基于电穿孔与机械挤压协同作用的微流控高通量细胞转染方法的研究

Transfection is an essential tool in both fundamental biological research and advanced medical applications such as gene therapy and genetic engineering for drug discovery. Recent advances in microfluidic transfection techniques are advantageous over conventional bench-top transfection methods regarding the benefits of miniaturization, low cost, easy handling and high precision. Yet these state-of-art microfluidic transfection platforms could suffer from issues inherently exhibited by a certain type of mechanism they exploit, for example, the low cell viability in electroporation or low throughput in microinjection. To tackle the issues posed by using a single transfection mechanism as well as to develop high performance transfection tools for the increasing demand from biomedical research and applications, we propose here a novel microfluidic transfection platform using electro-mechano-poration that enables simultaneous electroporation and mechanical squeezing of biological cells at high throughput. We will demonstrate a dual-functional constriction-microelectrode design based on the “electro-fluidic integration” concept, in which the electrode serves for electric field generation on one hand and squeezes cells with its readily built-in constrictions on the other hand. Microelectrodes with arrayed constrictions lead to highly parallel flow streams that facilitate high throughput cell poration. Meanwhile we will develop a straightforward fabrication process based on single mask for the proposed dual-functional design. To implement the function of the platform, we propose to separately demonstrate electroporation using 3D silicon microelectrodes for the first time and mechnoporation by squeezing cells through a novel ring-shape constriction. Then we propose to investigate the modulation of the combined electro-mechano-poration and therefore the entire system could benefit from the advantages while rejecting the issues of each individual mechanism that contributes to this collaborating effect. As a result, the platform is expected to exhibit excellent performance with considerably improved transfection efficiency and cell viability.

细胞转染技术是生命科学研究和基因治疗、基因工程制药等前沿医药领域的重要工具。近年来微流控技术与细胞转染技术的结合催生了一系列先进的转染方法，与传统的转染方法相比具有小型化、低成本、易操作和高精度等诸多优点。目前采用单一转染机制进行细胞转染的微流控器件仍存在一些缺陷，如电穿孔低细胞存活率、显微注射方法低通量等问题。针对单一转染技术的不足以及生物医药领域的高速发展对高性能转染工具的需求，申请者拟基于已有的工作积累，研究一种新型的利用电穿孔与机械挤压穿孔协同作用的高通量微流控细胞转染方法。拟设计的微结构基于“电极流道一体化”概念，在实现电穿孔和细胞挤压协同工作的同时简化了加工工艺，而阵列化设计则有利于提高通量。在研究中我们将首次探索立体硅电极的电穿孔作用和新型环状微孔结构对细胞的挤压穿孔作用，继而调控二者的协同作用来进行优势互补和避免单一机制的不足，实现细胞转染效率和存活率等关键性能参数的提升。

细胞转染是将外来核酸引入细胞以获得新的表型的过程，在基因治疗、药物筛选、疫苗开发等领域都有重要应用。电穿孔法具有毒性小，成本低，效率高等特点，但容易对细胞造成不可逆的损伤。机械穿孔法不依赖于电场，适用于广泛的细胞类型和转染材料，且细胞死亡率低，但转染效率较低。近年来，细胞转染技术逐渐向微型化技术发展，微流控电穿孔芯片能够很好地隔离单个细胞、聚焦电场、降低所需的电压。常用的电极形式有平行板电极、共面电极、金属丝电极和三维电极。平行板电极、共面电极都存在电场不均匀的问题，金属丝电极和三维电极虽然都能提供均匀电场，但它们分别存在着所需电压大和工艺复杂造价昂贵的缺点。微流控机械挤压穿孔的研究非常有限，现有器件存在微孔加工成本高且转染效率受限的问题。本研究实现了基于一体化流道电极的新型电穿孔和机械挤压协同作用的微流控转染芯片。提出的导电聚合物为材料加工而成的三维微电极细胞转染微流控芯片可通过软光刻倒模工艺实现，较现有的金属、硅基等三维电极具有明显优势，能够提供更加均匀的电场且极大简化了工艺、降低了成本。基于此创新性芯片工艺，本研究设计了单流道、高通量、电穿孔、协同作用穿孔的多种类型微流控芯片。研究中开展了多种细胞种类的转染实验。针对细胞转染，研究了器件设计、实验参数（流速、激励波形、幅值、频率等）对于转染效率和细胞活性的影响，并向细胞内成功递送多种材料，包括碘化丙啶、量子点和转染DNA质粒。本研究有潜力成为新型高通量且低成本的高效细胞转染器件，为生物医学研究提供有力工具。