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)化学测量与成像(CMI)计划和已建立的刺激竞争性研究(EPSCoR)计划的支持下，俄克拉荷马大学的杨志波和他的团队研究了可能检测和定量单细胞中一氧化氮(NO)的方法。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的调控，因为它是健康生物学中的重要信号分子；NO浓度的偏差也可能导致生物功能障碍；因此，迫切需要准确检测NO水平的方法，特别是在生命系统中。由于不同细胞中NO的浓度差异很大，因此需要在单细胞水平上进行有意义的研究。然而，检测和定量单个细胞中的NO是非常具有挑战性的，主要是因为其不稳定和低丰度(例如，细胞直径约为人头发的1/10)。杨博士和他的团队将设计一种微型设备，它可以与一种灵敏的分析工具--质谱仪(MS)相结合。该装置可以直接从单个细胞中提取NO，然后利用在线化学反应将其转化为稳定的分子，用于灵敏的检测和准确的MS定量。这一新技术可能为在单个水平上测量NO等氧化剂提供一种新的分析工具。暑期推广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发展。推广计划的产品(例如，讲座材料、课程和调查结果)将供其他高中和普通公众使用。此外，进行这项研究将为本科生和研究生提供专业发展。NO是一种小的生物活性分子，在许多与神经元信号、免疫反应和人类疾病相关的细胞功能中发挥重要作用。NO的功能与其在细胞内的丰度有关。由于细胞的异质性，几乎在所有的生物系统中都有报道，NO的丰度在不同的细胞之间有很大的差异。对单个细胞中NO的量化可以极大地提高我们对NO在生物系统中的功能和机制的理解。然而，这些研究是非常具有挑战性的，主要是因为单个细胞的尺寸非常小，以及NO的反应和扩散性质。该方案将化学反应与单细胞质谱仪(SCMS)相结合，以检测和定量单细胞中的NO。细胞系将被用作产生内源性和外源性NO的模型系统。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的测量。关键的诊断反应涉及氨氯地平(AML)的两电子氧化生成脱氢氨氯地平(DAM)。由于这一反应涉及从AML中简单地去除“H-H”元素，因此对DAM的观测是NO的间接测量，因此对原则上可能从AML中产生DAM的其他两电子氧化反应的控制将是重要的。也许最值得注意的是，作为这些研究的一部分，一种新的设备，细长的单探头(eSingle-Probe)正在开发中，将用于单细胞中NO的实时反应(RrSCMS)分析。该奖项反映了NSF的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。