Electrochemical monitoring of cellular signal transduction with a secreted alkaline phosphatase reporter system.
Electrochemical monitoring of cellular signal transduction with a secreted alkaline phosphatase reporter system.
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DOI：
10.1021/ac060737s
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发表时间：
2006-10
影响因子：
7.4
通讯作者：
Y. Torisawa;N. Ohara;K. Nagamine;S. Kasai;T. Yasukawa;H. Shiku;T. Matsue
中科院分区：
文献类型：
--
作者：
Y. Torisawa;N. Ohara;K. Nagamine;S. Kasai;T. Yasukawa;H. Shiku;T. Matsue
电化学监测细胞信号转导的三维（3-D）细胞培养条件下已被证明是通过结合基于细胞的微阵列与分泌型碱性磷酸酶（SEAP）报告系统。这些细胞经过基因工程改造，在核因子kappaB（NF kappaB）增强子元件的控制下产生SEAP，并将它们用小体积的胶原凝胶基质包埋在圆形硅微结构上。通过两种类型的电化学系统评估由NF κ B活化触发的细胞SEAP表达。首先，通过扫描电化学显微镜（SECM）连续原位监测芯片上的3-D细胞阵列的SEAP表达2天。由于基于SECM的测定能够评价细胞呼吸活性，因此可以同时测量细胞活力和信号转导。此外，我们已经开发了一种电极集成的细胞培养装置，用于并行评估细胞SEAP表达。检测器电极集成在硅微孔周围。将两种细胞固定在同一芯片上的微孔阵列上，以比较它们的SEAP活性。该电化学微器件可用于高通量检测多种细胞芯片中SEAP的表达活性。
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.