Monitoring the electrical activity within and between cells on a large scale in a living animal may seem like a task that's next to impossible. But that's precisely what researchers at Cambridge, Mass.-based Harvard University aim to do with the next iteration of nanoscale sensors in the shape of wires.

With the latest iteration published on in the July 1, 2019, issue of Nature Nanotechnology, the researchers updated their technique, so they were able to make hundreds of nanowires within a few weeks, and to use them to monitor intracellular activity in neurons and cardiac cells in a laboratory setting without any identifiable cellular damage.

The technology could eventually prove useful not just for monitoring a living brain at a cellular level, but also for developing more sophisticated closed-loop neural technologies and even as the basis for a brain-machine interface.

Into animals

That's a big step up from a prior version of the technology, which took weeks to create a few nanowires. Previous iterations were used to monitor cardiac cells, but this time both neurons and cardiac cells were successfully monitored. It's also an improvement as compared to a common research form of intracellular monitoring known as patch clamp electrodes; nanowires are 100 times smaller than patch clamp electrodes, thereby obviating the cellular damage and death that can be seen with the older technology.

Now, the researchers aim to improve upon the technology by enabling it to record for a longer period. Currently, the cells are slowly extruding the nanowires within about 10 minutes. Once the nanowires are adapted to remain more firmly anchored within the cell, the next step will be testing many nanowires simultaneously in a mouse model.

"First we need to further improve upon the technique. Right now, the major problem is that we cannot record for a very long time. Nanowires are very flexible, when they are inserted inside of the cytoskeleton, it then slowly pushes the nanowires out," Anqi Zhang, a graduate student in Harvard's Lieber Lab and one of the authors on this paper, told BioWorld.

"We need to make modifications to attach it to the inside of the cytoskeleton and make the interface more stable," she added. "Another adjustment would make the surface rougher, making the nanowire harder to push out. Then we are planning to use it in live animals."

If the researchers are successful, this could lead to a means for monitoring the activity within and between many cells in a living brain. That's an astonishingly difficult task that has required researchers using earlier technological approaches to coordinate across multiple labs in real-time.

Applications abound

Charles Lieber of Harvard's Department of Chemistry and Chemical Biology has long been involved in this research. His lab first reported earlier iterations of these nanowires starting in 2010.

Ultimately, he expects the nanowires will be used not only to monitor brain activity in a live animal without causing cellular damage, but it could also provide the basis for sophisticated closed-loop neuromodulation or even for a brain-machine interface that would enable direct connection between a wired human brain and an external device or computer.

But the immediate goal is to enable high-quality intracellular recording of a large number of neurons simultaneously in a live mouse. This approach is expected to offer a better approach to doing so than prior efforts.

"One of the advantages of this type of device is that it causes minimal damage to the cell membrane," said Zhang. "The reason is that the tip of the nanowire device is on the nanoscale. There are some other techniques already used for intracellular recording such as the patch clamp, but the tip of the patch clamp is about one to two micrometers."

"The nanowires we are using in this work are 15 nanometers in diameter, that's 100 times smaller than the patch clamp electrode," she continued. "In the previous work, the diameter of those nanowires was 80 nanometers, so now we can achieve even smaller diameter nanowires. Compared to other techniques, there is minimal damage to those cells."

Most methods for cellular monitoring are extracellular, meaning that they record from the outside of the cell. But intracellular monitoring offers a signal that is 1,000 times larger than can be achieved via extracellular monitoring. The better the signal, the more information can be gleaned – and the more precise it can be.

Untangled

The nanowires are first grown on a piece of substrate and develop like long, tangled hair. They are flipped upside-down onto the U-shaped forms. This enables the creation of hundreds of devices at the same time.

This time, rather than incorporating V-shaped nanowires one-by-one that required two or three weeks to make a couple of devices, researchers developed U-shaped versions. They took the noodle-like nanowires and combed them into U-shaped trenches to untangle them and deposit each nanowire into a neat position. Then, each nanowire is modified with a tiny transistor at the bottom.

Researchers were able to insert the nanowire into a cell and remove it repeatedly without killing the cell. But working in the context of a live brain will offer another challenge as well, the inability to see the cells they aim to monitor. It will be impossible to introduce the nanowires into cells in a live brain with the same precision as can be accomplished with ex vivo cells in the lab.

"It's a very cool thing to get an intracellular recording from every available device at the same time. But the problem of recording from inside of the brain of a live animal is that we can't really see the interface of the nanowires with the cells," observed Zhang.

"So, maybe some of the devices are not in contact with the cells; that's not controllable. But with the cell culture, we can see it under a microscope," she said. "We hope to use as many as possible, because this technique is very scalable and there's no limit to the number of devices we can make at the same time."