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
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
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DOI：
10.1021/ac800352j
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发表时间：
2008-07-01
影响因子：
7.4
通讯作者：
Takeyama, Haruko
中科院分区：
文献类型：
--
作者：
Matsunaga, Tadashi;Hosokawa, Masahito;Takeyama, Haruko
在这里，我们报告了一个高效率的单细胞捕获系统与聚（二甲基硅氧烷）（PDMS）微流控装置集成与微网，其应用于单细胞荧光原位杂交（FISH）分析。通过激光烧蚀在黑色聚（对苯二甲酸乙二醇酯）（PET）基底上制造包含10 × 10 15微腔的微网。空腔直径约为2 μ m。通过施加负压将哺乳动物细胞驱动并捕获到微腔上。捕获的细胞均匀地排列在微筛上，使得能够进行高通量显微镜分析。此外，我们开发了一种方法，PDMS表面改性，通过使用空气等离子体和共聚物普朗尼克F-127，以防止非特异性吸附的PDMS微通道。这种方法将细胞在微通道上的非特异性吸附降低到小于1%。当将细胞引入到与黑色PET微筛整合的微流体装置中时，大约70-80%的引入细胞被成功捕获。此外，对于mRNA表达分析，芯片上荧光原位杂交（例如，膜透化、杂交、洗涤）可以在集成装置上的微流体测定中进行。Ibis微流控装置已用于检测单个Raji细胞中β-肌动蛋白mRNA的表达。在血清供应或血清饥饿的细胞群中观察到β-肌动蛋白mRNA表达水平的差异。
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 mu m 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. Ibis 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.