高性能电化学发光应用于氧化应激相关疾病基础研究

Oxidative stress (OS), relates to series of diseases (included in respiratory system, cardiovascular system, degeneration and pathological changes of brain and pathological changes of endocrine system etc) which have become the worst diseases and pose threat to human health internationally and also in China, is a symptom of unbalanced oxygen metabolism in body. The excess produced and accumulated reactive oxygen species (ROSs) will attack all molecules and cells to destroy the organisms in the case of disease and finally lead to severe damages even death. Now, in clinical diagnosis, we can only detect those indirect indexes such as glutathione or superoxide dismutase to characterize the OS because we lack the direct detection means. This project which is based on our long-term researches proposes a highly sensitive response to ROSs with electrochemiluminescent (ECL) method combined with some advanced technologies such as enhancement by nano-materials, dual potentiostat excitation, AC lock-in amplification and photonic/electronic current multipilication, is in promising to detect the ROSs at the level of single cell or minute in-vivo samples. It constructs a technical platform suitable for pathological study, clinical diagnosis, therapy guidance or medicine discovery. This project is highly depended on the progress of synthesis of effective catalysts such as supported nano-materials, the surface functionalization, the development of solid-state reagentless ECL electrode and high performance ECL detection technology. Combined with the technologies as scaning electrochemical microscopy and microdialysis, we will provide a valuable approach for single cell or real-time quasi-in-vivo OS quantification for application in clinical researches.

氧化应激涉及多种疾病（如呼吸系统、心血管系统、脑退行病变、内分泌病变等），这些已经是国内外最常见和对生命威胁最严重的疾病。其本质是机体在疾病状态下氧的代谢平衡被破坏，导致过多活性氧的产生和积累，活性氧攻击所有的分子和细胞导致损伤及至最终死亡。在临床诊断中，由于缺乏直接的检测手段，目前主要检测间接的指标如谷胱甘肽和超氧化物歧化酶等。本项目在长期研究积累基础上，提出以电化学发光分析法实现对活性氧的高灵敏度响应，结合纳米增强、双恒电位激发、交流锁相放大和光电信号积等技术实现单细胞和微量活体采样水平上的检测，构成适合于病理研究、临床诊断、指导治疗和药物开发研究的一个技术平台。本项目将以研发高催化活性负载型纳米材料、基于纳米功能化表面的全固化无试剂电化学发光电极以及研究高性能的电化学发光检测技术实现对活性氧的高灵敏检测，并结合扫描电化学显微和微透析采样实现单细胞和实时准活体检测并应用于临床研究。

本研究按计划完成：（1）通过物理、数学方法提高ECL检测能力：i）实现了光学斩波/锁相放大功能，提高信/噪比；ii）双恒电位ECL检测技术，构建了两种微体积的双恒电位ECL池，分别应用于静态和动态监测体系，有效加强了ECL发射强度和分析灵敏度；iii）采用硅光电二极管替代光电倍增管，构建轻便型ECL检测装备；iv）多信道检测及信号处理，实现电化学和ECL两种信号的同时检测和数学计算，提升检测能力。（2）研究了多种不同成分和结构形态的纳米材料对鲁米诺ECL的增强作用：包括i）纳米金属材料如纳米Au、Pt、Ag及其合金，Au纳米囊，Pt纳米线等；ii）纳米TiO2，包括颗粒、纳米空壳、纳米管等；iii）复合纳米材料，上述纳米材料经复合可以得到协同作用而产生更好效果。纳米材料的使用使鲁米诺的ECL性能有很大的提高，从而达到更低的检出下限。研究了纳米发光材料，采用原位聚合或预聚合方式制备鲁米诺或鲁米诺/苯胺纳米材料，可将发光探针和纳米增强材料同时固着与电极表面，制备全固化ECL电极。（3）开发ECL传感器：包括 i）糖尿病：无创葡萄糖传感器、糖化血红蛋白传感器、beta-trophin传感器、糖尿病基因传感器、糖尿病二肽传感器、谷氨酸脱羧酶抗体传感器、C肽传感器等。ii）心血管疾病：胆固醇传感器、肌红蛋白传感器、C反应蛋白传感器等。iii）老年性智障：糖原合酶激酶（GSK-3β）传感器。（4）活性细胞/体液分析：将活细胞负载于经纳米TiO2功能化的ITO玻璃表面，获得单层分布的有限量活细胞界面，ECL响应反比于典型活性氧清除剂-白藜芦醇，表明ECL探针对上述活细胞界面上的氧代谢是敏感的，可应用于细胞氧化应激的监测；与微透析装置联用，实现了从大鼠尾部血管中采集活体体液，消除大分子血液成分对ECL测定的干扰，可应用于局部活体组织中特定成分的检测。发表SCI论文24篇，其中中科院一区论文6篇，二区论文8篇。申请专利4项，已授权3项。按期完成项目提出的各项研究成果目标。