基于液滴微流控的壳寡糖植物免疫诱导剂荧光检测  

Fluorescence detection of chitosan oligosaccharides plant immune elicitors based on droplets-microfluidics

作　　者：苍小鑫 赵小明[2] 钟润涛[3] 王文霞[2] 尹恒[2] 朱靖博[1] 丁燕[1] Cang Xiaoxin Zhao Xiaoming Zhong Runtao Wang Wenxia Yin Heng Zhu Jingbo Ding Yan(School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China Dalian Institute of Chemical Physics, Chinese Academic of Sciences, Dalian 116023, China School of Environmental Science and Engineering, Dalian Maritime University, Dalian 116026, China)

机构地区：[1]大连工业大学食品学院,大连116034 [2]中国科学院大连化学物理研究所,大连116023 [3]大连海事大学环境科学与工程学院,大连116026

出　　处：《农业工程学报》2017年第2期302-307,共6页Transactions of the Chinese Society of Agricultural Engineering

基　　金：国家自然科学基金(31370391);中科院STS计划(KFJ-SW-STS-143);大连市人才项目(2016RQ064);辽宁省教育厅项目(2016J008)

摘　　要：为了建立基于液滴微流控芯片平台的植物免疫诱导剂的荧光信号检测技术,该文设计制作了具有液滴生成结构的芯片,制备包裹烟草(BY-2)细胞的液滴,并对包裹了细胞的液滴数目进行统计分析。将包裹细胞的液滴孵育一氧化氮探针后使用质量浓度为50μg/m L壳寡糖处理,在荧光显微镜下观测其产生的荧光,利用荧光酶标仪对比采用96孔板和液滴承载的细胞的荧光变化趋势。结果显示,水相流速为100μL/h,油相流速为300μL/h时,微流控芯片产生的液滴尺寸适合包裹细胞,其中液滴包裹单细胞的比例达22.9%。经壳寡糖处理后的细胞在生成的液滴中产生了明显的荧光,且用96孔板和液滴承载的细胞的荧光变化趋势一致。研究结果为开发基于液滴微流控技术的植物免疫诱导剂的高通量筛选平台提供参考。The plant immune elicitors can induce a line of defense responses in plants and are identifed as new type of biological pesticides. At present stage, new plant immune elicitor is screening with potted plants or field experiments, which is time-consuming and high-cost. The droplet microfluidic technology, which is originated from analytical chemistry and owns micro-channel network structure, has properties of high throughput, high sensitivity, low consumption of reagent, no cross contamination and rapid reaction. These advantages provide a novel platform for screening plant immune elicitors. Chitosan oligosaccharides（COS） are obtained by degradation of chitosan. It was reported that COS could activate plant innate immunity, such as： stimulate H2O_2（hydrogen peroxide） production, induce defense response by NO（nitric oxide） pathway, make a synthesis of phytoalexin, impact the jasmonic acid / ethylene（JA/ET） signaling marker, trigger defense-related gene expression, cause changes in protein phosphorylation, activate mitogen-activated protein kinases（MAPKs）, and possess antimicrobial activity against bacteria and fungi in plant. Because of the advantages and the high solubility, nontoxicity, and biocompatibility, COS are considered as an effective plant immune elicitor by researchers. To preliminarily applying droplet microfluidic technology in plant immune elicitor screening, integrated microfluidic chip with droplets formation structure was designed and fabricated. COS were chosen as positive reagent, and BY-2 tobacco cells played as model plant cell. The flow rate of mobile phase was measured and established for BY-2 tobacco cells droplets formation, and the single cell encapsulating efficiency was calculated. Then COS and NO probe were dumped into the droplets with BY-2 tobacco cells, and the fluorescence intensity of NO probe from droplets was detected to evaluate the feasibility of screening the plant immune elicitor COS. To make the comparative analysis, the fluorescence intensity was comp

关 键 词：微流控 包裹 荧光 植物免疫诱导剂 烟草细胞 壳寡糖 

分 类 号：S4[农业科学—植物保护]