基于微纳技术的循环肿瘤细胞免疫检测新方法

A novel method for detecting circulating tumor cells immunity based on micro-nano technique

摘要:

本研究旨在探索一种高灵敏度、高特异性检测循环肿瘤细胞(circulating tumor cells, CTCs)的免疫检测新方法，以尽早地检出结直肠癌，提高该疾病的检出率。首先制备含有线性微柱结构的微芯片，通过在其表面孵育氧化石墨烯-链霉亲和素(graphite oxide-streptavidin, GO-SA)及偶联广谱一抗(antibody1, Ab₁)，即上皮特异性黏附分子(epithelial cell adhesion molecule, EpCAM)单克隆抗体以捕获CTCs。运用羧基化多壁碳纳米管(carboxylated multi-walled carbon nanotubes, MWCNTs-COOH)与结直肠癌相关抗体，即特异性二抗(antibody 2, Ab₂)偶联制备抗体复合物。在捕获CTCs的微芯片上孵育该抗体复合物，构建以Ab₁-CTCs-Ab₂为主体的超级三明治结构，通过电化学工作站检测并验证其高灵敏度和高特异性。结果发现，在免疫传感器的构建中结合应用微纳技术，极大地提高了CTCs的检测灵敏度和特异性。本研究验证了该免疫传感器应用于临床血样检测的可行性，并通过该免疫传感器对结直肠癌患者外周血中CTCs进行检测和计数。结果表明，基于微纳技术的超级三明治式免疫传感器为CTCs的检测提供了新的途径，对临床工作中的疾病诊断及病情实时监控方面均具有潜在的应用价值。

Abstract:

This study was to develop a new method for detecting circulating tumor cells (CTCs) with high sensitivity and specificity, therefore to detect the colorectal cancer as early as possible for improving the detection rate of the disease. To this end, we prepared some micro-column structure microchips modified with graphite oxide-streptavidin (GO-SA) on the surface of microchips, further coupled with a broad-spectrum primary antibody (antibody1, Ab₁), anti-epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody to capture CTCs. Besides, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) were coupled with colorectal cancer related antibody as specific antibody 2 (Ab₂) to prepare complex. The sandwich structure consisting of Ab₁-CTCs-Ab₂ was constructed by the microchip for capturing CTCs. And the electrochemical workstation was used to detect and verify its high sensitivity and specificity. Results showed that the combination of immunosensor and micro-nano technology has greatly improved the detection sensitivity and specificity of the immunosensor. And we also verified the feasibility of the immunosensor for clinical blood sample detection, and successfully recognitized detection and quantization of CTCs in peripheral blood of colorectal cancer patients by this immunosensor. In conclusion, the super sandwich immunosensor based on micro-nano technology provides a new way for the detection of CTCs, which has potential application value in clinical diagnosis and real-time monitoring of disease.