Innovating the world of MEMS by overcoming problems through ingenuity

Attempting a structure that inhibits ion diffusion

The Ion image sensor is a biosensor that combines of integrated circuit and sensor technology to directly image the distribution and movements of ions. By attaching an enzymatic membrane to the sensor and measuring the ions detected by the enzymatic reaction, it is possible to indirectly image neurotransmitters, making it an innovative sensor that will be useful for the advancement of brain science and early diagnosis of diseases.

At Toyohashi University of Technology, advancements in this research have been primarily led by professor Kazuaki Sawada, the inventor of the ion image sensor. He has already successfully visualized multiple neurotransmitters released from hippocampal slices taken from mice. Now, through industry-academia collaboration, the company is accelerating its efforts to commercialise the technology.

One of the key challenges in developing this ion image sensor has been ion diffusion. Chinatsu Kawakami, who started working on creating a sensor structure under professor Sawada, explains the reason in the following way.

"In order to measure the ion concentration, the measurement must be made in a solution. However, the ions diffuse in the solution. Therefore, the problem was that localized imaging could not be performed. Only an image with a vague, blurred outline could be obtained. Recently, a sensor was finally developed that can measure at the cellular level with a 2 μm pitch, but the close proximity of the pixels increases the effect of ion diffusion. Therefore, I started to work on creating a structure that inhibits ion diffusion."

At first, the instruction from professor Sawada was to create a well-shaped structure with a 2 μm pitch and a depth of 5 μm, using the photolithography technology used to fabricate integrated circuits. An enzyme membrane is formed on the sensor, and the well-shaped structure is formed above that. Ions that react to the enzyme are held in the well, which prevents lateral diffusion and allows high-definition images to be obtained.

"However, even if the well-shaped pattern can be made on the surface with photolithography, the effects of light diffraction, etc., prevented the imaging of depth. Also, even minor alignment deviations would result in all of the pixels being covered by the structure, causing it to be a failure. Through trial and error, I abandoned photolithography due to its limitations, so I looked for a more appropriate materialand found it: porous alumina membrane."

Successful high-definition imaging through the adoption of a porous alumina membrane

The porous alumina membrane is a thin membrane material with a self-organizing nanopore arrangement. The small pores have a diameter of 180 to 400 nm. Also, the deciding factor for the adoption of this material is its biocompatibility, as the membrane is used in biosensors.

Kawakami explained, "If this could be crimped to the structure, there would be multiple holes open for each pixel, and minor alignment deviations would not be a problem. A 54 μ㎡ enzyme membrane was formed on a semiconductor. This was exposed to light, and the enzyme membrane that degrades acetylcholine (ACh) was patterned at a 64 μm pitch. The porous alumina membrane was then crimped above that. As a result of this experiment, we proved that it is possible to inhibit ion diffusion and obtain a high-definition image."

When acetylcholine was dripped on a sensor without a structure, the ions generated by the enzyme reaction diffused, and as time elapsed, the pattern disappeared. In contrast, for a sensor with a structure, the square-shaped pattern of the ACh sensor part and the surrounding hydrogen ion sensor part was maintained even after time elapsed. For confirmation, the change in electric potential of the ACh sensor part and the hydrogen ion sensor part were investigated by plotting the electric potential, and it was understood that ions were only detected by the ACh sensor part when the sensor had a structure.

"Ultimately, we were successful in imaging with an enzyme membrane with a 16 μm pitch. Since the size of a single cell ranges from several μm to 50 μm, we were able to show that multiple types of neurotransmitters released by a single cell can be detected in high definition."

In the future, professor Sawada’s research team will will study the optimal thickness of the alumina film and increase the number of neurotransmitters that can be detected through animal experiments in collaboration with companies and research institutes.

Successful creation of micro-LED probes with a 3D printer

In actuality, Kawakami had difficulty coming up with the process for the structure during her fourth year as an undergraduate. She arrived at the idea for the porous alumina membrane during her first year as a graduate student. However, the challenge of successfully crimping the alumina still remained. In the end, she was able to adeptly attach the membrane by meshing a water-soluble photosensitive resin as a connecting layer. As it turned out, the final breakthrough success came just before the research grant meeting, when she finally succeeded in producing a higher resolution image.

"I was worried that I would finish my graduate degree without achieving any results. I was under pressure, because Professor Sawada had said that he wanted results no later than one month prior to the research brief, but I was relieved at getting a positive evaluation in the end."

Her inquiring mind and ability to act were also on display when she studied at KAIST (Korea Advanced Institute of Science and Technology) as her overseas internship during her final undergraduate year and the first year of her Masters. She was involved in research on optogenetics, a field of technology which is attracting attention as a way of controlling the functions of brain cells with light. In just 5 months, she succeded in creating a μm-sized LED probe (μLED) for irradiating cells with light.

"In order to directly emit light into brain cells, the probe needed to be created from a thin material that was more flexible than silicon and that would not cause damage. Generally, this would be created in a cleanroom at a semiconductor factory, but the professor wanted me to create it in the lab with a 3D printer. At first, I was instructed to create electrodes for the μLED by cutting and attaching aluminum foil. I thought this would be impossible, but I explored methods while using literature for reference. In the end, I printed a structure with grooves and turned it into a mold. I applied a silver paste and formed it with a blade to create the electrodes. Ultimately, I attached an μm-sized LED. This creation process was very simple."

At first, Kawakami was skeptical about creating with a 3D printer, but it turned out to be a simple and inexpensive process. The shape and size can be freely adjusted, and she became confident that this would be useful for on-site medical research in developing countries.

"At the start of my study abroad period, I had trouble speaking in English, and I was also the only girl in the lab. I felt a bit isolated, but as I produced results, I started to open up. I think my experience of making things on my own in KOSEN gave me confidence."

The results were published in "Advanced Functional Materials", an international, high-impact factor journal related to MEMS, and Kawakami was listed in the article as a key author of the paper. Also, a drawing of the probe designed by Kawakami was used on the cover of the journal.

She states, "I didn't think that I could achieve results like this as a graduate student. After graduation, I will work at Shimadzu to develop analytical machines and medical devices. I want to use my experiences from school to help me achieve my goal of developing medical devices." We look forward to her future activities.

Reference

Juhyun Lee, Kyle E. Parker, Chinatsu Kawakami, Jenny R. Kim, Raza Qazi, Junwoo Yea, Shun Zhang, Choong Yeon Kim, John Bilbily, Jianliang Xiao, Kyung‐In Jang, Jordan G. McCall, Jae‐Woong Jeong, “Optogenetic Probes: Rapidly Customizable, Scalable 3D‐Printed Wireless Optogenetic Probes for Versatile Applications in Neuroscience”, Advanced Function Materials 46/2020, 12 November 2020, https://doi.org/10.1002/adfm.202070305

Reporter's Note

Ms. Kawakami is the first student to appear in TUT Research Feature Story. The way she steadfastly pursued her dreams is an inspiration to us all. Aiming to become a world-class engineer, she joined the inaugural class of the Global Technology Architects Course (GAC) , established by Toyohashi University of Technology as a part of the Top Global University Project. She gained useful experiences along the way by living with international students on campus in Global House (a shared apartment style of student accomodation) and through studying abroad. Beyond language skills, she acquired the abilities required to be an internationally-minded person.

Additionally, she worked part time at a special nursing home for the elderly while she was a student. Based on her experience of living at home with a grandmother who required care, she keenly understands the need for very early diagnosis of Alzheimer's. We hope that she uses her characteristic persistence to achieve results for the development of medical devices. We're rooting for her future successes!