基于光电镊-微液滴芯片的细胞阵列操控与单细胞RNA测序研究

RNA-Sequencing (RNA-Seq) is a technology that can efficiently obtain expression patterns of protein as well as reveal mutant or missing gene by sequencing the cDNA fragments formed by mRNA reverse transcription. A high-throughput droplet-based microfluidics is an important for RNA-Seq at present. Nevertheless, the technique are limited to the identification of tumor markers from a single-cell RNA-Seq at high gene profiling resolution due to inflexible manipulation for micro-droplet, weak matching rate between cell and barcoded bead, and cell lysis with long time. Optoelectronic tweezers (OET), which have been studied in our group at present, not only provide high precise manipulation of tiny cell, but also induce photo-electroporation without biochemical reagent. Consequently, a novel OET-droplet microfluidic chip having some advantages, such as flexible parallel manipulation of cellular array, good matching rate of droplet assembly, fast cell lysis period, and low error rate of RNA-seq, has been presented. Furthermore, the theoretical model related to the optonic-electric-fluidic fields will be built. The fabrication of photosensitive thin film and microchannels could be optimized further. In addition, aiming at the main crucial parameters affecting high-throughput cell transport and transfection need to be done in detail. Finally, high effect parallel algorithm applied in gene comparison could be developed to analyze the mechanism of inducing genetic diversities so that the novel tool enable researchers to discover the relationship among vital activity, genovariation and disease.

RNA测序(RNA-sequencing, RNA-seq)是通过对mRNA反转录形成的cDNA片段进行测序，可以有效获取蛋白质的表达模式，揭示缺失或突变的基因片段。当前高通量液滴微流控是RNA测序的重要平台，然而液滴可控性低、细胞与编码磁珠配对率差、细胞裂解时间长等问题都严重限制了单细胞RNA测序基因图谱的高分辨率标志物识别。课题组前期研究结果表明光电镊技术不但实现微小细胞的精准控制，还能降低生化试剂的依赖自动诱导细胞发生光电裂解。因此，本项目创新性地提出一种光电镊-微液滴芯片，具有细胞阵列可控性高、液滴包裹配对率好、裂解速度快、RNA测序误码率低的优点。本项目拟建立光-电-流体学的多物理场理论模型、优化光敏薄膜和微通道的制备工艺、整合高通量细胞输运和转染的关键参数、开发并行基因对比算法、分析差异性基因表达的诱发机理，为探索生命活动、基因变异和疾病三者之间的内在联系提供新的研究工具。

随着我国国民经济条件的稳步飞跃，公众对早期疾病的重视程度也逐年提升。基因分析和诊断技术已开始纳入大众的医检范围中，降低基因测序费用、时间和环节成为了国内外学者共同需要解决的课题。液滴型微流控芯片可以高通量地生成相场均匀和化学稳定的液珠，实现细胞的有效封装和生化研究。光电镊芯片在微/纳粒子操控领域具有使用灵活和无创性好的优点。本项目一边详细探索液滴型微流控芯片的结构设计、理论模型和制备方法，一边认真研究光电镊芯片在细胞操控和裂解方面的电极化理论和实验平台建设。在此，项目制备得到了液滴和光电镊集成式芯片，实现了100μm，频率200Hz的液滴生成和细胞封装，达到了10~20μm的细胞操控和500ms~1.2s的细胞裂解目的，满足了细胞RNA测序的项目需求，具有潜在应用价值和科学意义。研究期间积累了两相流流体力学知识，掌握了液滴微流控芯片的制备流程和关键技术，熟悉了光电镊芯片与液滴微流控芯片的集成方法，培养了多名检测领域硕士研究生。依托项目经费资助，为人类早期疾病诊断和治疗提供了一种新颖的光-电-液分析平台。同时为生物芯片的发展提供了重要的技术积累，促进生物医学、信息光学、细胞药理学、基因组学、微/纳制造技术与计算机科学等学科间的交叉混合发展。