【作者】 王丹；

【导师】 李东浩；

【作者基本信息】 延边大学 ， 生物功能分子学-化学， 2022， 硕士

【摘要】 微纳尺度物质（纳米颗粒、蛋白质、外泌体、单细胞等）的分离与筛选是生物医药、材料科学、精准医学等领域的关键科学问题,为满足单细胞等微纳尺度物质的实际临床应用,亟需快速、高通量、连续、精准分离筛选平台。利用微纳尺度物质流动特性分离方法有惯性迁移分离、挤压流、水动力过滤及确定性位移分离等,虽然这些方法具有连续快速高通量优点,但由于微流体的难操控性使精准分离存在局限性。基于主动场辅助（如磁场、声场、光及电场辅助等）连续分离策略使其对微纳尺度物质的灵活操纵具有可行性。但这些连续分离的方法仍存在分离通道单一、目标物尺寸单一、分选效率及吞吐量低难富集的问题。综上在微流控芯片平台集成主动场的实时调控与流动场快速高通量特色可能是一种有效方案。因此基于上述技术缺陷及需求,迫切需要开发一种主动场辅助多通道且适用于多范围尺度目标物的连续分离与富集方法。本文提出了一种集聚焦、分离、富集的电场辅助流动场的连续分离方法,即正交芯片电泳分离方法。该方法使在流体驱动下的目标物在体系中被正交方向的流动相层流挤压实现线性聚焦,并在偏转电场驱动下迁移至分离通道,从而实现分离与富集。具体研究内容和成果为:（1）建立正交电泳分离体系分离理论。研究体系的分离及聚焦理论,基于三项关系探究分离体系中外加电场、流体流速及目标物质荷比等性质对偏转及分离行为影响,并通过理论模拟验证了电场、流动场分布及连续分离富集微纳尺度物质的可行性。（2）搭建正交电泳分离体系。该体系包括进样系统、分离系统、供电系统及监测系统。设计并制备具有不同功能性及多维度正交的微流控芯片电泳体系。其中分离系统为本技术的核心部分,设计利用流动场实现微纳尺度物质的鞘液聚焦进样装置模块,并通过探究电极位置及装置开闭性解决传统电泳技术中存在的气泡及大气压等对后续分离的干扰。（3）探究小分子与颗粒物在正交芯片电泳分离体系下聚焦、偏转及分离行为。通过流动场的可控操纵确定目标物的鞘液聚焦最佳流速比,通过调控电场参数确定目标物的偏转角度与电场强度及电迁移率的线性关系tanθ∝μ_e及tanθ∝E。探究正交电泳体系对相同尺寸5μm蓝色与绿色聚苯乙烯颗粒物分离性能、回收率、吞吐量,其在E=25-35 Vcm^-1下5μm蓝色与绿色聚苯乙烯颗粒物具有良好分离性能,回收率最高为85%,吞吐量为0.5 m L min^-1;正交电泳体系对不同尺寸3μm及5μm聚苯乙烯颗粒物在E=25-30 Vcm^-1具有良好分离性能,吞吐量为0.5 m L min^-1。（4）蛋白质等微纳尺度物质的在线分离。基于正交电泳体系尝试对蛋白质分离富集,并成功实现蛋白质HSA酶与FITC染料的在线、连续分离,为后续复合蛋白等的连续分离筛选提供新方法新平台。本论文提出的正交电泳分离体系有望实现不同微纳尺度物质的在线、连续、高通量分离富集。基于前期创新基础,可建立多维度正交芯片电泳体系,利用多电场调控颗粒物的定向迁移,对目标物实现在线分离与富集具有极大的可行性,并在后续细胞、外泌体等富集,细胞质谱等研发具有创造性的应用前景。更多还原

【Abstract】 The separation and screening of micro-objects（nanoparticles,proteins,exosomes,single cells,etc.）is a key scientific issue in the fields of biomedicine,materials science,and precision medicine.For applications,a rapid,high-throughput,continuous,and precise separation and screening platform is urgently needed.Separation methods using the characteristics of material flow at the micro-nano scale include inertial migration separation,squeeze flow,hydrodynamic filtration,and deterministic displacement separation.Although these methods have the advantages of continuous,rapid and high-throughput,the manipulation and separation of microfluidics make them highly effective.high challenge.The continuous separation strategy based on active field assistance（such as magnetic field,acoustic field,light and electric field assistance,etc.）makes it feasible for flexible manipulation of matter at the micro-nano scale.However,these continuous separation methods still have the problems of single separation channel,single target size,low separation efficiency and low throughput and difficulty in enrichment.In conclusion,it may be an effective solution to integrate the real-time regulation of the active field and the fast and high-throughput characteristics of the flow field on the microfluidic chip platform.Therefore,based on the above technical defects and requirements,it is urgent to develop an active field-assisted multi-channel continuous separation and enrichment method suitable for multi-scale targets.In this paper,a continuous separation method of electric field-assisted flow field integrating focusing,separation and enrichment,namely orthogonal chip electrophoresis separation method,is proposed.In this method,the target substance driven by the fluid is squeezed by the laminar flow of the mobile phase in the orthogonal direction in the system to achieve linear focusing,and is driven by the deflection electric field to migrate to the separation channel,thereby realizing separation and enrichment.The specific research contents and results are as follows:（1）Establish the separation theory of orthogonal electrophoresis separation system.The separation and focusing theory of the system was studied,and the effects of the properties of the applied electric field,fluid flow rate,and target material charge ratio on the deflection and separation behavior in the separation system were explored based on three relationships.Feasibility of collecting micro-and nano-scale substances.（2）Set up an orthogonal electrophoresis separation system.The system includes sampling system,separation system,power supply system and monitoring system.A microfluidic chip electrophoresis system with different functionalities and multi-dimensional orthogonality was designed and prepared.Among them,the separation system is the core part of the technology.The sheath liquid focusing injection device module that uses the flow field to realize micro-nano-scale substances is designed,and the problems of air bubbles and atmospheric pressure existing in traditional electrophoresis technology are solved by exploring the electrode position and device opening and closing.Interference from subsequent separations.（3）To explore the focusing,deflection and separation behavior of small molecules and particles in the orthogonal chip electrophoresis separation system.Through the controllable manipulation of the flow field,the optimal flow rate ratio of the sheath liquid focusing of the target object is determined,and the linear relationship tanθ∝μ_eand tanθ∝E between the deflection angle of the target object,the electric field strength and the electric mobility are determined by adjusting the electric field parameters.To explore the separation performance,recovery and throughput of 5μm blue and green polystyrene particles of the same size by the orthogonal electrophoresis system,it has good performance for 5μm blue and green polystyrene particles at E=25-35 Vcm^-1Separation performance,the highest recovery rate is 85%,and the throughput is 0.5 m L min^-1;the orthogonal electrophoresis system has good separation performance for 3μm and 5μm polystyrene particles of different sizes at E=25-30 Vcm^-1,and the throughput The amount is 0.5 m Lmin^-1.（4）Online separation of micro-and nano-scale substances such as proteins.Based on the orthogonal electrophoresis system,we tried to separate and enrich proteins,and successfully realized the online and continuous separation of protein HSA enzyme and FITC,which provided a new method and new platform for the subsequent continuous separation and screening of complex proteins.The orthogonal electrophoresis separation system proposed in this paper is expected to realize online,continuous,high-throughput separation and enrichment of different micro-and nano-scale substances.Based on the previous innovation foundation,a multi-dimensional orthogonal chip electrophoresis system can be established,and the directional migration of particles can be regulated by multi-electric fields,which has great feasibility for online separation and enrichment of targets.The research and development of cell mass spectrometry,etc.has creative application prospects.更多还原