基于微流控芯片液滴技术的糖链植物免疫诱导剂高通量筛选平台研制

The researches of plant glycobiology and plant immunology are a new scientific discipline and have played an increasingly important role in the international biology research realm. Since carbohydrate-based plant revulsant significantly improve plant immunity against plant diseases, it attracts attention in the glycobiology community and the advances in its research will also make great contributions in the safety of food production. At present, however, scarcity of plant revulsants used in agricultural production is not able to make them fulfill the actual demand. The main reasons behind such a restriction are the shortage of a feasible screening platform(s) to obtain new revulsants, and unclarity of the structure-activity relationship of carbohydrate-based plant revulsant. The droplet technique based on microfluidics have demonstrated advantages of flexibility, high throughput and speed, good heat and mass transfer performance, all of which are ideal for obtaining new carbohydrate-based plant revulsants and studying on their structures. And these specific aspects of microfluidics has not been reported yet. The team in which the applicantswork holds a rich accumulation in the research of microfluidics and has built an oligosaccharide bank with as many as 1000 samples. This project aims to build microfluidics-based screening platforms, on which droplets in nano- to pico-litre can be formed, mixed, reacted and detected. Each units of these functions can be combined and integrated in a flexible way. By applying oligosaccharides to a plant cell in a micro droplet and investigating the production of signaling molecules such as Ca2+ and NO, we are able to study the effects of different kinds or concentrations of carbohydrate-based plant revulsants . A study of plant revulsants as above in parallel can be achieved, thus high-throughput screening can be made. This platform also allows large sample size and rapid analysis in the structure-activity relationship of the carbohydrate-based revulsants study.

植物免疫及植物糖生物学是新兴学科和国际热点研究领域，糖链植物免疫诱导剂能显著提高植物免疫力, 是近期植物保护领域的研究热点, 也将对食品安全作出重要贡献。目前被用于农业生产的植物免疫诱导剂种类很少，难以满足实际需求。制约瓶颈主要是筛选平台缺乏。基于微流控芯片的液滴技术操控灵活，通量大，速度快，又有优良的传热传质性能，应当是植物免疫诱导剂规模筛选的理想平台，而此相关工作却未见报道。 申请者及其所在课题组已建立数量多达1000余种的寡糖库，又在微流控芯片领域有丰富积累，本申请拟自行搭建基于微流控芯片液滴技术筛选平台，实现液滴生成、融合、反应、检测等功能单元的灵活组合和规模集成，通过不同寡糖和同类植物细胞在单个液滴中的共存，检测并比较不同种类、不同浓度寡糖作为诱导剂激发植物细胞产生信号分子NO和Ca2+的能力，实现寡糖类植物免疫诱导剂的高通量筛选。

本项目以在农业生产中发挥重要作用的糖链植物免疫诱导剂为材料，利用液滴微流控芯片技术，开展“基于微流控芯片液滴技术的糖链植物免疫诱导剂高通量筛选平台研制”。在项目研究过程中，针对核心研究内容，研究组搭建了微流控芯片制作、液滴控制及检测平台, 建立和突破了相关实验技术，如液滴包裹植物细胞技术、萘酰亚胺型NO荧光探针合成技术、NO、H2O2和Ca2+荧光探针在包裹植物细胞液滴的装载技术等。本项目获得的成果如下：合成一个溶酶体定位的荧光探针，以萘酰亚胺为荧光团，吗啉作为溶酶体定位基团，以邻苯二胺作为NO的识别功能团，实现了在细胞的溶酶体内识别NO的功能；设计制作出了适合烟草BY-2细胞液滴芯片；确定了包裹BY-2细胞液滴的大小及制备条件；确定了烟草植物细胞在液滴中的包裹率符合泊松分布；确定了烟草BY-2细胞装载NO、H2O2和Ca2+荧光探针的条件；研究出了装载Ca2+荧光探针的烟草细胞液滴的制备技术；测定了微液滴中BY-2细胞与孔板中BY-2细胞经壳寡糖诱导后荧光检测一致性；搭建液滴微流控芯片平台；应用微流控芯片初筛了植物免疫诱导剂，经过验证是可行的。