Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices

Tatsuro Nakao, Yutaka Kazoe, Emi Mori, Kyojiro Morikawa, Takemichi Fukasawa, Ayumi Yoshizaki, Takehiko Kitamori

Research output: Contribution to journalArticle

Abstract

Analysis of proteins released from living single cells is strongly required in the fields of biology and medicine to elucidate the mechanism of gene expression, cell-cell communication and cytopathology. However, as living single-cell analysis involves fL sample volumes with ultra-small amounts of analyte, comprehensive integration of entire chemical processing for single cells and proteins into spaces smaller than single cells (pL) would be indispensable to prevent dispersion-associated analyte loss. In this study, we proposed and developed a living single-cell protein analysis device based on micro/nanofluidics and demonstrated analysis of cytokines released from living single B cells by enzyme-linked immunosorbent assay. Based on our integration method and technologies including top-down nanofabrication, surface modifications and pressure-driven flow control, we designed and prepared the device where pL-microfluidic- and fL-nanofluidic channels are hierarchically allocated for cellular and molecular processing, respectively, and succeeded in micro/nanofluidic control for manipulating single cells and molecules. 13-unit operations for pL-cellular processing including single-cell trapping and stimulation and fL-molecular processing including fL-volumetry, antigen-antibody reactions and detection were entirely integrated into a microchip. The results suggest analytical performances for countable interleukin (IL)-6 molecules at the limit of detection of 5.27 molecules and that stimulated single B cells secrete 3.41 IL-6 molecules per min. The device is a novel tool for single-cell targeted proteomics, and the methodology of device integration is applicable to other single-cell analyses such as single-cell shotgun proteomics. This study thus provides a general approach and technical breakthroughs that will facilitate further advances in micro/nanofluidics, single-cell life science research, and other fields.

Original languageEnglish
Pages (from-to)7200-7208
Number of pages9
JournalAnalyst
Volume144
Issue number24
DOIs
Publication statusPublished - 2019 Dec 21
Externally publishedYes

Fingerprint

Nanofluidics
Cytokines
Equipment and Supplies
Molecules
proteomics
Single-Cell Analysis
Processing
Proteins
protein
Interleukin-6
Cells
Antigen-antibody reactions
life science
Immunosorbents
Pressure control
flow control
Lab-On-A-Chip Devices
antigen
Nanotechnology
Flow control

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy
  • Electrochemistry

Cite this

Nakao, T., Kazoe, Y., Mori, E., Morikawa, K., Fukasawa, T., Yoshizaki, A., & Kitamori, T. (2019). Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices. Analyst, 144(24), 7200-7208. https://doi.org/10.1039/c9an01702j

Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices. / Nakao, Tatsuro; Kazoe, Yutaka; Mori, Emi; Morikawa, Kyojiro; Fukasawa, Takemichi; Yoshizaki, Ayumi; Kitamori, Takehiko.

In: Analyst, Vol. 144, No. 24, 21.12.2019, p. 7200-7208.

Research output: Contribution to journalArticle

Nakao, T, Kazoe, Y, Mori, E, Morikawa, K, Fukasawa, T, Yoshizaki, A & Kitamori, T 2019, 'Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices', Analyst, vol. 144, no. 24, pp. 7200-7208. https://doi.org/10.1039/c9an01702j
Nakao, Tatsuro ; Kazoe, Yutaka ; Mori, Emi ; Morikawa, Kyojiro ; Fukasawa, Takemichi ; Yoshizaki, Ayumi ; Kitamori, Takehiko. / Cytokine analysis on a countable number of molecules from living single cells on nanofluidic devices. In: Analyst. 2019 ; Vol. 144, No. 24. pp. 7200-7208.
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