High-efficiency single-cell entrapment and fluorescence in situ hybridization analysis using a poly(dimethylsiloxane) microfluidic device integrated with a black poly(ethylene terephthalate) micromesh

Tadashi Matsunaga, Masahito Hosokawa, Atsushi Arakaki, Tomoyuki Taguchi, Tetsushi Mori, Tsuyoshi Tanaka, Haruko Takeyama

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 μm in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. This microfluidic device has been employed for the detection of β-actin mRNA expression in individual Raji cells. Differences in the levels of β-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.

Original languageEnglish
Pages (from-to)5139-5145
Number of pages7
JournalAnalytical chemistry
Volume80
Issue number13
DOIs
Publication statusPublished - 2008 Jul 1
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

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