Imaging of Cell-Array by Scanning Electrochemical Microscopy

在本项目中，我们研究了两个主题：1)活细胞的微图案化，2)单个细胞的活性测量。这些研究课题可以结合为“微图案化活细胞生物检测”，这将导致细胞芯片技术的发展。本课题取得的主要成果如下：1)细胞微图案的制备：利用PDMS制作的微残桩，采用微接触印刷法，在玻片上成功制备了粘附性动物细胞的微图案。使用纤维连接蛋白等细胞黏附蛋白作为墨水材料。成功地构建了HeLa细胞、鸡心肌细胞和PC12细胞在单个细胞水平上的细胞网络。2)微图案化细胞的活性评价：利用扫描电化学显微镜(SECM)系统对微图案化的HeLa细胞的呼吸活性进行成像。发现形状受限细胞的呼吸活性高于自由铺展细胞。3)构图细胞网络活性的评价：对微构图心肌细胞进行细胞内钙离子成像。微图案化的心肌细胞通过形成缝隙连接形成电性共轭网络。在芯片上实现了辛醇、河豚毒素和咖啡因对心脏组织模型的药理作用。通过应用微氟器件系统，还尝试了对图案化单元的局部剂量。

In the present project, we investigated two topics: 1) the micropatterning of living cells, 2) activity measurements of single cells. These research topics can be combined as the "bioassay using micropatterned living cells", which would lead to the development of cell-chip technology. The results obtained in this project can be summarized as follows,1) Preparation of cellular micropatterns: We succeeded to prepare micropatterns of adhesive animal cells on a glass slides by the micro-contact printing method using PDMS-made microstumps. The cellular adhesive protein such as fibronectin was used as the ink material. HeLa cells, chick cardiomyocytes and PC12 nuronal cells were successfully patterned to form cellular networks on a single cell level. The application of PEG molecules as the cell resistive modification works well so as to maintain the cell pattern for more than 5 days.2) Activity evaluation of the micropatterned cells: The respiratory activity of the micropatterned HeLa cells was imaged by using the scanning electrochemical microscopy (SECM) system. It was found that the respiration activity of the shape-restricted cells is higher than that of the freely spreading cells.3) Evaluation of the activity of the patterned cellular networks: The intracellular Ca2+ imaging was conducted for the micropatterned cardiac myocytes. The micropatterned cardiac myocytes were found to form the electrically conjugated network via forming the gap junctions. It was achieved to observed the pharmacological effects of octanol, tetrodotoxyn, and caffein for the cardiac tissue model on the chip. The local dosing to the patterned cells has also been attempted by applying the microfuluidic device systems.