Electrochemical single-cell gene-expression assay combining dielectrophoretic manipulation with secreted alkaline phosphatase reporter system.
Scanning electrochemical microscopy (SECM) was used for the analysis of single-cell gene-expression signals on the basis of a reporter system. We microfabricated a single-cell array on an Indium tin oxide (ITO) electrode comprising 4 x 4 SU-8 microwells with a diameter of 30microm and a depth of 25microm. HeLa cells transfected with plasmid vectors encoding the secreted alkaline phosphatase (SEAP) were seeded in the microwell at a concentration of 1 cell per well by positive-dielectrophoresis (pDEP). A pDEP pulse of 3.0Vpp and 1MHz was applied between the microwell array/ITO electrode and an ITO counter electrode located on the top of the flow-cell assembly of the microdevice. The electrochemical responses of the individual HeLa cells transfected with SEAP were significantly larger than those of the wild-type HeLa cells. The electrochemical response of the transfected single cells was statistically distinguishable from that of wild-type HeLa cells. The size of the wells and the material of the single-cell array were optimized in order to evaluate the tumor necrosis factor alpha (TNF-alpha)-induced activation process of nuclear factor kappa B (NFkappaB) that was used as the model for on-chip monitoring of cellular signal transduction.
Murata
Tatsuya
T
Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan.
Yasukawa
Tomoyuki
T
Shiku
Hitoshi
H
Matsue
Tomokazu
T
D000469:Alkaline Phosphatase / Q000235:genetics*; D001681:Biological Assay / Q000379:methods*; D004563:Electrochemistry / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism*
D009363:Neoplasm Proteins; D000469:Alkaline Phosphatase