基于液滴技术的烟草细胞免疫应激信号的检测

Detection of Immunological Stress Signal for BY-2 Cell Based on Droplet Microfluidic

基于液滴微流控芯片技术,用壳寡糖(chitosan oligosaccharide,COS)处理悬浮培养的烟草细胞(BY-2cell),对烟草细胞产生的免疫应激信号分子(H_2O_2和Ca^(2+))进行荧光检测。用50μg/mL的壳寡糖水溶液分别刺激装载过氧化氢和钙离子荧光探针的烟草细胞,在荧光显微镜下观察到烟草细胞产生的荧光;以液滴微流控芯片为实验平台,研究发现水相流速为100μL/h,油相流速为300μL/h时,液滴尺寸为300μm,适合烟草细胞的包裹,在该条件下将COS与荧光探针孵育后的细胞包裹在液滴中,在荧光显微镜下观察到液滴中的荧光;后用荧光酶标仪检测96孔板和液滴承载的细胞的荧光强度,结果显示1h内两者的变化趋势一致。试验为液滴微流控芯片在植物免疫诱导剂和生物农药方面的研究提供了参考。

Based on droplet microfluidics technology,we detected immunological stress singling mole cules （H2O2 and Ca2＋） secreted by BY-2 cells under simulation of chitosan oligosaccharide （COS） in the paper.BY-2 cells labeled with H2DCF-DA and Fluo-3AM were stimulated by 50 μg/mL COS solution,and fluorescence was detected.We optimized our parameters for this droplet microfluidics platform and could achieve ideal encapsulation of BY-2 cells at an aqueous phase flow rate of 100 μL/h,an oil phase flow rate of 300 μL/h,and a droplet diameter of 300 μm.Under the optimized conditions,COS and fluorescence-labeled BY-2 cells encapsulated in droplets and fluorescence was observed.Then the fluorescence intensity of droplet-encapsulated BY-2 cells were measured by microplate reader and the unencapsulated cells in 96-well plate were taken as the control group.The results indicated that within 1 h,the trend of the intensity changed similarly,and this could provide the guidance for microfluidics-based research in plant immune inducer and biopesticides.