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Open AccessArticle

Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes

1
Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan
2
Department of Applied Chemistry, Faculty of Science and Engineering, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan
3
Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan
4
Immunotherapy Division, Shizuoka Cancer Center Research Institute, Shizuoka 411-8777, Japan
*
Authors to whom correspondence should be addressed.
Micromachines 2020, 11(7), 628; https://doi.org/10.3390/mi11070628
Received: 20 May 2020 / Revised: 23 June 2020 / Accepted: 26 June 2020 / Published: 27 June 2020
(This article belongs to the Special Issue Micro and Nano Devices for Cell Analysis)
Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0–20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells. View Full-Text
Keywords: single-cell analysis; peptide nucleic acid (PNA) probe; cell microarray; single nucleotide mutation; T790M mutation; lung cancer; epidermal growth factor receptor (EGFR) single-cell analysis; peptide nucleic acid (PNA) probe; cell microarray; single nucleotide mutation; T790M mutation; lung cancer; epidermal growth factor receptor (EGFR)
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Shigeto, H.; Yamada, E.; Kitamatsu, M.; Ohtsuki, T.; Iizuka, A.; Akiyama, Y.; Yamamura, S. Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes. Micromachines 2020, 11, 628.

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