神经元及神经环路中重要生理活性物质的光学传感体系研究

The research on neutral circuits has been a hot topic in recent years. In neurons or neutral circuit systems, many important bioactive species, such as transmitters (e.g. NO and H2S), reactive oxygen species (ROS) and redox enzymes, are closely related with the functions of neutral circuits. Thus, monitoring of these molecules may provide a useful way for tracing the neutral circuits. However, many of the bioactive species still lack efficient detection and sensing methods. One of the possible routes to solving these problems is to develop new spectroscopic probes with excellent properties, and then to use them to construct new methods for spectroscopic sensing and tracing of the bioactive species in neutral circuits and neurons. The current proposal will focus on the research of the above topic. Firstly, we will concentrate on the rational design and synthesis of long-wavelength fluorophores with new structures, and then prepare new fluorescent probes by utilizing the specific reactions of the target molecules, such as the Michael addition-cyclization reaction of H2S, the selective cleavage of the ether bond by HClO and the selective reduction of the nitro group by nitroreductase in the presence of NADH; secondly, highly sensitive and selective methods will be established based on the specific reactions of probes with the target; and finally, we will apply these methods to the detection of the related bioactive species such as gaseous transmitters, ROS and redox enzymes in neurons and neutral circuits. It is hoped that the selective and sensitive detection of these species in neurons and neutral circuits may help to disclose their molecular mechanisms.

神经环路的研究已成为近年来的一个热点和前沿领域。在神经元及神经环路中，重要的生理活性物质如信号分子（NO、H2S）、活性氧物种及氧化还原酶（硝基还原酶）等与神经环路的功能密切相关。因此，对这些分子进行传感与监测将有助于实现神经环路和神经元的可视化示踪。然而，上述许多物质目前尚缺乏其可行的检测方法。解决这些问题的有效途径之一可能是设计性能优良的光学探针，进而构建神经元和神经环路中相关物种的光学传感与示踪新方法。本申请即以此为题展开深入研究。首先将借助H2S可发生迈克尔加成-环化反应、HClO可切断醚键以及硝基还原酶可催化还原芳硝基等特异的化学反应，以新型BODIPY等母体为平台设计荧光探针，再利用识别基团对目标物的特异性反应，实现目标物的光学传感与示踪，并尝试用于神经元及脑神经环路中上述物质的可视化成像分析。该项目的成功实施将有助于揭示这些物质在神经环路或神经元中的生成与分子作用机制。

本项目通过合理的分子设计，以萘酰亚胺、罗丹明、resorufin等为荧光母体，分别制备了几种与神经活动密切相关的重要生理活性物质如ROS、氧化还原酶等的荧光探针,以实现细胞水平这些生理活性物种的选择性光学传感与示踪检测，从而揭示出这些物质在神经环路系统中特定条件（如缺氧）下的变化规律。首先以萘酰亚胺为荧光母体，分别制备了酪氨酸酶、单胺氧化酶A及硝基还原酶的荧光探针，这些研究提供了简便、可视化地检测细胞中这些酶代谢的新方法，同时也揭示了一些外界诱导导致的酶活性的变化规律。进一步，提出了新的酪氨酸酶识别基团3-羟基苯基，并将其引入到稳定的半菁荧光骨架中，设计合成了一种检测酪氨酸酶的近红外荧光探针。该探针能有效避免活性氧物种的干扰，并应用于细胞以及活体斑马鱼中酪氨酸酶的成像分析。接下来，为了发展更高灵敏度的酪氨酸酶荧光探针，制备了以3-羟基苯基为识别基团，resorufin为荧光母体的酪氨酸酶荧光探针。该反应对酪氨酸酶具有高选择性和高灵敏度的特点。利用该探针，对不同细胞中酪氨酸酶的含量水平进行了成像，揭示了不同细胞中酪氨酸酶表达水平的差异。在小分子活性硫与活性氧物种的荧光探针方面，首先制备了一种可区分检测谷胱甘肽和半胱氨酸的荧光探针，并利用该探针对血清中半胱氨酸和谷胱甘肽的浓度分别进行了测定；其次，将亚细胞器线粒体的靶向基团引入到稳定的罗丹明骨架中，分别制备了具有线粒体靶向功能的HClO和ONOO-荧光探针；结合荧光共聚焦成像技术，研究并揭示出细胞线粒体在外界刺激下HClO和ONOO-浓度的变化。在纳米探针方面，以富电子的间苯二胺聚合物作淬灭剂，设计合成了具有低背景信号的复合物荧光探针，进而构建了高灵敏度的透明质酸酶检测方法。该方法不仅用于结直肠癌患者血清中透明质酸酶的灵敏测定，而且还用于在不同浓度As(VI)影响下血清中透明质酸酶活性的检测。