动物毒素多肽享有“上帝药方”的美称，但产量极低、针对性筛选策略严重匮乏，挖掘率尚不足0.1%。如何低样耗、高通量地对微量毒素进行高效快速筛选是当前毒素资源开发中亟待解决的技术难题。前期研究发现At-line筛选模式可将分离分析-结构鉴定-活性评价一体化，微流控技术具有低样耗、高通量和高灵敏度等优点，为解决该难题提供了一种新思路。本项目拟通过液滴微流控技术将纳升液相及高密度微阵列芯片联接，实现动物毒素的超微量及高分辨分离分析和馏分收集；将基于微阵列芯片的抗血栓整体活性评价和质谱数据相结合，实现毒素多肽高内涵结构和活性鉴定，最终建立一个微型化At-line抗血栓毒素多肽高效筛选平台，应用于东亚钳蝎微量毒素中抗血栓多肽的筛选。该平台在显著降低样品消耗、提高分析通量的同时，有望实现高内涵检测，提高毒素多肽研发效率，为抗血栓毒素多肽的分离筛选提供强有力的工具，对推动动物毒素资源开发意义重大。

With potent activity, high selectivity and rich structural diversity, animal venoms, comprising complex mixtures of peptides and proteins, are considered as important resources for new antithrombotic drug development. However, only less than 0.1% of the animal venoms have been explored so far, which is attributed to the lack of suitable screening assays for very low amount of venoms. The conventional activity-guided fractionation assay (AGF), which involves bioactivity determination with subsequent identification and purification of the bioactive compounds, is high sample and time consumption, high cost and low screening efficiency. Therefore, it is of great interest to develop a low sample consumption, high resolution, high sensitivity and high-throughput screening (HTS) assay for the animal venoms study. This study aims at the development, optimization and application of a miniaturized at-line screening platform, which consists of nano-liquid chromatography (nano-LC) for the separation of crude venom components in combination with parallel post-column anticoagulation screening and high-resolution mass spectrometry (MS) for the identification of bioactive venom peptides. A robust droplet-based microfluidic device will be developed for nanoliter (nL) fractionation of nano-LC eluate stream onto a microarray at high frequency and sub-peak resolution. The nano-LC eluate stream is compartmentalized by immiscible perfluorinated oil into nL or pL droplets inside a micro T-junction. The post-column anticoagulation screening is a time-course spectrophotometric measurement based on high density microarray. It kinetically measures the clotting profile of bovine plasma incubated with Ca2+ and nL fractions. After a systematical optimization and evaluation, a low sample consumption, high resolution and rapid at-line platform will finally be developed for screening antithrombotic peptides in Buthus martensii venom. The miniaturized at-line screening platform based on droplet-based microfluidics has many advantages over conventional AGF, such as low sample consumption, low cost, high throughput etc. The animal venoms study based on this platform will be beneficial to significantly reduce the sample consumption and improve the screening efficiency. This research not only provides a powerful tool for screening novel antithrombotic peptides, but also shows great meaning in promoting the development of animal venoms.