脑是人体结构最精密，功能最复杂的器官。脑功能的正常运作依赖于脑内紧密联系的神经化学分子对特定神经元在空间和时间上的精确调控。在复杂生理与病理过程中，脑内神经活性物质组成的分子网络实时变化，其中任一分子的变化异常均会致使神经系统失常，甚至引起脑疾病的发生。因此，对于神经化学信号的精准检测与应用极具意义，有助于加深人类对脑的认知、为脑疾病的诊断与治疗提供理论基础与工具。然而，无论是对脑内神经化学信号的传感还是调控，均受限于少量电化学活性好的神经活性分子，例如多巴胺、抗坏血酸等；尚存大量重要的神经活性物种的电化学检测与调控面临选择性的关键问题。本研究报告针对这些难以选择性检测与调控的重要神经活性分子（如活性氧、含硫物质等），利用单原子催化剂的可设计性和优良的催化活性，构筑了高选择性电化学传感界面，实现高选择性传感分析；结合课题组已发展的单细胞分析平台，探索调控方法的介导分子在单细胞层次的神经调控机制，为神经化学信号的调控方法提供理论基础。具体研究工作简述如下：

（1）半胱氨酸的电化学高选择性传感方法研究：半胱氨酸（L-Cys）是调控脑内氧化还原氛围的重要分子，与帕金森综合症（PD）、阿尔兹海默症（AD）等脑疾病的发生发展相关，因此其含量的动态监测对神经生理病理分子机制的研究具有重要意义。然而，L-Cys的活体电化学传感易受结构相似的谷胱甘肽（GSH）的干扰。本研究制备并筛选了具有不同配位环境的FeN_n单原子催化剂（SAC），利用其配位环境对调控含硫物质吸附强弱的，提高了L-Cys传感器抗GSH干扰的能力。利用选择性最高的FeN₃C₁-SAC，结合GRP传感方法，实现了L-Cys的高选择性传感。本研究为高选择活体电化学传感分析方法的研究提供了新的思路。

（2）硫化氢的电化学高选择性传感方法研究：硫化氢（H₂S）是脑内重要的气体递质分子，与诸多神经退行性疾病相关，其含量的动态监测对于认识和了解神经生理病理过程的分子本质具有重要意义。然而，H₂S的电化学氧化电位与脑内其他物质的氧化还原电位相近，且与抗坏血酸（AA）等还原型物质共同变化。本研究设计制备并筛选金属位点，选择一种含有NiN₄位点的SAC，选择性地加快了H₂S氧化过程中决速步骤的电子转移速率，提高了H₂S传感的选择性。将该SAC修饰于双极化CFE（bipolar-CFE，BCFE）阳极，结合原电池氧化还原电势法（GRP），克服了H₂S氧化产物吸附电极致使的稳定性差的挑战。应用该H₂S传感方法，实现了对DJ-1基因敲除（DJ-1 KO）鼠与野生型（WT）鼠的H₂S释放行为的检测，发现其释放持续时长存在差异。该电化学界面的构筑结合了GRP与SAC可设计的优势，实现了H₂S的高选择性、高稳定性活体传感，为脑神经化学研究提供了工具。

（3）过氧化氢调控神经递质释放的机制研究：过氧化氢(hydrogen peroxide, H₂O₂)作为一种重要的扩散性信使和动态神经调节因子，在中枢神经系统(central nervous system, CNS)中具有调节多巴胺(dopamine, DA)的释放和神经元兴奋性的功能。无论是神经系统中或是在任一细胞内，H₂O₂的生理病理作用都取决于它的浓度。然而，实时产生的低生理水平H₂O₂在神经生理过程中的神经功能尚不清楚。利用单囊泡电化学方法，我们发现细胞外纳摩尔水平的H₂O₂可以促进培养的PC12细胞DA的囊泡释放。此外，我们推测这种促进作用是由于在低生理水平的H₂O₂的胞吐过程中打开了K_ATP通道，增加了囊泡释放初始融合孔的大小。对低生理浓度H₂O₂神经调节作用的研究，为H₂O₂神经化学信号的调控写入坚实的理论基础。

The brain is the most precise and complex organ in the human body, responsible for regulating various functions. Its normal operation relies on the precise spatial and temporal regulation of specific neurons through tightly connected neurochemical molecules. In both physiological and pathological processes, the molecular network of neuroactive substances in the brain undergoes real-time changes, where any abnormal alteration can lead to nervous system disorders and even brain diseases. Therefore, achieving accurate reading and writing of neurochemical signals is crucial for deepening our understanding of the brain's cognition as well as providing theoretical basis and tools for diagnosing and treating brain diseases. However, both sensing and regulating neurochemical signals in the brain face challenges due to a limited number of neuroactive molecules with good electrochemical activity (e.g., dopamine and ascorbic acid), which poses a key problem regarding selectivity. In this thesis, we aim to address this issue by leveraging single-atom catalysts' designability and excellent catalytic activity to construct a highly selective electrochemical sensing and control interface specifically targeting important physiological active molecules that are difficult to selectively analyze in vivo electrochemically (such as reactive oxygen species and sulfur-containing substances). By combining this approach with our developed in vivo in situ analysis platform, we will explore the neuroregulatory mechanism mediated by these molecules at the single-cell level while also investigating its applicability in studying physiological models.

(1) Highly selective electrochemical L-Cysteine sensing method in vivo: Cysteine (L-Cys), serving as an important molecule that regulates the redox atmosphere in the brain, is directly related to pathological processes such as Parkinson's disease (PD) and Alzheimer's disease (AD). Monitoring of its dynamics is of great significance to take an in-depth look into the cerebral pathological processes. Traditional electrocatalysts and electrochemical sensing methods are susceptible to the interference from glutathione (GSH) that shares similar structure with L-cys. We have designed and synthesized FeN_n-SACs with different coordination environments. Different coordination environment was capable for regulating the adsorption abilities between biothiol and SACs, resulting in a greatly improved selectivity towards L-cys against GSH. The combination of FeN₃C₁-SAC with GRP sensing method realizes the highly selective sensing of L-cys, opening a new modulating paradigm in the design of selective electrochemical in vivo sensing strategies.

(2) Highly selective electrochemical hydrogen sulfide sensing method in vivo: Hydrogen sulfide (H₂S) is an important gasotransmitter in the brain, which is directly related to many neurodegenerative diseases. Monitoring of its dynamics is of great significance to the understanding of neurophysiological and neuropathological processes. However, the electrochemical oxidation potential of H₂S is similar to the redox potential of other neurochemicals in the brain, and the level of H₂S changes together with reducing neurochemicals such as ascorbic acid (AA). We have designed and synthesized a SAC with NiN₄ catalytic sites, which selectively accelerates the electron transfer during H₂S oxidation. The SAC was modified on the bipolar-CFE (BCFE) anode and combined with the galvanic redox potentiometry (GRP) to overcome the challenge of poor stability caused by the adsorption of H₂S oxidation products on the electrode. Using this H₂S sensing method, we detected the H₂S release behavior of DJ-1 knockout (DJ-1 KO) mice and wild type (WT) mice, and found that the duration time of H₂S release was different. The construction of the electrochemical interface combines the advantages of the design of GRP and SAC, realizes the high selectivity and high stability of H₂S in vivo sensing, and provides a tool for brain neurochemistry research.

(3) Mechanism research of the neurochemical regulating function of Hydrogen peroxide: hydrogen peroxide (H₂O₂), as an important diffusive messenger and dynamic neuroregulator, it has the function of regulating the release of dopamine (DA) and neuronal excitability in the central nervous system (CNS). Whether in the nervous system or in any cell, the physiological and pathological effects of H₂O₂ depend on its concentration. However, the low levels of real-time produce physiological H₂O₂ in the process of neurophysiological neural function is unknown. Using a single-vesicle electrochemical approach, we show that extracellular nanomolar levels of H₂O₂ can promote the vesicular release of DA from cultured PC12 cells. Furthermore, we speculate that this promoting effect is due to the opening of K_ATP channels during exocytosis of low physiological levels of H₂O₂, increasing the size of the initial fusion pore for vesicle release. Studies on the neuromodulation of H₂O₂ at low physiological concentrations provide a solid theoretical basis for the regulation of H₂O₂ neurochemical signals.