COP-PDMS微流控芯片的制备及在太赫兹对肠道上皮细胞生物效应中的应用

Fabrication of cyclic olefin polymer and polydimethylsiloxane co-bonded microfluidic device and its appliactions in terahertz biological effects on intestinal cells

用于细胞培养的常规培养皿无法直接用于激光共聚焦显微镜的观察,在很大程度上限制了细胞水平上的太赫兹生物效应研究的开展.本文采用了对太赫兹吸收率低的材料——环烯烃聚合物(COP),与聚二甲基硅氧烷(PDMS)进行共键合,借用软蚀刻、光刻、等离子清洗和高温高压孵育技术将两种材料整合成新型微流控芯片,并利用该微流控芯片探索太赫兹对肠道细胞的生物效应.该微流控芯片可实现肠上皮细胞Caco-2的动态培养,原位免疫荧光染色及激光共聚焦显微镜的直接观察,为太赫兹细胞水平的生物效应研究提供了高效便捷的实验环境.研究中将微流控芯片中培养的肠上皮细胞Caco-2经15 mW/cm^(2)的0.1 THz波辐照10 min,免疫荧光染色结果显示细胞贴附生长的形态改变,胞间紧密连接蛋白ZO-1及桩蛋白Paxillin的水平及分布发生变化,提示太赫兹对细胞的胞外连接及胞间连接均可产生影响,可减弱细胞与培养界面的黏附.本研究设计制备的COP-PDMS共键合微流控芯片为探索太赫兹对细胞的生物效应提供了便捷有效的平台,有望在未来进一步用于太赫兹对细胞分子效应的实时研究.

The current research on the terahertz biological effects at a cellular level is limited by the conventional petri dishes used for cell culture,which cannot be directly used for confocal laser microscopy.In this research,the cycloolefin polymer(COP),a material that possesses low terahertz absorption rate but excellent optical property for microscopy,is bonded to polydimethylsiloxane(PDMS),thereby developing a novel COP-PDMS microfluidic device by using the techniques of soft etching,photolithography,plasma cleaning,high-temperature and high-pressure incubation.The bonding strength of resulting device is tested by using a push-tension meter.The results indicate that the developed device shows a bonding strength as strong as the device fabricated by quartz and PDMS,which is thought as the tightest binding in the multiple types of microfluidic device.In addition,by perfusing the device chamber at a high flow rate(200μL/s)and long-term time-course(2 weeks),which simulates the dynamic shearing stress occurring in in-vivo organs and tissues,this COP-PDMS microfluidic device can still maintain the original shape and sealing property,indicating that this device qualifies the requirements of the following dynamic cell culture.The biological effects of terahertz on the cells are explored by using this COP-PDMS microfluidic device mentioned above.In this device,we develop the dynamic culture of intestinal epithelial cells Caco-2 with a perfusion rate of 0.05μL/s,which meets the findings of the in-vivo gastrointestinal lumen shearing stress.The Caco-2 cells are then irradiated with 0.1 THz wave with the power of 15 mW/cm^(2) for 3 days,and the irradiation duration is 10 min per day.The biological effects of terahertz irradiation on the intercellular tight junction protein ZO-1,the Paxillin relating to the cell adhesion and migration,and the cytoskeletal microfilament protein F-actin of Caco-2 cells are detected in the device directly using the technique of immunofluorescence staining.The results show that the morphology of cell adhesion as well as the level and distribution of ZO-1 and Paxillin are changed.In brief,the protein expression of ZO-1 and Paxillin are induced more by the terahertz irradiation,while the F-actin is not influenced by the irradiation.As can be seen from the F-actin results,the cells without terahertz irradiation show a spread and outward shape with regular smooth cell edge while a contraction and burr shape of cell edge are shown after irradiation,suggesting that the cell adhesion is weakened after irradiation.Even though the expression level of F-actin is consistent,the changed morphology indicates that terahertz may regulate the interaction and aggregation among actin proteins in cells.Interestingly,the ZO-1 presents diffuse distribution in the cells and its location on the cell membrane is not obvious,that is,a large amount of ZO-1 expresses not only on the cell membrane but also in the intracellular matrix after the irradiation.The expression of Paxillin is enhanced after terahertz irradiation,and some cells show local aggregation and distribution of Paxillin.These indicate that the terahertz irradiation might affect the biomolecular mechanism of synthesis and distribution of protein.The COP-PDMS co-bonded microfluidic device developed in this study provides a convenient and effective platform for exploring the biological effects of terahertz irradiation on cells,and is expected to be further used for real-time research on the effects of terahertz on cells and molecules in the future.