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

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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.