体液样品快速平衡活体固相微萃取研究

The aim of the project is fast equilibrium in vivo sample preparation of biofluidics. There are three major problems existed in current in vivo solid phase microextraction methods, i.e. long extraction time, tedious quantization process, and limited recovery to hydrophilic compounds. Thus, fast mass transfer and chemically selective extraction are chosen as two key scientific challenges, and fast equilibrium in vivo solid phase microextraction is proposed as the optimal research strategy. This project will be carried out in three related aspects. (1) The dynamic in-tube SPME (DIT-SPME) will be designed to produce fast reciprocating chaotic flow of sample solution. The hydrophilic, thin and porous extraction phase will be electrospun to decrease the mass transfer resistance. Thus, the fast equilibrium SPME method and the corresponding in vitro standard calibration method will be established. (2) The composite functional extraction surface will be developed. Various extraction modes including ion exchange, hydrophilic interaction, chemical affinity, and size exclusion will be developed, and the extraction efficiency to hydrophilic compounds will be improved largely. Surface biocompatibility is also pursued to inhibit protein adsorption and cell adhension. Then, DIT-SPME can be applied in vivo. (3) With the acidic phytohormones like gibberellic acids, etc., in succulent tissues of tomato and aloes, endogenous nucleosides and peptides, and exogenous medicines and environment pollutants in biofluids of rabbits and rats as model compounds, in vivo DIT-SPME will be carried out. Combining with LC-MS/MS, the in vivo chemical information can be obtained with a temporal and/or spatial resolution, which is unprecedented by most traditional in vitro sample preparation methods. We believe that the research proposed in this project, and related studies about fast mass transfer, device design and fabrication, surface chemical modification, in vivo sample preparation will set a concrete foundation for in vivo analysis.

本项目以体液活体样品制备为目的，优选固相微萃取方法，针对萃取时间长、定量难度大、亲水性化合物回收率低三个重要问题，聚焦分子传质和化学选择性两个科学问题，发展快速平衡活体固相微萃取材料和方法，开展以下研究：（1）设计制备动态管内固相微萃取器件，强化样品相湍流传质，制备亲水、薄壁、大孔静电纺丝萃取相，降低萃取相传质阻力，实现快速平衡萃取和外标法准确定量；（2）发展功能化表面修饰方法，引进静电、亲水、尺寸排阻等作用，重点提高亲水化合物的萃取回收率，发展生物相容性修饰方法，抑制生物大分子和细胞粘附，推进活体萃取应用；（3）以模式植物（番茄、芦荟）多汁组织中赤霉素等酸性激素，模式动物（兔子、大鼠）体液中内源性核苷、多肽，以及外源性药物、环境污染物为目标分子，活体萃取样品制备，并结合色谱-质谱分离鉴定手段，获取活体化学分子的时空动态信息，发展活体样品制备方法，为活体分析积累重要科学技术基础。

本项目针对固相微萃取的主要问题：萃取时间长、定量难度大、亲水性化合物回收率低，发展超快速平衡固相微萃取方法，做出如下结果：（1）针对溶液中小分子有机物，发展烧结多孔钛快速过滤固相微萃取方法（ST-SPME），研制生物相容性、亲水、超分散纳米电纺丝云微萃取材料（NC-SPME），实现30秒内平衡微萃取，为生物体液样品快速制备提供了一种新思路。（2）针对微量植物样品中的痕量植物激素，发展了枪头固相微萃取方法（PT-SPE）、微分散固相萃取方法（μMSPD），结合高灵敏衍生、超高效分离，对5毫克植物样品中的5种油菜素甾醇，检出限达27-94 amol；对0.5毫克植物样品中的8种赤霉素，检出限达10.1-72.3 amol，对拟南芥单叶片赤霉素检测的空间分辨率达到2 mm × 2 mm。（3）针对尿液中的核苷，制备杂合TiO2-ZrO2纳米颗粒自组装修饰多孔二氧化硅微球（TiO2-ZrO2/SiO2），材料具有更强、更多的Lewis酸位点，用于生理条件下尿液中核苷的高选择性萃取，实现了100uL尿液中42种核苷的定量检出。（4）针对极性SPME萃取相大量吸附水的问题，发展了PDMS复合固相萃取材料，减少95%以上水的吸附残留，保护了后续色谱分离与检测系统，促进SPME-热解析-GC/MS快速联用，进一步推动固相微萃取进入环境监测实际应用。（5）另外，在多肽定量分析、色谱分离材料、纳米磁性萃取材料、VOCs超快速分析等方面做出了一些探索工作。本项目对固相微萃取材料、器件、定量方法的研究，提高了微萃取方法的选择性、稳定性和萃取速度，将推动微萃取分析方法的发展和实际应用。项目共发表SCI文章13篇，授权发明专利16项，申请发明专利6项，达到项目考核指标要求。