Electrophoretic cell manipulation and electrochemical gene-function analysis based on a yeast two-hybrid system in a microfluidic device.
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.
Yasukawa
Tomoyuki
T
Graduate School of Environmental Studies, Tohoku University; 6-6-11-604, Aramaki-Aoba, Aoba, Sendai 980-8579 Japan. yasu@sci.u-hyogo.ac.jp
Nagamine
Kuniaki
K
Horiguchi
Yoshiko
Y
Shiku
Hitoshi
H
Koide
Masahiro
M
Itayama
Tomoaki
T
Shiraishi
Fujio
F
Matsue
Tomokazu
T
D004563:Electrochemistry / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D005800:Genes, Fungal; D044085:Microfluidics / Q000295:instrumentation*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020798:Two-Hybrid System Techniques