Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device.
Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device.
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DOI：
10.1021/ac800143t
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发表时间：
2008-03
影响因子：
7.4
通讯作者：
T. Yasukawa;K. Nagamine;Yoshiko Horiguchi;H. Shiku;Masahiro Koide;T. Itayama;F. Shiraishi;T. Matsue
中科院分区：
文献类型：
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作者：
T. Yasukawa;K. Nagamine;Yoshiko Horiguchi;H. Shiku;Masahiro Koide;T. Itayama;F. Shiraishi;T. Matsue
为了进行基于单细胞的基因功能分析，开发了一种具有分析室阵列的新型微流控装置。使用该装置进行了一系列分析过程，包括单细胞的电泳操作和基因功能的电化学测量。制造具有微流体通道（150微米宽，10微米高）和分析室（100 × 20 × 10微米（3））的聚（二甲基硅氧烷）微结构，并在具有Au微电极阵列的玻璃基板上对准。两个微电极定位在分析室中被用作工作电极的电泳操作的细胞和电化学测量。携带β-半乳糖苷酶报告基因的酵母菌株（酿酒酵母Y190）用于证明该装置可以检测该酶。通过使用放置在主通道上的接地电极的电泳，将流过主通道的靶细胞引入腔室中。当细胞用17 β-雌二醇处理时，基因表达被触发产生β-半乳糖苷酶，催化对氨基苯基-β-D-吡喃半乳糖苷水解形成对氨基苯酚（PAP）。通过循环伏安法和安培法在装置的室中的单细胞水平检测酶促产生的PAP。还应用发生器-收集器模式电流分析法来放大源自被捕获的单细胞中的基因表达的电流响应。电化学测量后，捕获的细胞很容易释放的室使用电泳力。
A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.