Cyborg Zebrafish

Art Courtesy of Courtney Johnson

The human body is a machine. At a fundamental level, it depends on electrical currents within cells and electrical signals between cells to function. So, in a sense, the body produces its own electricity. But what happens if this power gets unplugged? This is the problem that researchers have faced in trying to cure diseases such as Parkinson’s, Alzheimer’s, epilepsy, and depression. In the brain—where electrical signaling is paramount—any small unplugging can throw off the system of electrical currents and quickly lead to improper function. Without a way to fix an improper pattern of electrical signaling, that part of the brain will slowly lose its charge and fizzle out, leading to these diseases. 

A team led by organic bioelectronics researchers Xenofon Strakosas and Hanne Biesmans of Linköping University in Sweden may be on track to develop a viable solution to this problem. They have targeted this issue of restoring dysfunctional electrical pathways by harnessing the body’s chemistry to form an electrode, or electrical conductor that helps produce a current, within the brain. “The really cool thing about this electrode is that it is a soft polymer that forms in situ, or within the brain, unlike metal electrodes that are harsh and rigid, and require open skull surgery,” Biesmans said. Any implant in the human body that does not belong there runs the risk of causing inflammation and inducing an immune response that will try to fight the implant. Current standards for inducing artificial electrical currents in the brain, such as gold electrodes, are not optimal. Biesmans’ team’s primary goal was to develop a ‘softer’ alternative that could be formed within the body. 

Their approach was to create a gel mixture that, once injected into the brain, could self-assemble into a polymer that was able to restore electrical activity. There’s a popular saying that ‘the answer you seek is within you,’ and that’s the advice that the researchers followed. Previous researchers at Stanford had used an outside solution—genetic modification—to produce soft electrodes. However, this pathway comes with its own problems when it comes to human application due to ethical concerns over modifying human DNA. “With our simple injectable gel, there’s no need for genetic modification. And in the long term, maybe there is also no more need for open skull surgeries,” Biesmans said.

This gel cocktail concoction is composed of monomers, or building blocks, as well as enzymes that will be used to make the polymer electrode. The powerful part of this research is that it takes advantage of the enzymatic breakdown of two types of biological sugars found in the body to assemble these monomers into a polymer electrode. First, glucose or lactate—two types of sugars in the body—are converted into hydrogen peroxide by common enzymes known as oxidases. Next, hydrogen peroxide is used by horseradish peroxidase (HRP), a naturally occurring enzyme, to start the polymerization process. And that’s it—a simple, two-step process links together the monomer components from the injected gel to form a soft electrode directly in the body.

But even the coolest products still need to be tested for quality, and that’s why Biesmans and her team came up with a set of seven criteria ranging from fluidity to biocompatibility and stability to assess the electrodes. The first test they ran assessed the effectiveness of their injected electrode gel on 0.6 percent agarose gels, which is a well-known model for simulating brain chemistry and conditions. “In the early stages of research, if you don’t know much, you don’t want to go straight to zebrafish or animal models because you don’t know what works yet,” Biesmans said. In this initial stage of testing, the researchers went through at least fifty to sixty different injectable electrode gels to find a few that were promising enough to continue working with. “Some gels were too thick and did not even make it into the agarose gels,” Biesmans said. 

The team then moved on to demonstrating the conductivity, stability, and biocompatibility of the gel and the formed electrode. It would have been ideal to test these long-term effects with zebrafish, but this was not possible. “Our ethical permits did not allow us to keep the zebrafish alive for more than three days, so we had to find a different way,” Biesmans said. Instead, the researchers used electrode arrays to test for electrical currents maintained by the electrode polymer. They also exposed the polymers to harsh sound energy and live cell conditions to ensure that the polymer would not degrade. Both tests showed excellent results and confirmed the stability of the gel.

These tests showed that the gel performs well, but is it safe to use? This was the team’s next question and perhaps the trickiest because of the potency of glucose oxidase enzymes. These enzymes can quickly produce lots of hydrogen peroxide, which can kill cells at very high concentrations. “We had to find an optimal balance between lactate oxidase and HRP enzymes so that we could get rid of the hydrogen peroxide as fast as it was being produced,” Biesmans said. Finally, the team had a breakthrough and found that a twenty-seven to one ratio of HRP to lactate oxidase worked best. “We were finally able to tune the amount of hydrogen peroxide so that we are not creating more problems than we are fixing,” Biesmans said.

The final hurdle for Biesmans and her team was to test their gel in two live models: zebrafish and medicinal leeches. In zebrafish, the gel was introduced in the tailfin first, followed by the brain and heart. The gel turns deep blue when successfully polymerized, and the team saw this beautiful color in all three locations. Their hard work had finally resulted in a working electrode, without noticeable side effects on the zebrafish. One of Biesmans’ favorite experiments was soaking zebrafish hearts in the gel cocktail. “I really like that the polymer formed around the arteries, where you find glucose and lactate. It’s not covering the entire heart. That was a nice demonstration of the specificity of this gel,” she said. 

Leeches were another nice proof of concept for demonstrating the effectiveness of this polymer electrode since they are easy to visualize. “Leeches have one central nerve, and if you stimulate the nerve, it will contract immediately. So, you get instant visual proof of whether your stimulation worked,” Biesmans said. A key finding from the leech model was that the polymer electrodes produced a gentler, tissue-friendly current as opposed to the stronger, harsher current produced by gold electrodes. 

Even with the success of this initial experiment, there is still much work to be done. Biesmans and her research team plan to further optimize the current gel, test new versions of gels with different monomer building blocks, and introduce their current gel in mice models. Biesmans is also working on trying to induce a similar gel-based polymerization in single cells, as opposed to tissues. “Overall, we are working on building up this toolbox of monomers and techniques for so many different applications,” Biesmans said. These applications include treating various diseases requiring electrical stimulation by forming polymer electrodes at many different sites with disparate properties.

This is just the beginning of exploring electrical patterns in the brain. While this team focused on restoring electrical activity, there are also projects such as Elon Musk’s Neuralink program that seek to use machines to interpret the meaning of the brain’s electrical signals. Perhaps this research will lead us towards a future of not just cyborg zebrafish, but cyborg humans that fully utilize the brain-machine interface. This makes organic bioelectronics a highly interdisciplinary field, and for Biesmans, this has been one of her favorite aspects of the work. “It’s interesting and fun to work with all these collaborators from different disciplines together. So, let’s see where it brings me,” Biesmans said.