This paper was funded by the National Institutes of Health (R01AR074234, R21EB026824, R01 AR073822-01), the Brigham Research Institute Stepping Strong Innovator Award, and AHA Innovative Project Award (19IPLOI34660079).

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the institutional Animal Care and Use Committee (IACUC) of Brigham and Women&#39;s Hospital.
1. GelMA synthesis
<ol>
	<li>Dissolve 10 g of gelatin in 100 mL of Dulbecco&#39;s phosphate-buffered saline (DPBS) using a magnetic stirrer at 50 &#176;C.</li>
	<li>Add 8 mL of methacrylic anhydride slowly while stirring the gelatin prepolymer solution at 50 &#176;C for 2 h. Dilute the reacted gelatin solution with preheated DPBS at 50 &#176;C.</li>
	<li>Transfer the diluted solution into dialysis membranes (molecular weight cutoff = 12&#8211;14 kDa) and place them into deionized (DI) water. Perform dialysis at 40 &#176;C for about 1 week.</li>
	<li>Filter the dialyzed GelMA prepolymer solution using a sterile filter (pore size = 0.22 &#181;m) and transfer 25 or 30 mL of the solution into 50 mL tubes and store at -80 &#176;C for 2 days.</li>
	<li>Freeze-dry the frozen GelMA prepolymer solution using a freeze dryer for 5 days.</li>
</ol>
2. Preparation of poly(ethylene glycol) diacrylate (PEGDA) prepolymer solution
<ol>
	<li>Dissolve 200 mg (20% of total solution) of PEGDA (MW = 1,000) with 5 mg (0.5% of total solution) of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (photo-initiator, PI) in 1 mL of DPBS.</li>
	<li>Incubate the prepolymer solution at 80 &#176;C for 5 min.</li>
</ol>
3. Preparation of GelMA-coated CNT dispersed stock solution
<ol>
	<li>Dissolve 80 mg of GelMA (used as a biosurfactant) in 4 mL of DPBS and then add 20 mg of COOH functionalized multiwalled carbon nanotubes (MWCNTs) into the GelMA prepolymer solution.</li>
	<li>Sonicate the MWCNT-laden GelMA prepolymer solution for 1 h (0.66Hz, 100 Watt). 
	NOTE: During the sonication process, the solution must be immersed in a water bath at ~15 &#176;C to prevent evaporation of solvent due to the rise in temperature.</li>
</ol>
4. Preparation of 1 mg/mL CNT containing 5% GelMA prepolymer solution
<ol>
	<li>Dissolve 50 mg of GelMA and 5 mg (0.5% of total solution) of PI in 0.8 mL of DPBS at 80 &#176;C for 10 min.</li>
	<li>Add 0.2 mL of the prepared CNT stock solution (step 3). Vortex and incubate the solution at 80 &#176;C for 10 min.</li>
</ol>
5. Preparation of a 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) coated glass slide
<ol>
	<li>Wash the glass slides (thickness = 1 mm, size = 5.08 cm x 7.62 cm) with pure ethanol.</li>
	<li>Stack the cleaned slides vertically in a 250 mL beaker and spread 3 mL of TMSPMA on top of them using a syringe. Cover the beaker with aluminum foil to prevent evaporation of TMSPMA.</li>
	<li>Incubate the slides in an 80 &#730;C oven for 1 day.</li>
	<li>Wash the coated glass slides by dipping them into pure ethanol, then dry.</li>
	<li>Store the coated glass slides wrapped in aluminum foil at room temperature (RT). 
	NOTE: Try to minimize touching the surfaces of the TMSPMA-coated glass slides.</li>
</ol>
6. Fabrication of the flexible Au microelectrodes
<ol>
	<li>Design a shadow mask using computer-aided design (Supplementary File 3).</li>
	<li>Fabricate and purchase a shadow mask.</li>
	<li>Wash the glass slide (thickness = 1 mm, size = 3 cm x 4 cm) with acetone and dry with a compressed air gun.</li>
	<li>Attach the shadow mask to the glass substrates using double sided tape, then put them in an E-beam evaporator and wait until the chamber pressure reaches at least 10-6 Torr. 
	NOTE: The two pieces of tape were placed manually on the support at a distance short enough to host the glass and large enough to fit the entire pattern. This step takes around 45&#8211;60 min.</li>
	<li>Deposit a 200 nm thick Au layer by E-beam evaporator (e.g., with Denton EE-4, vacuum = 10-6 Torr, power = 2.6%, rate = 2 &#197;/s) and cut the fabricated microelectrodes using a dicing saw machine (electrodes size = 7.38 mm x 8.9 mm x 200 nm).</li>
</ol>
7. Fabrication of an Au microelectrode-integrated micropatterned multilayered hydrogel scaffold
NOTE: The result of this procedure is a membrane where a micropatterned PEGDA hydrogel is in the bottom layer, a micropatterned CNT-GelMA hydrogel is on top, and the Au microelectrodes are between the two layers. This configuration ensures a better flexibility to the electrode and limits the risk of breaking.
<ol>
	<li>Design and fabricate two photomasks to create the micropatterned PEGDA (1st photomask) and the CNT-GelMA hydrogel (2nd photomask) layers. See Supplementary File 2&#8211;3. The design can be done by using CAD software. 
	NOTE: See Figure 2B, E.</li>
	<li>Place 50 &#956;m spacers made by stacking one layer of commercial invisible tape (Thickness: 50 &#956;m) on a TMSPMA coated glass. Pour 15 &#956;L of 20% PEGDA prepolymer solution on top of the TMSPMA coated glass, then cover with gold microelectrode. Place the 1st photomask for the glass slide (micropatterned PEGDA) on top of the gold microelectrode and expose the whole construct to UV light (200 W mercury vapor short arc lamp with 320&#8211;390 nm filter) at 800 mW of intensity and 8 cm distance for 110 s. 
	NOTE: See Figure 1A.</li>
	<li>Add DPBS to surround the glass slide and detach the micropatterned PEGDA hydrogel together with the Au microelectrodes from the uncoated glass substrate carefully after 5&#8211;10 min to obtain the glass slide that has the micropatterned PEGDA hydrogel with the Au microelectrodes. 
	NOTE: See Figure 1B. Due to the TMSPMA coating, the construct is transferred from the uncoated glass substrate to the TMSPMA-coated one. Detach carefully because the Au microelectrodes can break easily during this step (Figure 3).</li>
	<li>Place 100 &#956;m spacers made by stacking two layers of commercial transparent tape (thickness = 50 &#181;m) on the bottom of a Petri dish. Deposit a drop of 20 &#956;L CNT-GelMA prepolymer solution between the spacers and then flip the glass slide obtained in 7.3 and fix it onto the dish with adhesive tape.</li>
	<li>Rotate the device upside down and place the 2nd photomask on top of the glass slide. Expose under UV light at 800 mW of intensity and 8 cm distance for 200 s. 
	NOTE: See Figure 1C. Alignment of the 2nd mask is important.</li>
	<li>Wash the obtained scaffold with DPBS and with cell culture medium that includes 10% fetal bovine serum (FBS).</li>
	<li>Leave them overnight in the 37 &#176;C incubator before seeding the cells.</li>
</ol>
8. Neonatal rat cardiomyocytes isolation and culture
<ol>
	<li>Isolate hearts from 2-day-old Sprague-Dawley rats following protocols approved by the Institute&#39;s Committee on Animal Care8.</li>
	<li>Put the heart pieces on the shaker overnight (around 16 h) in 0.05% trypsin without EDTA in HBSS in a cold room.</li>
	<li>Collect the heart pieces with a pipette gun and minimize the amount of trypsin, then put them in a 50 mL tube with 10 mL of warm cardiac media (10% FBS, 1% P/S, 1% L-glutamine).</li>
	<li>Swirl slowly (~60 rpm) in a 37 &#176;C water bath for 7 min. Remove the media carefully from the tube with a 10 mL pipette and leave the heart pieces in the tube.</li>
	<li>Add 7 mL of 0.1% collagenase type 2 in HBSS and swirl in a 37 &#176;C water bath for 10 min.</li>
	<li>Mix with a 10 mL pipette 10x gently to disrupt the heart pieces. Remove the media from the tube with a 1 mL pipette.</li>
	<li>Add 10 mL of 0.1% collagenase type 2 in HBSS and swirl quickly (~120&#8211;180 rpm) in a 37 &#176;C water bath for 10 min, then check if the heart pieces are dissolving.</li>
	<li>Mix with a 10 mL pipette, then repeat with a 1 mL pipette to break the last heart pieces.</li>
	<li>Once the solution looks homogeneous, place a 70 &#181;m cell strainer on a new 50 mL tube and pipette the solution 1 mL at a time on strainer.</li>
	<li>Centrifuge the heart cell solution at 180 x g for 5 min at 37 &#176;C. 
	NOTE: If there are still some heart pieces or mucus which did not dissolve, repeat steps 8.7&#8211;8.9 again.</li>
	<li>Carefully remove all the liquid above the cell pellet and resuspended the cells in 2 mL of cardiac media.</li>
	<li>Add 2 mL of cardiac media from the tube wall carefully to resuspend the cells and avoid breaking them.</li>
	<li>Add the suspended cells into a T175 flask with warm cardiac media drop by drop. Put the flask in a 37 &#176;C incubator for 1 h to allow cardiac fibroblasts to attach to the bottom. 
	NOTE: At this preplating step, the cardiac fibroblasts will attach to the flask while the cardiomyocytes will remain in the suspension medium.</li>
	<li>Collect the media from the flask that contains the cardiomyocytes and put it into a 50 mL tube.</li>
	<li>Count the cells, then centrifuge at 260 x g for 5 min at 37 &#176;C.</li>
	<li>Resuspend and seed the cells on top of the fabricated soft robot in step 7. Pour specific volume of cardiac media with the cardiomyocytes at a concentration of 1.95 &#215; 106 cell/mL drop by drop onto the entire surface of the device.</li>
	<li>Incubate the samples at 37 &#176;C and change the media with 5 mL cell culture media with 2% FBS and 1% L-glutamine on the first and the second days after seeding. Change the media every time the color of the media shifts.</li>
</ol>
9. Cell staining for alignment analysis
<ol>
	<li>Remove the media and wash with DPBS for 5 min at RT.</li>
	<li>Fix the cells using 4% paraformaldehyde (PFA) for 20 min at RT. Then wash with DPBS for 5 min at RT.</li>
	<li>Incubate the cells with 0.1% triton in DPBS at RT for 1 h. Wash 3x with PBS for 5 min at RT.</li>
	<li>Incubate the cells with 10% goat serum in DPBS at RT for 1 h.</li>
	<li>Incubate the cells with a primary antibody (sarcomeric &#945;-actinin and connexin-43) in 10% goat serum in DPBS at 4 &#176;C for ~14&#8211;16 h.</li>
	<li>Wash 3x with DPBS for 5 min at RT. Incubate the cells with the secondary antibody in 10% goat serum in DPBS at RT for 1 h.</li>
	<li>Wash 3x with DPBS for 5 min at RT, then counterstain cells with 4&#39;,6-diamidino-2-phenylindole (DAPI) in DI water (1:1,000) for 10 min at RT. Wash 3x with DPBS for 5 min at RT.</li>
	<li>Take fluorescence images using an inverted laser scanning confocal microscope.</li>
</ol>
10. Actuator testing and behavior evaluation
<ol>
	<li>Spontaneous beating of the cardiomyocytes on the soft robot
	<ol>
		<li>Incubate bioinspired actuators at 37 &#176;C for 5 days and refresh the media on day 1 and 2 and when necessary (i.e., when the media turns yellow). Use an inverted optical microscope to take images daily (5x and/or 10x). Record cell movements using video capture software on the microscope&#39;s live window for 30 s at 20 frames per second (5x and/or 10x) when the contractile activity starts (generally around day 3).</li>
		<li>At day 5, detach the membranes by gently lifting from the edge with a cover slide. 
		NOTE: If the cells show a strong beating behavior, the membranes will detach by themselves due to the mechanical action of the contractions.</li>
	</ol>
	</li>
	<li>Bulk electrical signal stimulation
	<ol>
		<li>Using a 3 cm spaced PDMS as a holder, affix two carbon rod electrodes with platinum (Pt) wire in a 6 cm Petri dish filled with cardiac media. Then carefully transfer the soft robot into the Petri dish.</li>
		<li>Apply a square waveform with 50 ms pulse width, DC offset value 0 V, and peak voltage amplitude between 0.5 and 6 V. The frequency varies between 0.5, 1.0, and 2.0 Hz with a duty cycle between 2.5%, 5%, and 10%, respectively. Record macroscale contractions using a commercially available camera.</li>
	</ol>
	</li>
	<li>Electrical stimulation with the Au microelectrodes
	<ol>
		<li>After fabrication of Au microelectrode-integrated multilayered hydrogel scaffold, attach two copper wires to the Au electrodes though an external square port using silver paste.</li>
		<li>Cover the silver paste with a thin layer of PDMS precured at 80 &#176;C for 5 min. Then put the samples on a hot plate at 45 &#176;C for 5 h to fully crosslink the PDMS.</li>
		<li>After seeding cardiomyocyte, apply a square wave electrical stimulus on the copper wires with DC offset value 1 V, peak voltage amplitude between 1.5 and 5 V, and frequencies of 0.5, 1.0, and 2.0 Hz respectively.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Using this method, we were able to successfully fabricate a batoid fish-like bioinspired soft robot with an integrated self-actuating cardiac tissue on a multilayer structured scaffold that is controlled by embedded Au microelectrodes. Due to two distinct micropatterned hydrogel layers made of PEGDA and CNT&#8211;GelMA hydrogels, the bioinspired scaffold showed good mechanical stability and ideal cell alignment and maturation. The PEGDA pattern layer, which serves as a cartilage joint of the skeletal architecture in a st...

Modern state-of-the-art soft robots can mimic the hierarchical structures and muscle dynamics of many living organisms, such as the jellyfish1,2, sting ray2, octopus3, bacteria4, and sperm5. Mimicking the dynamics and architecture of natural systems offers higher performances in terms of both energetic and structural efficiency6. This is intrinsically related to the soft nature of natural tissue (e.g., skin or muscle tissue with a Young&#39;s modulus between 104&#8722;109 Pa) which allows for higher degrees of freedom and superior deformation and adaptability when compared with standard engineered actuators (e.g., a Young's modulus usually between 109&#8722;1012 Pa)6. Cardiac muscle-based soft-actuators, especially, show superior energy efficiency due to their self-actuation as well as their potential for autorepair and regeneration when compared to a mechanically based robotic system7. However, the fabrication of soft robots is challenging due to the necessity of integrating different components with different physical, biological, and mechanical properties into the one system. For example, engineered synthetic systems need to be integrated with living biological systems, not only providing them with structural support but also influencing and modulating their actuation behavior. In addition, many microfabrication methods require harsh/cytotoxic processes and chemicals that decrease the viability and function of any living components. Therefore, new approaches are necessary to enhance the functionality of the soft robots and to control and modulate their behavior.
To successfully integrate living components with good viability, a hydrogel-based scaffold is an excellent material to create the body of a soft robot. A hydrogel's physical and mechanical properties can easily be tuned to create microenvironments for living components such as muscle tissues8,9. Also, it can easily adopt various microfabrication techniques, resulting in the creation of hierarchical structures with high fidelity1,2,10. Flexible electronic devices can be incorporated into the soft robot to control its behavior with electrical stimulation. For example, optogenetic techniques to engineer electrogenic cells (e.g., cardiomyocytes), which show a light-dependent electrophysiological activation, have been used to develop a polydimethylsiloxane (PDMS)-based soft robotic sting ray guided by light that was able to recreate the undulatory movement of the fish in vitro2. Although optogenetic techniques have shown excellent controllability, the work presented uses electrical stimulation, a conventional and traditional simulation method. This is because electrical stimulation via flexible microelectrodes is easy and simple compared to optogenetic techniques, which require extensive development processes11. The use of flexible electronic devices can allow for long-term stimulation and standard/simple fabrication processes as well as tunable biocompatibility and physical and mechanical properties12,13.
Here, we present an innovative method to fabricate a bioinspired soft robot, actuated by the beating of engineered cardiac muscle tissue and controlled by electrical stimulation through embedded flexible Au microelectrodes. The soft robot is designed to mimic the muscle and cartilage structure of the sting ray. The sting ray is an organism with a relatively easy to mimic structure and movement compared to other swimming species. The muscles are recreated in vitro by seeding cardiomyocytes on an electrically conductive hydrogel micropattern. As previously reported, incorporating electrically conductive nanoparticles such as CNT in the GelMA hydrogel not only improves the electrical coupling of the cardiac tissue, but also induces an excellent in vitro tissue architecture and arrangement8,9. The cartilage joints are then mimicked using a mechanically robust PEGDA hydrogel pattern that acts as the mechanically robust substrate of the whole system. Flexible Au microelectrodes with a serpentine pattern are embedded in the PEGDA pattern to locally and electrically stimulate the cardiac tissue.

<table><tbody><tr><td>250 mL Beaker</td><td>PYREX</td><td>1000-250CNEa</td><td></td></tr><tr><td>2-Hydroxy-4&#039;-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>Sigma-Aldrich</td><td>410896</td><td></td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>Milipore</td><td>M6514</td><td></td></tr><tr><td>37&amp;deg; Water bath</td><td>VWR</td><td>W6M</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>50mL Conical Centrifuge Tubes</td><td>Falcon</td><td>14-959-49A</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>80&amp;deg; incubator</td><td>VWR</td><td>1370GM</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A11029</td><td></td></tr><tr><td>Alexa Fluor 594 goat anti-rabbit IgG (H+L)</td><td>Invitrogen</td><td>A11037</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Invitrogen</td><td>A12379</td><td></td></tr><tr><td>Antibiotic/Antimycotic solution</td><td>ThermoFisher Scientific</td><td>15240062</td><td></td></tr><tr><td>Anti-Connexin 43/GJAI antibody</td><td>Abcam</td><td>ab11370</td><td>Rabbit polyclonal</td></tr><tr><td>Anti-Sarcomeric &amp;alpha;-actinin</td><td>Abcam</td><td>ab9465</td><td>Mouse monoclonal</td></tr><tr><td>Benchtop Freeze Dryers</td><td>Labconco</td><td>77500-00 K</td><td></td></tr><tr><td>Biosafety cabinet</td><td>Sterilgard</td><td>A/B3</td><td></td></tr><tr><td>Carbon rod electrodes</td><td>SGL Carbon Group</td><td>6971105</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Forma Scientific</td><td>3110</td><td></td></tr><tr><td>Collagenase, Type II, Powder</td><td>Gibco</td><td>17-101-015</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 880</td><td></td></tr><tr><td>COOH Functionalized Carbon Nanotubes</td><td>NanoLab</td><td>PD30L5-20-COOH</td><td></td></tr><tr><td>Dicing saw machine</td><td>Giorgio Technology</td><td>DAD-321</td><td></td></tr><tr><td>DMEM, High Glucose</td><td>Gibco</td><td>11-965-118</td><td></td></tr><tr><td>DPBS without Calcium and Magnesium</td><td>Gibco</td><td>14-190-144</td><td></td></tr><tr><td>E-beam evaporator</td><td>CHA</td><td>57367</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10-437-028</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td>Type B, 300 bloom from porcine skin</td></tr><tr><td>Glass slide</td><td>VWR</td><td>48382-180</td><td></td></tr><tr><td>HBSS without Calcium, Magnesium or Phenol Red</td><td>Gibco</td><td>14-175-079</td><td></td></tr><tr><td>Inverted optical microscope</td><td>Olympus</td><td>CK40</td><td></td></tr><tr><td>Magnetic hotplate</td><td>Corning</td><td>PC-420</td><td></td></tr><tr><td>methacrylic anhydride</td><td>Sigma-Aldrich</td><td>276695</td><td>Contains 2,000ppm topanol A as inhibitor</td></tr><tr><td>Nunc EasYFlask 175cm&lt;sup&gt;2&lt;/sup&gt;</td><td>ThermoFisher Scientific</td><td>159910</td><td></td></tr><tr><td>Olicscope</td><td>Siglent</td><td>SDS1052DL+</td><td></td></tr><tr><td>Paraformaldehyde Aqueous Solution -16%</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>PDMS SYLGARD 184</td><td>Sigma-Aldrich</td><td>761036</td><td></td></tr><tr><td>Photomask</td><td>Mini micro stencil inc</td><td></td><td></td></tr><tr><td>Platinum wire</td><td>Alfa Aesar</td><td>AA43014BU</td><td></td></tr><tr><td>Polyethylene glycol dimethcrylate</td><td>Polysciences Inc.</td><td>15178-100</td><td></td></tr><tr><td>Regenerated Cellulose Dialysis Tubing</td><td>Fisherbrand</td><td>21-152-14</td><td></td></tr><tr><td>Silver Epoxy Adhesive</td><td>MG Chemicals</td><td>8330S</td><td></td></tr><tr><td>Stericup Quick Release-GP Sterile Vacuum Filtration System</td><td>Millipore</td><td>S2GPU02RE</td><td></td></tr><tr><td>Ultra sonicator</td><td>Qsonica</td><td>Q500</td><td></td></tr><tr><td>UV Curing System</td><td>OmniCure</td><td>S2000</td><td></td></tr><tr><td>Vortex mixer</td><td>Scientific Industry</td><td>SI-0246A</td><td></td></tr><tr><td>Waveform generator</td><td>Agilent</td><td>33500B</td><td></td></tr><tr><td>Wrap Aluminium foil</td><td>Reynolds</td><td>N/A</td><td></td></tr></tbody></table>

bioinspired soft robot with incorporated microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

Flow diagram of the steps for developing the Au microelectrode-incorporated bioinspired soft robot 
The aim of the soft robot design was to build a membrane capable of actuating a swimming movement with minimal complexity. The structure must be able to sustain strong flexions repeatedly over time (about 1 Hz) and be able to keep its shape while achieving a strong beating. By selectively photo crosslinking the polymer using photomasks, we fabricated a hierarchically structured scaffold comprised of a...

Watch this Scientific Journal Video about Bioinspired Soft Robot with Incorporated Microelectrodes at JoVE.com

Bioinspired Soft Robot with Incorporated Microelectrodes

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

bioinspired-soft-robot-with-incorporated-microelectrodes

Research

JoVE Journal

Bioengineering

8.7K Views.  Harvard Medical School. A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Video: Bioinspired Soft Robot with Incorporated Microelectrodes

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Fabrication of Carbon-Based Ionic Electromechanically Active Soft Actuators

Ionic electromechanically active capacitive laminates are a type of smart material that move in response to electrical stimulation. Due to the soft, compliant and biomimetic nature of this deformation, actuators made of the laminate have received increasing interest in soft robotics and (bio)medical applications. However, methods to easily fabricate the active material in large (even industrial) quantities and with a high batch-to-batch and within-batch repeatability are needed to transfer the knowledge from laboratory to industry. This protocol describes a simple, industrially scalable and reproducible method for the fabrication of ionic carbon-based electromechanically active capacitive laminates and the preparation of actuators made thereof. The inclusion of a passive and chemically inert (insoluble) middle layer (e.g., a textile-reinforced polymer network or microporous Teflon) distinguishes the method from others. The protocol is divided into five steps: membrane preparation, electrode preparation, current collector attachment, cutting and shaping, and actuation. Following the protocol results in an active material that can, for example, compliantly grasp and hold a randomly shaped object as demonstrated in the article.

This article describes a fast and simple manufacturing process of ionic electromechanically active composite materials for actuators in biomedical, biomimetic and soft robotics applications. The key fabrication steps, their importance for the actuators&#39; final properties, and some of the main characterization techniques are described in detail.

Ionic electromechanically active capacitive laminates are a type of smart material that move in response to electrical stimulation. Due to the soft, ...

Engineering

Manufacturing, Control, and Performance Evaluation of a Gecko-Inspired Soft Robot

This protocol presents a method for manufacturing, control, and evaluation of the performance of a soft robot that can climb inclined flat surfaces with slopes of up to 84&#176;. The manufacturing method is valid for the fast pneunet bending actuators in general and might, therefore, be interesting for newcomers to the field of actuator manufacturing. The control of the robot is achieved by means of a pneumatic control box that can provide arbitrary pressures and can be built by only using purchased components, a laser cutter, and a soldering iron. For the walking performance of the robot, the pressure-angle calibration plays a crucial role. Therefore, a semi-automated method for the pressure-angle calibration is presented. At high inclines (&#62; 70&#176;), the robot can no longer reliably fix itself to the walking plane. Therefore, the gait pattern is modified to ensure that the feet can be fixed on the walking plane.

This protocol provides a detailed list of steps to be performed for the manufacturing, control and evaluation of the climbing performance of a gecko-inspired soft robot.

This protocol presents a method for manufacturing, control, and evaluation of the performance of a soft robot that can climb inclined flat surfaces with ...

Four-Dimensional Printing of Stimuli-Responsive Hydrogel-Based Soft Robots

The present protocol describes the creation of four-dimensional (4D), time-dependent, shape-changeable, stimuli-responsive soft robots using a three-dimensional (3D) bio-printing method. Recently, 4D printing techniques have been extensively proposed as innovative new methods for developing shape-transformable soft robots. In particular, 4D time-dependent shape transformation is an essential factor in soft robotics because it allows effective functions to occur at the right time and place when triggered by external cues, such as heat, pH, and light. In line with this perspective, stimuli-responsive materials, including hydrogels, polymers, and hybrids, can be printed to realize smart shape-transformable soft robotic systems. The current protocol can be used to fabricate thermally responsive soft grippers composed of N-isopropylacrylamide (NIPAM)-based hydrogels, with overall sizes ranging from millimeters to centimeters in length. It is expected that this study will provide new directions for realizing intelligent soft robotic systems for various applications in smart manipulators (e.g., grippers, actuators, and pick-and-place machines), healthcare systems (e.g., drug capsules, biopsy tools, and microsurgeries), and electronics (e.g., wearable sensors and fluidics).

This manuscript describes a 4D printing strategy for fabricating intelligent stimuli-responsive soft robots. This approach can provide the groundwork to facilitate the realization of intelligent shape-transformable soft robotic systems, including smart manipulators, electronics, and healthcare systems.

The present protocol describes the creation of four-dimensional (4D), time-dependent, shape-changeable, stimuli-responsive soft robots using a ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

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.

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 ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Microfluidic Channel-Based Soft Electrodes for Capacitive Pressure Sensing

Microfluidic Channel-Based Soft Electrodes and Their Application in Capacitive Pressure Sensing

Flexible and stretchable electrodes are essential components in soft artificial sensory systems. Despite recent advances in flexible electronics, most electrodes are either restricted by the patterning resolution or the capability of inkjet printing with high-viscosity super-elastic materials. In this paper, we present a simple strategy to fabricate microchannel-based stretchable composite electrodes, which can be achieved by scraping elastic conductive polymer composites (ECPCs) into lithographically embossed microfluidic channels. The ECPCs were prepared by a volatile solvent evaporation method, which achieves a uniform dispersion of carbon nanotubes (CNTs) in a polydimethylsiloxane (PDMS) matrix. Compared to conventional fabrication methods, the proposed technique can facilitate the rapid fabrication of well-defined stretchable electrodes with high-viscosity slurry. Since the electrodes in this work were made up of all-elastomeric materials, strong interlinks can be formed between the ECPCs-based electrodes and the PDMS-based substrate at the interfaces of the microchannel walls, which allows the electrodes to exhibit mechanical robustness under high tensile strains. In addition, the mechanical-electric response of the electrodes was also systematically studied. Finally, a soft pressure sensor was developed by combining a dielectric silicone foam and an interdigitated electrodes (IDE) layer, and this demonstrated great potential for pressure sensors in soft robotic tactile sensing applications.

Author Spotlight: Microfluidic Channel-Based Soft Electrodes and Their Application in Capacitive Pressure Sensing  

Flexible electrodes have a wide range of applications in soft robotics and wearable electronics. The present protocol demonstrates a new strategy to fabricate highly stretchable electrodes with high resolution via lithographically defined microfluidic channels, which paves the way for future high-performance soft pressure sensors.

Flexible and stretchable electrodes are essential components in soft artificial sensory systems. Despite recent advances in flexible electronics, most ...