Combining Chemical Reaction with Single Cell Mass Spectrometry for Real-time Quantification of Nitric Oxide (NO) Inside Live Single Cells

将化学反应与单细胞质谱法相结合，实时定量活单细胞内的一氧化氮 (NO)

• 批准号: 2305182
• 负责人: Zhibo Yang
• 金额: $ 41万
• 依托单位: University of Oklahoma Norman Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2305182&HistoricalAwards=false
• 关键词: Combining; Chemical; Reaction; Single; Cell

With support from the Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry (CHE) and the Established Program to Stimulate Competitive Research (EPSCoR), Zhibo Yang and his group at the University of Oklahoma are investigated methods for the possible detection and quantification of nitric oxide (NO) in single cells. NO is a small molecule important for human health and diseases. The production and concentration of NO is tightly regulated as this is an important signaling molecule in healthy biology; deviations in NO concentration can also potentially lead to biological dysfunction; hence methods to accurately detect NO levels, particularly in living systems are in high demand. Because the concentrations of NO in cells are very different from cell to cell, meaningful studies need to be performed at the single-cell level. However, detecting and quantifying NO in single cells is very challenging, primarily because of its instability and low abundance (e.g., a cell diameter is ~1/10 of that of human hair). Dr. Yang and his group will design a microscale device that can be coupled to a sensitive analytical tool, mass spectrometry (MS). This device can directly extract NO from single cells, and then use online chemical reactions to convert it into a stable molecule for sensitive detection and accurate quantification using MS. This new technique can potentially offer a new analytical tool for the measurement of oxidants such as NO at the single level. The summer outreach program is expected to provide lesson development for science teachers at Oklahoma high schools through school-university-community collaborations. The products (e.g., lecture materials, lessons, and survey results) from the outreach program will be accessible by other high schools and general public. In addition, conducting the research will provide professional development for undergraduate and graduate students.NO is a small bioactive molecule playing important roles in numerous cell functions that are relevant to neuronal signaling, immune response, and human disease. The functions of NO are related to its abundance in cells. Due to cell heterogeneity, which has been reported in nearly all biological systems, the abundance of NO significantly varies from cell to cell. Quantification of NO in individual cells could substantially improve our understanding of the functions and mechanisms of NO in biological systems. However, these studies are very challenging, primarily because of the extremely small size of single cells and the reactive, diffusive nature of NO. This proposal combines chemical reactions with single cell mass spectrometry (SCMS) to detect and quantify NO in single cells. Cell lines will be used as model systems to produce endogenous and exogenous NO. An established single-probe SCMS experimental setup will be combined with off-line chemical reactions for NO measurement. The key diagnostic reaction involves the two-electron oxidation of amlodipine (AML) to dehydroamlodipine (DAM ). Since this reaction involves the simple removal of the elements of "H-H" from AML, the observation of DAM is an indirect measure of NO, and it will be important to control for other two-electron oxidation reactions that could, in principle, produce DAM from AML. Perhaps most notably, as part of these studies, a new device, the elongated single-probe (eSingle-probe), is being developed and will be used for real-time reactive (rrSCMS) analysis for NO in single cells.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在化学系 (CHE) 化学测量和成像 (CMI) 项目和刺激竞争性研究既定项目 (EPSCoR) 的支持下，俄克拉荷马大学杨志博和他的团队研究了可能的检测方法和单细胞中一氧化氮 (NO) 的定量。 NO 是一种对人类健康和疾病很重要的小分子。 NO 的产生和浓度受到严格调控，因为它是健康生物学中重要的信号分子； NO 浓度的偏差也可能导致生物功能障碍；因此，准确检测一氧化氮水平的方法，特别是在生命系统中，非常需要。 由于细胞中 NO 的浓度因细胞而异，因此需要在单细胞水平上进行有意义的研究。然而，检测和定量单细胞中的 NO 非常具有挑战性，主要是因为它的不稳定和丰度低（例如，细胞直径约为人类头发直径的 1/10）。杨博士和他的团队将设计一种微型设备，可以与灵敏的分析工具质谱（MS）相结合。该装置可以直接从单细胞中提取NO，然后利用在线化学反应将其转化为稳定的分子，以便利用MS进行灵敏检测和准确定量。这项新技术有可能为单一水平的 NO 等氧化剂的测量提供一种新的分析工具。夏季外展计划预计将通过学校-大学-社区合作为俄克拉荷马州高中的科学教师提供课程开发。外展计划的产品（例如讲座材料、课程和调查结果）将可供其他高中和公众使用。此外，开展这项研究将为本科生和研究生提供专业发展。NO是一种生物活性小分子，在与神经信号、免疫反应和人类疾病相关的众多细胞功能中发挥着重要作用。 NO的功能与其在细胞中的丰度有关。由于几乎所有生物系统中都存在细胞异质性，NO 的丰度因细胞而异。对单个细胞中 NO 的定量可以极大地提高我们对生物系统中 NO 功能和机制的理解。然而，这些研究非常具有挑战性，主要是因为单细胞的尺寸极小以及 NO 的反应性、扩散性。该提案将化学反应与单细胞质谱 (SCMS) 相结合，以检测和定量单细胞中的 NO。细胞系将用作模型系统来产生内源性和外源性NO。已建立的单探针 SCMS 实验装置将与离线化学反应相结合，用于 NO 测量。关键的诊断反应涉及氨氯地平 (AML) 到脱氢氨氯地平 (DAM) 的双电子氧化。 由于该反应涉及从 AML 中简单去除“H-H”元素，因此观察 DAM 是 NO 的间接测量，并且控制原则上可能产生 DAM 的其他双电子氧化反应非常重要来自反洗钱。也许最值得注意的是，作为这些研究的一部分，正在开发一种新设备，即细长单探针 (eSingle-probe)，并将用于单细胞中 NO 的实时反应 (rrSCMS) 分析。该奖项反映了通过使用基金会的智力价值和更广泛的影响审查标准进行评估，NSF 的法定使命被认为值得支持。