用于三维癌细胞培养微环境原位检测的微型生物传感器构筑方法研究

The three-dimensional culture of cancer cells is close to the body growth environment, which provides a better understanding of the behavior and regulation mechanism of cancer cells in vivo. The cells microenvironment contains important information of cells, but the lack of effective sensors to detect the microenvironment of three-dimensional cells has hindered its research in this field. Based on the platinum wire with a diameter of less than 50 µm, a new construction method of cellular level micro-biosensor is proposed for in situ detection of three-dimensional cancer cells microenvironment, according to the road of “microelectrode packaging and modification”, “assembly of sensors in the cellular level” and “in situ detection of cells microenvironment”. Firstly, the key problems, such as monomer molecular deposition and electropolymerization, were studied for insulation packaging and sensitive materials modification of platinum wire. And the influence rule of functional film microstructure on its properties was explored. Secondly, a design idea of multi-stage alignment of microelectrodes was proposed and a methodology to form a biosensor by microelectrodes in the three-dimensional cells level was constructed. Finally, the mechanism of sensor measurement was studied and the method of in situ detection of glucose/lactate in the microenvironment of BT474 culture of breast cancer cells was explored. The results would provide accurate data support for the biological model based on three-dimensional cells information expression, with great values in drug screening and cancer research.

癌细胞三维立体培养接近在体生长环境，为更准确认识体内癌细胞行为和调节机制提供了条件。细胞生长微环境包含重要细胞信息，然而因缺乏有效传感器对三维癌细胞微环境检测，使其在该领域的研究受到阻碍。本项目以直径≤50微米铂丝为基础电极，基于“微电极封装修饰”→“在细胞微环境内装配形成传感器”→“对细胞微环境原位检测”的思路，研究一种细胞水平上的微型生物传感器构筑方法，实现三维癌细胞微环境原位在线检测。具体研究铂丝电极绝缘封装和敏感材料修饰中的单体分子沉积、电聚合等关键问题，探求功能薄膜微观结构对其性能的影响规律；提出微电极多级对准的设计思想，建立微电极在三维细胞微环境内精密装配形成传感器的方法学；研究传感器检测机理，开展乳腺癌球体细胞BT474培养微环境中葡萄糖/乳酸浓度原位在线检测实验。研究成果为基于三维细胞信息表达的生物模型提供精准数据支持，在药物筛选和癌症研究方面具有重要价值。

细胞模型是进行药物筛选的主要模型之一，三维细胞模型比二维细胞模型更接近细胞生长的自然环境，可以为细胞分析提供更精确的信息，然而因缺乏有效传感器对三维癌细胞微环境检测，使其在该领域的研究受到阻碍。本项目研究了一种微型生物传感器，用于三维细胞微环境检测。重点研究了传感器微电极绝缘封装和敏感材料修饰中的单体分子沉积、电聚合等关键技术，实现了铂丝微电极绝缘封装，并在暴露的尖端修饰纳米材料和固定酶分子等敏感材料，制备出高灵敏的工作电极，电极响应电流密度达0.51 A/m2。提出了一种多级对准的方法，在细胞水平上装配微电极形成微型生物传感器。实验测试了微型生物传感器的重要性能，在三维细胞培养微环境原位在线检测实验结果表明微型生物传感器对葡萄糖在浓度0 - 30 mM范围内表现出良好的线性响应信号，线性相关系数0.99，灵敏度为0.5 nA/mM，可满足在细胞培养微环境中对葡萄糖浓度的测量。由于良好的酶固定化技术和低的工作电位，在抗干扰实验中，尿酸、抗坏血酸、乳酸作为干扰剂对传感器测量葡萄糖浓度的影响不大。研究成果为基于三维细胞信息表达的生物模型提供精准数据支持，在药物筛选和癌症研究方面具有重要价值。