Bioinspired Soft Robot with Incorporated Microelectrodes

Our new method of fabricating electrically controlled bioinspired actuator can not only overcome the limitation of existing biohybrid actuator, but also strongly enhance the performance of cell-based actuator. Using this low cost and easy-to-handle technique, the actuation behavior of bioinspired soft robot can be controlled and tuned leading to real-time stimulation. Our new method can potentially be extended toward the application of wireless powered implantable flexible electronic device for heart regeneration.

This method can also serve as a new platform for the study of the local electrical stimulation of cell latent constructs. Begin by dissolving 80 milligrams of GelMA in four milliliters of Dulbecco's PBS. Then add 20 milligrams of carboxylic acid functionalized multiwalled carbon nanotubes and sonicate the solution for one hour at 660 millihertz and 100 watts.

To create micropatterned PEGDA, place one layer of 50 micrometer thick commercial transparent tape to one TMSPMA coated glass slide, pour 15 microliters of 20%PEGDA prepolymer solution on top of the coated glass slide, then cover it with gold microelectrodes. Place the first photomask on top of the slide and expose the entire construct to 200 watts of ultraviolet light at 800 milliwatts of intensity and an eight centimeter distance for 110 seconds. At the end of the UV exposure, put the glass slide into Dulbecco's PBS.

Add a 20 microliter drop of the carbon nanotube GelMA prepolymer solution between the spacers. After five to 10 minutes, carefully detach the micropatterned PEGDA hydrogel and the gold microelectrodes from the uncoated glass substrate and place the slide upside down onto the spacers. Fix the slide to the dish with adhesive tape and flip the entire assembly upside down.

Place the second photomask onto the glass slide and expose the assembly to UV light as demonstrated for 200 seconds. At the end of the exposure, wash the scaffold one time with fresh Dulbecco's PBS and one time with cell culture medium supplemented with 10%fetal bovine serum. Then place the scaffold in fresh medium in a new Petri dish in a 37 degree Celsius incubator overnight.

After cardiomyocyte isolation from two-day-old neonatal rat hearts according to standard protocols, resuspend the cells at a 1.95 times 10 to the six cells per milliliter of cardiac medium concentration and seed the cells onto the fabricated soft robot in droplets. When the entire surface of the device has been covered, incubate the samples at 37 degrees Celsius for five days, replacing the culture supernatant with five milliliters of fresh cell culture medium supplemented with 2%fetal bovine serum and 1%L-glutamine on the first and the second days after seeding. To assess the spontaneous beating of the cardiomyocytes on the soft robot, starting on day three of culture, place the robot onto an inverted optical microscope stage and use a 5X or 10X objective and video capture software to image the cardiomyocyte activity for 30 seconds at 20 frames per second.

On day five, use a cover slide to gently lift the membranes at the edge. Using a three centimeter spaced PDMS as a holder, affix two carbon rod electrodes with platinum wire to a six centimeter Petri dish filled with cardiac medium and carefully transfer the soft robot to the dish. Then apply a square waveform with a 50 millisecond pulse width, direct current offset value of zero volts, and peak voltage amplitude between 0.5 and six volts.

For electrical stimulation with the gold microelectrodes, after fabrication of multilayered construct, use silver paste to attach two copper wires to the gold electrodes through an external square port and cover the paste with a thin layer of PDMS pre-cured at 80 degrees Celsius for five minutes. Then place the sample on a hot plate at 45 degrees Celsius for five hours to fully crosslink the PDMS. After seeding cardiomyocyte on the wires connected soft robot, apply a square wave electrical stimulus on the copper wires with the direct current offset value of one volt, a peak voltage amplitude between 1.5 and five volts, and frequencies of 0.5, one, and two hertz respectively.

These soft robots were designed by biomimicking the patterns of two different aquatic animals, the starfish and the manta ray. The cardiomyocyte seeded carbon nanotube GelMA layers exhibited different beating behaviors according to the pattern distances. To prevent irreversible complete rolling of the soft robot during the dynamic beating of the cardiomyocytes, the pattern spacing of the PEGDA hydrogel support layer was optimized to 300 micrometers.

In these frames acquired from contraction recordings, a manta ray shaped actuator can be clearly observed bending the wings as expected with the tail balancing the structure by straightening when the wings were robustly closing in the middle. Some of the membranes demonstrate a rotating movement while contracting due to misaligned micropatterned carbon nanotube GelMA and PEGDA hydrogels. Cardiac tissue on micropatterned PEGDA and carbon nanotube GelMA patterns can also be visualized by F-actin DAPI confocal imaging.

Partial uniaxial sarcomere alignment and interconnected sarcomere structures can also be observed on the patterned areas by confocal microscopy as well as well-interconnected sarcomere structures of cardiac tissues located directly above the microelectrodes. The excitation threshold voltage is different at different frequencies of electrical stimulus through the external carbon electrode or copper wire connected to the gold electrode. The UV crosslinking process of PEGDA and the GelMA micropatterning using photomasks is important for obtaining high quality gold microelectrode incorporating multilayers covered.

Bioprinting can be used for fabricating micropatterned hydrogel and flexible electrode. We used bioprinting to obtain geometrically well-defined soft robot in a rapid, inexpensive, and high throughput manner. Our method can potentially contribute to the development of the wireless electrical stimulation of soft robot through integration of flexible electronic device directly into hydrogel-based scaffold.

Carbon nanotube and organic solvents should always be handled inside a hood as the carbon nanotube fibers can make their way into the lungs posing a risk for cancer development.