基于LAPS/SPIM的单细胞电化学成像与传感技术及其在细胞凋亡研究中的应用

Physiological activities of cells are usually coupled with electron and/or electroactive species transferring. It has been shown that cancer cell biological behaviors, including cell adhesion, spreading, proliferation and apoptosis, are always companioned with the alterations in electrochemical properties such as cell surface charge and impedance. Therefore, the development of label-free and non-invasive biosensors for cell electrochemical sensing is particularly important in cancer cell research. Conventional electrochemical methods lake spatial information, making it impossible to investigate cellular heterogeneities, which is of particular importance in cancer precise diagnosis and prognosis. Light-addressable potentiometric sensor (LAPS)/scanning photo-induced impedance microscopy (SPIM) overcomes these problems. Based on an electrolyte-insulator-silicon (EIS) field-effect structure, multi-sites on LAPS/SPIM chips can be addressed by scanning a focused light beam across the sensor substrates, providing information of surface potentials and impedance with lateral resolution. In this project, small peptides of cyclic-Arg-Gly-Asp (RGD)fK modified and self-assembled monolayers (SAMs) insulated LAPS/SPIM combined with microfluidics will be used to consolidate functions of single cell analysis and two-dimensional imaging of electrochemical properties together. To specific, by using the integrated cell-based biosensor, the changes of the local surface charge and impedance due to the attachment of the target cell can be observed by measuring local photocurrents. The cell heterogeneous properties at single cell level of breast cancer cells will be analyzed via high-resolution LAPS/SPIM imaging. Furthermore, as cell death results in dramatic electrochemical property changes, cell apoptosis processes induced by typical anti-cancer drugs such as tamoxifen will be recorded using LAPS/SPIM. The correlation between electrochemical properties of single cells and their drug-induced cell apoptosis processes will be investigated and determined. The proposed technique has the advantages that capable of high-resolution imaging and real-time single cell sensing with muti-channels and multi-parameters, which are not accessible to other electrochemical techniques. The results of this project will provide a novel method for the study of cancer physiopathology basis and impact in the fields of drug screening, drug efficacy evaluation, cancer precise diagnosis as well as personalized treatments.

如何从单细胞水平多参数获取肿瘤细胞电化学特性与图像信息是目前细胞电化学传感领域亟待解决的一个难题。我们拟在前期工作基础上，将高分辨、高灵敏光寻址电位传感器和扫描光诱导阻抗显微成像技术联用，结合微流控芯片，构建无损免标记多通道集成单细胞成像与传感平台；以人乳腺癌细胞为模型，对不同亚型细胞进行光电流检测和成像，使用点接触细胞-芯片耦合模型分析单细胞阻抗和表面电位信息，从单细胞水平评估细胞的电化学异质性；实时在线监测单个乳腺癌MCF-7细胞在不同浓度抗癌药物及其组合作用下电化学响应信号及图像信息的变化，研究药物诱导细胞凋亡动力学过程和非均向特性，揭示肿瘤单细胞电化学特性与药物诱导细胞凋亡过程的重要联系。本项目开发的细胞电化学成像技术能从单细胞水平综合检测与分析肿瘤细胞的异质性及细胞凋亡的动态过程，为细胞病理生理基础研究提供新的技术手段，对肿瘤精准诊断、个性化治疗和指导用药等领域具有重要意义。

细胞病理生理活动与细胞电化学信号的表达息息相关，从单细胞水平研究不同细胞个体之间电化学行为的区别与联系，避免群细胞的平均效应导致的细节信息丢失，对细胞生物学状态、功能及演化规律的准确表征具有重要意义。如何从单细胞水平多参数获取细胞电化学特性与图像信息是目前细胞电化学传感领域亟待解决的一个难题。光寻址电位传感器（LAPS）和扫描光诱导阻抗显微成像（SPIM）作为具有空间分辨率的电化学检测技术，具有无损、免标记、制作工艺简单和使用寿命长等优势。本项目首先按计划进行了RGD修饰蓝宝石-硅基LAPS/SPIM用于单细胞成像的可行性研究。由于传统LAPS场效应结构对电荷的敏感距离受到德拜常数的局限，难以实现单细胞的高灵敏成像监测。在本研究中我们通过去除绝缘层扩大表面电荷敏感距离至微米级。分别采用ITO-电解液和α-Fe2O3-电解液界面构建了高分辨LAPS/SPIM成像系统, 成功用于单个肿瘤细胞的电化学成像，分辨细胞异质性，并实时成像研究了抗癌药物他莫昔芬诱导MCF-7乳腺癌细胞凋亡过程，从电化学角度解析了细胞凋亡的形态及贴附性能的变化，揭示肿瘤单细胞电化学特性与药物诱导细胞凋亡过程的重要联系。此外，基于LAPS/SPIM，我们还研究了多种新型光电化学成像和检测方法：包括调制光诱导电化学用于免标记多通道DNA传感与检测；扫描电化学光度传感器用于免标记活细胞成像及实时光学定量分析；光寻址方波伏安法用于生物分子的检测及阻抗二维成像等。本项目开发的光电化学成像技术不仅能从单细胞水平研究细胞的生物学行为，还可用于高通量生物分子的检测与分析，为生物医学传感与检测提供新的技术手段，对肿瘤精准诊断、个性化治疗和药物筛选等领域具有重要意义。在本项目支持下，发表SCI学术论文9篇，申请国家发明专利1项，参加国内学术会议2次，参与培养博士研究生2人、硕士研究生1人。