非电活性物质的比率型电化学分析新方法的研究

The ratiometric analysis has been attracting considerable attentions in anlytical chemistry because of their sensitivity and built-in correction for environmental effects. However, so far, there is few report about application of ratiometric pricinple in electrochemistry and electroanalysis. In this project，in view of the vital role of electroinactive molecules in understanding and revealing their target in living process, we proposed a novel ratiometric electrochemical sensor through integration of highly sepcific recognition elements and a inner reference elemetns with highly electrochemical stablity on nanosurface, in which the specific elements is contributed to the high selectivity of sensor, and the inner reference serve as a probe to monitor the change of electrode performance, thereby reducing or diminishing the determination error between the sample and real cell. Meanwhile, the conductive micropattern is in site prepared on nanostructural surface by applying remote photocatalytical method. Then, ratiometric probes are confined on the micropattern. Site-selective cell adhension methdod is untilized to in situ cultivate cell on micropattern based on functional ratiometric electrochemical probe. As a consequence, the electrochemical inactive species released from the cell will be collected, and then remarkably enlarge the sensing signal, enhancing the sensitivity and detect limit and realizing the in situ, and real-time monitoring the electrochemical inactive species in cell. These investigations will not only propose a new ratiometric electrochemical senosor for highly selective and accurate detection electrochemical inactive species, but also provide a practical platform for electroanalysis of electrochemical inactive species in single cell.

对测定环境变化敏感、可自我矫正的特点，让比率型分析方法正在成为分析化学研究中的热点领域之一。但是，目前为止基于此原理的电化学分析仍未见报道。本项目以在生命过程中具有重要作用的非电活性分子为测定目标，拟通过合成高选择性的识别配体和稳定的内参比分子，设计新型的、高选择性、高准确性的比率型电化学探针。同时，采用非接触性光催化法，在导电性纳米基底，原位制备导电性微阵列，然后组装比率型探针分子，实现其功能化。再结合细胞原位固定的生物技术，在比率型微电极阵列界面实现单细胞的选择性固定和培养，实现单细胞非电活性物质的原位、实时、动态的电化学分析一体化。本研究不仅建立了一种高选择性、高准确度的非电活性分子的比率型电化学传感器，而且为单细胞非电活性物质的高通量电化学分析提供了一种切实可行的研究模式。

非电活性物质金属离子、氨基酸及pH等在脑缺血、阿兹海默症等诸多脑疾病中具有十分重要的作用。本项目针对细胞及活体检测中干扰物质多、体内外检测误差大等关键科学问题，通过合理设计并合成多种金属离子的高选择性识别配体，结合新型的具有高比表面的纳米界面及性能稳定的内参比分子，构建了一系列电流及电位型的比率型电化学传感，实现多种金属离子及pH的高选择性、高灵敏度、高准确度的分析。基于比率型电化学传感的概念，我们进一步发展了可同时实现电位及电流双信号输出的金属离子及pH同时分析的比率型电化学分析方法。最终，结合微电极技术，成功将新构建的电化学探针应用于脑缺血、阿兹海默症等脑疾病模型鼠鼠脑中目标分子的在体分析。结果表明脑缺血会导致脑环境的酸化。此外，相较于正常鼠，阿兹海默鼠脑中的铜离子平衡会发生明显的变化，呈现增加的趋势，而半胱氨酸呈现减少的趋势。通过本项目的实施，不仅提出了多种有效地、适用于鼠脑中金属离子、氨基酸及pH的新型电化学分析方法，而且为在细胞及活体中其他生理物质的在体分析，提供了多种切实可行的研究途径，为探索并解析脑缺血及阿兹海默等脑疾病的分子机制提供了可靠而有用的数据。

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