nickel powder. This layer performs multiple functions. It is magnetic, and therefore allows the biorobot to be anchored at the bottom of the medium, during cell seeding, with a magnet. The nickel mixture is of higher density than the MB-PDMS and medium, and ensure an upright position of the biorobot while floating. The weight of this layer generates a restoring torque on the biorobot at any pitch and roll. Also, the volume ratio between the Ni-PDMS and the MB-PDMS maintains the submersion depth. The presented protocols would be highly useful to researchers interested in characterizing the beating force of muscle cells and tissues, as well as those who wish to build swimming biorobots.
The seeding of the functionalized biological actuator and biorobot devices, the mechanical and biochemical characterization of the cells, and the quantitative analysis of the device function are described in detail in Part 2 of this two-part article as well as in the recent work15.
1. Calculate Mass of PDMS and Additives
2. Fabrication of Biological Actuators on a Stationary Base
NOTE: See Figure 1a.
3. Fabrication of Biorobots (Figure 1b)
4. Functionalization of the Devices
NOTE: Below, we describe the process of preparing the devices for cell seeding.
The biological actuator and biorobot have very similar fabrication processes, as the biorobot is a natural extension of the biological actuator (Figure 1). The biological actuator was developed first to establish techniques required for the biorobot, to analyze the force generated by the cells, and to characterize the cell maturation mechanically and biochemically, both of which are described in detail in Part 2 of this two-part article as well as in our recently published work15.
The spring constant of the actuator was assessed and tuned for a large change in radius of curvature of the cantilever during full contraction of the cardiomyocyte sheet. Then, we designed the biorobot while giving special consideration to its stability, control during cell seeding, and ease of locomotion. Initially, a few designs were chosen, as shown in Figure 2b-2d, with different properties to assess which attributes contribute the most to the design requirements. Biorobots were designed and tested with short, long, and wide cantilevers, as well as with multiple cantilevers to test the effect of changes in the actuator on biorobot function. We also considered different sizes of the floating base. The geometry of the base was maintained as a triangle as it creates the asymmetry that would result in a directional movement.
The stability of the biorobot was a critical component in the design process. The top MB-PDMS layer was used to provide buoyancy to the device, while the bottom Ni-PDMS layer was used for stability and magnetic control. Owing to a higher density, the base layer made of nickel provides the biorobot the ability to keep itself upright and return to its original position after exposure to external disturbances; shown in Figure 3. The layer also provides sufficient weight to keep the device afloat at the appropriate level. In this study, we provide an alternate approach, to develop a biorobot, which focuses on varying the properties of the mechanical backbone to create a self-stabilizing structure. Unlike other biorobots in the literature, the biorobot developed here can maintain its own pitch, roll and immersion depth. These parameters can be tuned by varying the mixing ratios of each composite material and their volume. However, there is a limit to total thickness of each base within the device beyond which the stability is compromised.
The following equation can describe the height of the biorobots above the surface of the medium:
where HNi, HMb, ρmedium, ρMb, and ρNi are thickness of Ni-PDMS, thickness of MB-PDMS, density of the medium, density of MB-PDMS, and density of Ni-PDMS, respectively (See Figure 3b). The height of the biorobots is one critical factor that affects the maximum load it can carry and its stability. Additional weight loaded on the base will lower the biorobots into the media and a larger volume of the base will be submerged. The additional volume to be submerged has a density lower than that of the medium and produces extra buoyancy to lift the added weight. Hence, to increase the maximum carrying load we need to increase h as much as possible. Nevertheless, the stability of the biorobot will be decreased as h increases. For maximum stability, the center of weight of the base should be as low as possible. However, increasing h would place the center of weight of the biorobot close to or above the medium, destabilizing the biorobot. Hence, detailed analysis is required to optimize the stability and the maximum carrying load simultaneously before modifying the base structure of the biorobot.
To determine the right thickness of each composite layer, various mixing ratios were tested with Ni-PDMS, and MB-PDMS. The maximum and minimum densities that could easily be mixed were 0.648 g/cm3 for MB-PDMS and 1.64 g/cm3 for Ni-PDMS, as shown in Figure 3a. All biorobot heights were designed so that the restoring moment of a biorobot at any tilting angle would be strong enough to bring it back to the horizontal position. A triangular shape was used to reduce hydrodynamic drag. The final dimensions are shown in Figure 3d. Using a computer script, the stability was numerically analyzed and proven to have a strong restoring moment using the two-layered method, as shown in Figure 3e. See table of materials and supplementary information for the computer program used.
Figure 1: Process Flow for the Fabrication of the Biological Actuator and Biorobot. Each drawing represents the steps in the materials and methods in protocol sections 2 and 3 for biological actuator and biorobot fabrication. PDMS cantilevers are fabricated by spin-coating and laser engraving. Then the cantilevers are attached to a stationary base with a glass bead for the biological actuator (a) or to a self-stabilizing floating base for the biorobot (b). Please click here to view a larger version of this figure.
Figure 2: Dimensions of the Biological Actuator and Biorobots that are Fabricated in this Study and the CAD Files for Engraving Both the Biological Actuator and Various Types of Biorobots. (a) Biological actuator. (b) Double-arm cantilever biorobot. (c) Wide-arm cantilever biorobot. (d) Single-arm biorobot. (e) CAD drawing of biological actuator for laser engraving. (f) CAD drawing of biorobots for laser engraving. Please click here to view a larger version of this figure.
Figure 3: Mixing Densities for Ni-PDMS and MB-PDMS and Stability of the Biorobots. (a) Mixing ratios and resulting densities. (b) The densities and heights of the bases in relation to the media. (c) The rotation and restoration of the biorobot when tilted. The misalignment between the center of gravity (CG) and center of buoyancy (CB) generates a rotating moment. This moment will either restore the biorobot or cause it to tilt further. (d) The dimensions of the single arm biorobot in millimeter scale. (e) Restoring force was simulated for the single arm biorobot shown in part (c) under tilt conditions in (b) using two layers (Ni-PDMS and MB-PDMS) versus single layer (MB-PDMS). The graph shows that a single layer biorobot will not restore itself if it is tilted over 45°, whereas the dual layered biorobot will have always positive restoring force, keeping the biorobot upright. Please click here to view a larger version of this figure.
Various locomotion mechanisms can be found among aquatic swimmers16. The locomotion mechanism of the biorobot in this study uses fin-based locomotion, specifically ostraciiform locomotion. Ostraciiform swimmers propel themselves by wagging a tail (cantilever) and having a rigid body (layered base)16. Fish such as the boxfish and cowfish use this type of locomotion. Ostraciiform swimmers are typically slow and have inefficient body dimensions. Although ostraciiform swimming lacks velocity, this form of swimming allows engineers to implement various functionalities (such as dynamic stability) on the base or body. The biorobot design developed in this study is based on a solid base for floatation and stability, with a self-actuating cantilever as the propelling mechanism. One of the most important steps in the fabrication of the biorobot in this study is the thin film PDMS and laser engraving process to form the cantilever. Without a clean cantilever, the right mixture of PDMS (for elasticity), correct thickness (for spring constant) and dimensions (having sufficient area for confluent adhesion of cardiomyocytes to produce motion), the biorobot will not operate. Moreover, it is also necessary to remove all bubbles from the cantilever surface through ultrasonication to create a viable surface for cardiomyocyte attachment.
The developed PDMS composite materials, MB-PDMS and Ni-PDMS can be used to precisely control the submersion depth and successfully produce the dynamic stability of the biorobots. The mass density of these materials can be finely tuned, as shown in Figure 3a. Furthermore, these materials do not show any negative effects on the maturation and contraction of the cardiomyocytes as we have shown in our recent work15. Hence, the developed materials can be widely used to implement a self-stabilizing and floating structure for biorobots and other applications.
Although the current protocol was able to build a self-stabilizing swimming biorobot, it has a few limitations. First, as the cantilever is manually peeled off from the wafer, the cantilever may be deformed during the process and the repeatability of the biorobot performance is affected. This can be addressed by using a water-dissolving sacrificial layer instead of the photoresist layer, so that the cantilever can easily be removed from the wafer; larger cantilevers can be used as well for higher power. Second, the procedure mainly relies on manual operations. The fabrication procedure can be streamlined for higher efficiency. For instance, the assembly process including the cardiomyocyte seeding can be modified so as to conduct it on a wafer level instead of individual device level. Lastly, the shape of the triangular base of the biorobot can be optimized to increase the directionality and stability of swimming.
Biorobots that harness the power generated by living muscle cells are of considerable interest as an alternative to traditional fully artificial robots. This protocol uses soft lithography and bio-MEMS techniques to produce a self-stabilizing, swimming biorobot. The particular design can be further refined. The efficiency of the actuator could be increased by patterning alignment cues for the cardiomyocytes on the cantilever surface. This will promote cell orientation and can increase the force generation of the cariomyoctyes17. The dimensions could also be varied or multiple cantilever arms could be attached, to further increase the net force from synchronized contractions. As described earlier, the multiple-layer base allows for tailoring of the height of the biorobot above the media surface. This determines the maximum carrying load and stability. Furthermore, we can substitute or add conductive materials to the cantilever in order to facilitate electrical stimulation. Electrical stimulation can be used to control the contraction rate of cells and the speed of the biorobots. We believe that the presented methods can be used to develop highly efficient biorobots for applications such as small package delivery.
The authors have nothing to disclose.
M. T. Holley is supported by the Graduate Fellows program of the Louisiana Board of Regents and C. Danielson is supported by Howard Hughes Medical Institute Professors Program. This study is supported by NSF Grant No: 1530884. The authors would like to thank the support of the cleanroom at the Center for Advanced Microstructures and Devices (CAMD).
Polydimethylsiloxane (PDMS) | Dow Corning | 184 sil elast kit 0.5kg | Sylgard 184 |
Nickel Powder | Sigma-Aldrich | 266981-100G | |
Phenolic microballoons | US Composites | BJO-0930 | |
Silicon wafers | 4 inch diameter | ||
PWM101 light-duty spinner | Spin- coater | ||
Positive photoresist (S1808) | Dow Corning | DEM-10018197 | |
Hotplate | |||
Vacuum chamber | |||
M206 mechanical convection oven | Convection oven | ||
Laser engraver | Universal Laser System | VLS2.30 | Utilizes a 10W, 10.6 µm wavelength, CO2 Laser |
Universal Laser Systems Application | Universal Laser System | Application for running the VLS 2.30 | |
Matlab | MathWorks | Numerical analysis program | |
Scotch Tape | Scotch Brand | ||
Solid-glass beads | Sigma-Aldrich | Z265926-1EA | Soda-lime glass, diameter 3 mm |
Scale | Mettler Toledo | EL303 | |
BD-20AC Laboratory Corona Treater | Electrotechnic Products | 12051A | Corona Discharger |
Ultrasonic Bath 1.9L | Fisher Scientific | 15-337-402 | 40 kHz industrial transducer |
Fibronectin from bovine plasma | Sigma-Aldrich | F1141 | |
Dulbecco’s Phosphate Buffer (PBS) | Sigma-Aldrich | D1408-100ML | |
Dulbecco’s Modified Eagle Medium (DMEM) | Hyclone Laboratories | 16750-074 | With 4500 mg/L glucose, 4.0 mM L-glutamine, and 110 mg/L sodium pyruvate. |
Fetalclone III serum | Hyclone Industries, GE | 16777-240 | Fetal bovine serum |
Penicillin-G sodium salt | Sigma-Aldrich | P3032 |