title: Electrochemical monitoring of cellular signal transduction with a secreted alkaline phosphatase reporter system.
abstract: Electrochemical monitoring of cellular signal transduction under three-dimensional (3-D) cell culture conditions has been demonstrated by combining cell-based microarrays with a secreted alkaline phosphatase (SEAP) reporter system. The cells were genetically engineered to produce SEAP under the control of nuclear factor kappaB (NFkappaB) enhancer elements, and they were embedded with a small volume of a collagen gel matrix on a pyramidal-shaped silicon microstructure. Cellular SEAP expression triggered by NFkappaB activation was assessed by two types of electrochemical systems. First, SEAP expression of a 3-D cell array on a chip was continuously monitored in situ for 2 days by scanning electrochemical microscopy (SECM). Since the SECM-based assay enables the evaluation of cellular respiratory activity, simultaneous measurements of cellular viability and signal transduction were possible. Further, we have developed an electrode-integrated cell culture device for parallel evaluation of cellular SEAP expression. The detector electrode was integrated around the silicon microhole. Two kinds of cells were immobilized on the array of microholes on the same chip for comparative characterization of their SEAP activity. This electrochemical microdevice can be applied to evaluate the SEAP expression activity in multiple cellular microarrays by a high-throughput method.
authors.affiliation: Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan.
authors.forename: Yu-Suke
authors.initials: YS
authors.lastname: Torisawa
authors.forename: Noriko
authors.initials: N
authors.lastname: Ohara
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Nagamine
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kasai
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Yasukawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shiku
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Matsue
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D001943:Breast Neoplasms / Q000473:pathology; D002450:Cell Communication / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D004563:Electrochemistry; D004566:Electrodes; D005260:Female; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D008853:Microscopy; D016328:NF-kappa B / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D012825:Silicon / Q000737:chemistry; D013997:Time Factors
chemical_list: D016328:NF-kappa B; D000469:Alkaline Phosphatase; D012825:Silicon