Research Article | Open Access

Volume 2024 |Article ID 0162 | https://doi.org/10.34133/plantphenomics.0162

Microfluidic Device for Simple Diagnosis of Plant Growth Condition by Detecting miRNAs from Filtered Plant Extracts

Yaichi Kawakatsu,^1,11 Ryo Okada,^2,11 Mitsuo Hara,^3,11 Hiroki Tsutsui,² Naoki Yanagisawa,^4,5 Tetsuya Higashiyama,^4,5,6 Akihide Arima,⁷ Yoshinobu Baba,^7,8,9 Ken-ichi Kurotani,¹ Michitaka Notaguchi ^1,2,10

¹Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
²Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
³Department of Molecular and Macromolecular Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan
⁴Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
⁵Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan.
⁶Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
⁷Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
⁸Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
⁹Institute of Quantum Life Science, National Institutes for Quantum Science and Technology (QST), Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan
¹⁰Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
¹¹These authors contributed equally to this work

Abstract

Plants are exposed to a variety of environmental stress, and starvation of inorganic phosphorus can be a major constraint in crop production. In plants, in response to phosphate deficiency in soil, miR399, a type of microRNA (miRNA), is up-regulated. By detecting miR399, the early diagnosis of phosphorus deficiency stress in plants can be accomplished. However, general miRNA detection methods require complicated experimental manipulations. Therefore, simple and rapid miRNA detection methods are required for early plant nutritional diagnosis. For the simple detection of miR399, microfluidic technology is suitable for point-of-care applications because of its ability to detect target molecules in small amounts in a short time and with simple manipulation. In this study, we developed a microfluidic device to detect miRNAs from filtered plant extracts for the easy diagnosis of plant growth conditions. To fabricate the microfluidic device, verification of the amine-terminated glass as the basis of the device and the DNA probe immobilization method on the glass was conducted. In this device, the target miRNAs were detected by fluorescence of sandwich hybridization in a microfluidic channel. For plant stress diagnostics using a microfluidic device, we developed a protocol for miRNA detection by validating the sample preparation buffer, filtering, and signal amplification. Using this system, endogenous sly-miR399 in tomatoes, which is expressed in response to phosphorus deficiency, was detected before the appearance of stress symptoms. This early diagnosis system of plant growth conditions has a potential to improve food production and sustainability through cultivation management.