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.
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 microm 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 beta-actin mRNA expression in individual Raji cells. Differences in the levels of beta-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. tmatsuna@cc.tuat.ac.jp
Tadashi
T
Matsunaga
Masahito
M
Hosokawa
Atsushi
A
Arakaki
Tomoyuki
T
Taguchi
Tetsushi
T
Mori
Tsuyoshi
T
Tanaka
Haruko
H
Takeyama
D000199:Actins / Q000235:genetics; D002051:Burkitt Lymphoma / Q000737:chemistry / Q000235:genetics / Q000473:pathology; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011093:Polyethylene Terephthalates; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics
D000199:Actins; D004129:Dimethylpolysiloxanes; D011093:Polyethylene Terephthalates; D012333:RNA, Messenger; D011092:Polyethylene Glycols; C013830:baysilon