纸电化学发光免疫传感芯片的构建及应用研究

Paper-based electrochemiluminescence immunoassay (PECLIA) has attracted much attention from researchers which has the powerful functions of ECLIA and the inexpensive portable, multi-channel detection characteristics of paper chips. However, the PECL platform currently cannot achieve high sensitivity and stability, and the continuous processing of separation, transportation, and detection. This project proposes to customize the functional materials for PECL based on the structure-response relationship which is independent to the natural pattern of specific materials. To reveal the mechanism between PECL electrochemical behavior and signal enhancement, the amplification effect of assemble of the functionalized materials on the biological molecules and signals would be explored. Furthermore, the multilayer screen print technology would be used to construct the high-aspect ratio three-dimensional microchannels and the porous planar electrode through the layer-by-layer solidification. The intrinsic link between the process parameters and the chip performance would be revealed over the effect of the process parameters on the apparent paper chips. Then, it is hoped that the novel PECL immunosensor chips in the integration of sample separation, conveying and detection can be developed through combining the super-hydrophobic membrane modification technology and the proposed methods. The developed PECL immunosensor chips can be applied to the real-time sensitive identification of the highly pathogenic influenza virus of four subtypes, and can provide a novel platform for ECL research and pathogen detection.

纸电化学发光免疫分析(PECLIA)可兼具ECL的强大分析功能和纸芯片低廉、便携、可多通道检测等特性而备受关注。然而，PECLIA平台构建中存在样品高灵敏检测及分离、输送、检测过程连续化的技术难题。鉴于此，本项目拟采用结构-反应性关系且不遵循具体材料的固有模式，定制功能材料，通过探索功能材料复合组装对生物分子有序、高效固定及对生物信号放大的作用，揭示PECLIA电化学行为与信号增强的内在关联机制；拟采用丝网印刷技术进行多层印刷、逐层固化构建高深宽比的圆环式三维纸芯片微通道和多孔平面电极，研究工艺参数对纸芯片表观的影响，揭示工艺参数与芯片性能指标之间的内在联系，并通过超疏水膜修饰技术及气动驱动控制液流，以构建集样品分离、输送、检测于一体的PECLIA传感芯片，实现对高致病流感病毒四种亚型的高灵敏即时鉴定，为ECL基础研究和病原体检测提供一个崭新平台和方式。

本项目旨在构建一种纸电化学发光免疫传感芯片，使之应用于病原体的快速、灵敏检测。本项目基本按照原研究计划进行，结合新出现的问题与国内外相关研究最新进展，对部分研究计划与内容作了适当的调整和扩展。已开展的工作如下：(1) 研究了网版质量、机器参数设置、油墨材料等影响丝网印刷电极、微通道的因素，并优化了实验条件，针对印制的微通道重现性较低的问题，开展了单通道纸基电极的研究并完成了条件优化；(2)根据纸基电极结构及反复实验结果，设计制作了三种纸芯片电化学发光测试装置，装置机械采用灵活设计，电路采用特色设计保证装置高灵敏度、可靠性和稳定性；(3) 开展生物探针的固定和信号放大方法研究，实现了二氧化硅纳米微球、磁性石墨烯表面稳定修饰及钌标二抗的条件优化；(4) 基于研制的第二代纸芯片电化学发光测试装置成功完成了模式抗体IgG、临床标本乙肝表面抗原、丙肝抗体、梅毒螺旋体抗体的电化学发光检测；（5）在研制的第三代便携式纸芯片电化学发光分析装置的基础上，并完成了与罗氏全自动电化学发光免疫分析仪乙肝表面抗原检测结果的比较。所取得的研究成果形成学术论文4篇，其中SCI收录3篇，申请国家发明专利1项、授权实用新型专利3项；培养硕士生2人，基本完成了任务计划书中的研究内容和预期目标。本项目设计的纳米材料稳定修饰技术、纸电化学发光分析装置及检测技术，有望为后续开发基于纸基电化学发光装置的灵敏、特异、快速、经济且适于现场分析的病原体诊断新平台提供有益的新思路。