CMOS-Based Amperometric Sensor Utilizing Redox-Cycling Technique for Selective Detection of Dopamine from PC12 Spheroids
Authors: Abe Hiroya , Ino Kosuke , Kanno Yusuke , Inoue Kumi Y. , Kunikata Ryota , Suda Atsushi , Matsudaira Masahki , Shiku Hitoshi , Matsue Tomokazu 
Published: February 2020

Monitoring neurotransmitters, such as dopamine, released from neural cells in the brain is crucial to reveal the mechanism of intercellular communication. Since a released amount of dopamine is closely related to Alzheimer’s disease and Parkinson’s disease, quantitative estimation method of the released amount of dopamine from the cells is required. Among various methods, the electrochemical technique is a major method to monitor dopamine. The conventional electrochemical monitoring of dopamine is basically relay on a single microelectrode setting near a cell. However, this method can only obtain very local information. To detect wider area, the scanning electrochemical monitoring of dopamine is used but it cannot simultaneously obtain information from multiple points. To make an image of dopamine released from relatively big tissue-like samples, we previously developed an CMOS-based amperometric electrode array called Bio-LSI device [1]. The Bio-LSI has 400 micro-disk electrodes with 250 µm pitch. In the report, we successfully obtained the real-time images of dopamine released from the aggregates (ca. 500 µm in diameter) of pheochromocytomas of the rat adrenal medulla (PC12) cells, which is often used as a model of neural cells [2]. To detect the dopamine from the actual brain tissue, it is known that the ascorbic acid interfere the detection of dopamine, because the ascorbic acid is secreted from brain tissues much higher concentration than dopamine. In this study, we developed a novel electrochemical device incorporating the Bio-LSI and an IDA for selective imaging of dopamine release from tissues in the presence of ascorbic acid. We used Bio-LSI as a substrate of the device. We made 20×18 interdigitated electrode arrays (IDAs) by patterning the electrodes on the LSI surface. According to the report described by Niwa et al. [3] the IDAs amplify the amperometric signal of dopamine but does not amplify the signal of ascorbic acid, because dopamine is a reversible redox species which induces the redox cycling at IDA, but ascorbic acid is an irreversible redox species which does not induce the redox cycling at IDA (Fig. 1A).
The device was fabricated by photolithography technique (Fig. 1B). The electrodes were made by Pt spattering. The width and the interval of the IDA were 5 µm. The 20×18 IDAs were arrayed with 250 µm pitch. The oxidation electrodes of IDAs on a same low were connected each other with a lead line connected to the oxidation voltage application pad (Fig. 1C). The reduction electrodes of IDAs were independently connected to the pad nearest to the each detection point. PC12 spheroids with 450 ~ 500 µm in diameter were obtained by the hanging drop method. A PC12 spheroid was simulated by accommodating on a device filled with a high K+solution (Fig. 1B). By applying an oxidation (+0.60 V vs. Ag/AgCl) and reduction (0.00 V vs. Ag/AgCl) potential to the each electrode, we successfully obtained the dopamine released image from the PC12 spheroid (Fig. 1D). This result shows that we can detect dopamine using our device incorporating the Bio-LSI and an IDA. We are now trying to the imaging of dopamine in the presence of ascorbic acid. The detail of the results with the advantage of the use of the IDA electrodes will be discussed in the presentation. Our device will be a promising tool for imaging a dopamine released from a brain tissue to reveal the mechanism of intercellular communication.
References
[1] K. Y. Inoue, M. Matsudaira, R. Kubo, M. Nakano, S. Yoshida, S. Matsuzaki, A. Suda, R. Kunikata, T. Kimura, R. Tsurumi, T. Shioya, K. Ino, H. Shiku, S. Satoh, M. Esashi and T. Matsue, "LSI-based amperometric sensor for bio-imaging and multi-point biosensing," Lab Chip, 12, 3481, 2012
[2] H. Abe. K. Ino, C.-Z. Li, Y. Kanno, K. Y. Inoue, A. Suda, R. Kunikata, M. Matsudaira, Y. Takahashi, H. Shiku, T. Matsue, “Electrochemical imaging of dopamine release from three-dimensional–cultured PC12 cells using LSI-based amperometric sensors” Anal. Chem., 87, 6364, 2015
[3] O. Niwa, M. Morita and H. Tabei, ”Highly sensitive and selective voltammetric detection of dopamine with vertically separated interdigitated array electrodes.”, Electroanalysis, 3, 163-168, (1991)

Figure 1