基于光磁多信号联合检测的循环肿瘤细胞亚群分析方法研究

Distribution monitoring and sorting of circulating tumor cells (CTC) subpopulations could gain more insight into the clinical value of diagnosis and treatment of cancer. Currently, there are few methods developed for analysis of CTC subpopulations, and moreover the method are limited by disadvantages of labor intensive, time consuming and usually unable to sort each CTC subpopulation. To address the above issues related to CTC subpopulations assay, we propose a new strategy for the simple and rapid differentiation of CTC subtypes in peripheral blood based on the association assay of multiple optical-magnetic signals and by taking advantages of the series of optical-magnetic multifunctional nanomaterials and multiplex specific protein markers on the surface of CTC. Firstly, by employing fluorescent and magnetic nanomaterials such as quantum dots, Fe3O4 and so on, a series of multifunctional nanomaterials based detecting probes with adjustable and controllable optical-magnetic properties will be fabricated controllably and characterized fully. Secondly, we will establish a analysis model for differentiating cell subpopulations by investigating the dependence relationship and change trend between the information of protein types and abundance on the cell surface and the information of optical-magnetic signals (such as saturation magnetization, magnetophoretic time, fluorescent wavelength and intensity of target cells, and external magnetic field strength) within a capillary coupled flow analysis system associated with magnetic separation techniques. Then, a new method based on the association of optical-magnetic signals for analysis CTC subpopulation will be established by taking artificial CTC samples as model along with mechanical perturbation techniques and signal amplification technology, and finally the method will be subjected to clinical sample analysis after its fully optimization. By sample based on the different optical and magnetic properties of target cells, our strategy for CTC subpopulation assay is independent with the aid of large-scale instrument and complicated analysis system. Thus, we proposed a simple, convenience and rapid method for the analysis of CTC subpopulations, which is expected to provide possible new methods and technologies for the diagnosis and treatment of cancer.

循环肿瘤细胞（CTC）亚群分离分析具有重要的癌症诊治研究价值。目前CTC亚群的分析方法有限且存在“操作繁琐耗时、不能纯化分离”等不足。针对上述问题，本项目拟以光磁纳米材料和CTC表面多元特异蛋白标志物为基础、以光磁多信号联合分析为技术突破口，开展CTC亚群简便、快速分离与灵敏分析的基础研究。借助量子点、Fe3O4等荧光和磁性材料构建系列光磁性质可控的纳米探针；在毛细管荧光流通分析系统中基于上述探针并结合磁分离技术，研究细胞表面蛋白种类/丰度信息与可测量的“饱和磁化强度/荧光波长/荧光强度和外磁场强度”等光磁信号信息的依存关系，建立细胞亚群分析模型；以人工模拟外周血CTC为样本，基于机械微扰和信号放大等技术建立分析方法并进行临床样本分析。本研究同时基于靶细胞光磁多信号差异进行其亚群分析，无需大型仪器或复杂系统辅助，快速、简便、经济，有望发展具有临床应用前景的CTC亚群分析新方法。

肿瘤细胞异质性导致肿瘤增殖能力、侵袭能力和药物敏感性的差异，最终影响肿瘤患者的诊断、治疗和疾病进展。因此，肿瘤细胞亚群分析对于肿瘤的诊断和个体化治疗具有重要意义。针对目前肿瘤细胞亚群分离分析方法的不足及现有方法缺陷，本项目完成了如下工作：.（1）基于荧光量子点和磁性纳米粒子、通过金属配位组装及白细胞膜包覆策略构建仿生免疫荧光-磁性探针；以Her2表达量不同的乳腺癌细胞为研究模型，通过与仿生探针识别后具备不同磁响应性能，在恒定外磁场作用下建立基于磁性响应分辨的肿瘤细胞亚群简单快速分析方法。（2）基于荧光量子点和磁性纳米粒子、通过包埋-自组装联合及白细胞膜包覆策略制备不同磁响应性能仿生免疫荧光-磁性探针；以三种表面标志物种类及表达量差异化的肿瘤细胞作为分析模型，通过与具有不同磁响应性能仿生免疫荧光-磁性探针识别后，在恒定外磁场作用下建立基于磁性响应分辨的肿瘤细胞亚群分析方法。本研究工作的科学意义在于：成功实现了基于物质磁性大小进行细胞亚群分离的简单（仅需一个磁分离器）、快速（3分钟内可实现三种不同细胞亚群分离）的新模式，与现有的流式细胞术相比无需大型仪器、与微流控分析相比无需复杂系统，有望成为临床细胞亚群分析的有效技术手段。