混合动电微流控芯片分离捕获循环肿瘤细胞的研究

Cancer has a devastating personal and societal impact. Despite medical advances, malignancy currently account for over 7600,000 annual deaths in the world. In recent decades, vast resources have been allocated to the preclinical and clinical development of novel chemotherapeutic and biologic agents, yielding hundreds of promising new therapeutic targets and strategies. Paradoxically, the therapeutic "bottle-neck" has shifted and no longer stems simply from a lack of treatments; rather, there is a widening gap between the growing armamentarium of novel agents and the available biomarkers needed to inform the use of those agents and to monitor treatment response. Traditional disease indices are ill-suited to guide patient management: Biopsy of the primary tumor often does not predict the genotype and phenotype of recurrent/metastatic disease; moreover, serial radiographic imaging does not measure specific drug-on-target effects, nor does it reflect the more subtle tumoristatic impact of many new biologic agents. For these reasons, efficient new technologies are urgently needed to enable more sophisticated patient evaluation and to allow more rapid and highly predictive assessment of response to therapy. Circulating tumor cells (CTCs), the cancer cells shed by solid tumors into the peripheral blood of cancer patients, can cause distant metastasis and tumor recurrence which are the main causes of death of malignant tumor. CTC capture and analysis allows repeated, minimally-invasive, real-time disease sampling and provides invaluable prognostic and predictive data for individualized patient management and improved therapeutic outcome. To date, enumeration of CTCs has provided prognostic and predictive insights in advanced breast, colorectal, and prostate cancer. We will develop a new technology - hybrid electrokinetic microfluidics - an "on-chip" platform which exploits cells' innate structural and electrical properties to concentrate and isolate them from other cells in solution. Using the EK-chip, we have already successfully demonstrated the manipulation of various pathogens. EK sorting offers the triple advantage of rapid high-throughput processing, universal applicability to all CTCs regardless of cell surface markers, and capture of live cells that can be further studied; moreover, this compact, "on-chip" approach capitalizes on components and technologies that we have already developed and validated for non-cancer applications. In this current project, we propose to test and validate EK sorting technology for the purpose of CTC capture and analysis from clinical samples. Specifically, the project will consist of the following aims: Develop hybrid electrokinetic blood matrix management for isolating circulating tumor cells. Adapt hybrid electrokinetic manipulation to a high throughput system for circulating tumor cell isolation in conductive biological fluids. Validate the biological and clinical relevance of EK-captured cancer cells.

恶性肿瘤致死的主要原因是少量肿瘤细胞获得侵袭能力进入血液循环，造成病灶远处转移和复发。因此，在外周血中捕获并检测循环肿瘤细胞(CTC)具有重要临床应用价值。目前采用的免疫磁珠分选法仅适用于上皮肿瘤细胞；介电泳法要求较低电导率。至今尚未见同时具备广谱、活体、高电导率环境的CTC捕获方法。本课题首次提出用混合动电微流控技术分离捕获CTC的方法，它是通过加工在芯片通道内的微电极在高电导率流体中产生多种动电力，同时基于细胞内在结构和电性质差异，借助混合动电力和芯片通道网络操控细胞，从而将目标细胞富集并捕获或将它们与溶液中其它细胞分离的微流控芯片技术。通过研究电极、通道结构，电场、生理电导液（包括血液、白细胞液、尿液等）对细胞的作用规律，建立广谱、高效的CTC活体捕获方法，进一步研究用该方法捕获癌细胞的生物学和临床相关性，为体外早期诊断，个体化治疗，肿瘤复发监测以及肿瘤新药物开发提供新技术、新方法。

本课题研究了基于动电作用结合微流控技术分离捕获循环肿瘤细胞（CTC）的方法，它是通过加工在芯片通道内的微电极在高电导率流体中产生多种动电力，同时利用不同种类细胞内在结构和电性质差异操控细胞，从而将目标细胞富集并捕获或将它们与溶液中其它细胞分离的微流控芯片技术；开发了一种透明胶带雕刻微流控芯片模具的快速制备方法；初步探索了基于表面拉曼增强光谱（SERS）信号放大的微流控生物传感分析方法。.首先，用单分散聚苯乙烯(Polystyrene, PS)微球模拟不同种类和大小的细胞，并探讨了微粒定向运动的动电学原理，为进一步操控生物细胞提供了理论基础。为此，制作了一种以玻璃为基片的聚二甲基硅氧烷 (Polydimethylsiloxane, PDMS)微流控芯片，该芯片使用的是Ti-Au-Ti三层夹心式、三个电极相平行的微电极设计。通过施加一个交流电场，对不同尺寸的微粒进行高效分离的研究。以羧基修饰的聚苯乙烯微球为例，研究结果表明，对于直径分别为5 μm、10 μm和25 μm的三种PS微球（分别模拟白细胞、红细胞和癌细胞），在电压为11 V，频率为1 MHz时，可以达到大球和另外两种尺寸较小微球的快速有效分离。并且不同电性的聚苯乙烯微球也可以实现分离，分离效率均可达90%以上。同时发现，相邻电极中间位置层流区域的形成，对微球分离起到关键作用。在高电导率介质中研究多种动电力对不同尺寸微目标的作用规律，对于最终实现实际生物样品的高效动电操纵具有重要意义。.其次，在PS微球分离电场条件基础上，对血细胞和乳腺癌细胞的分离进行了研究，结果表明，细胞分离和微球分离现象一致，证明用微球模拟细胞的研究路线是正确的。进一步研究了适合细胞分离的电场条件，得出了癌细胞和血细胞分离的最佳电场条件，其电压是15 V，频率300 kHz，直流偏压为1 V DC。同时证明了细胞分离也与细胞尺寸、内在结构和电性质差异有关。.最后，将微流控生物反应器与表面增强拉曼光谱相结合，构建了一种多功能智能探针--发夹型靶向识别探针DNA循环放大SERS检测miRNA-203的方法。该方法检测miRNA-203的线性浓度范围为1.0 × 10-14~1.0 × 10-7M，检测限可达6.3 fM（S/N=3），实现了miRNA-203的灵敏检测。该方法操作简便快速，灵敏度高，选择性好，为后续专一检测CTC做出了有益探索。