Instrumental optimization and operation for the analysis of reactive oxygen species and glutathione in single cells and small volume samples

The remarkable chemical diversity of the biological landscape requires information rich approaches to broaden our understanding of the heterogeneity that exists within the tissues and cells of organisms. It seems intuitive that different tissues will exhibit stark heterogeneity in terms of the chemical composition compared to one another, but this chemical diversity even exists between neighboring cells within the same tissue section thus highlighting the need for single cell analysis. Capillary electrophoresis (CE) as the separation mechanism coupled with laser-induced fluorescence (LIF) detection is a particularly powerful combinatory technique for single cell analysis due to its ability to probe both mass and volume limited samples, produce high separation efficiencies, and detect and quantitate low abundance analytes. This study hyphenates a single-beam optical trap with CE and multichannel LIF detection. Cell localization is achieved with the trap’s utilization of light’s ability to impart momentum on the order of 10-12 N, and then a micromanipulator controlled using a computer allows for the positioning of the capillary inlet near the cell. Single cell injections can also be attained via the positioning of the capillary inlet near a cell using a micromanipulator followed by aspiration of the cell using a pressure gradient. Cell lysis is achieved within the capillary inlet via hydrodynamic injection. The sample is then separated using -30 kV in a CE separation and detected via LIF. The system enables injections of individual HL-60 cells that have been stained with the fluorescent dye naphthalene-2,3-dicarboxaldehyde (NDA) to impart fluorescence to the ubiquitous scavenger molecule glutathione and dihydrorhodamine-123 (DHR-123), which reverts to its fluorescent form of Rh-123 upon exposure to reactive oxygen species (ROS). The detection of glutathione derivatized with NDA allows for a quantitative measure of the cellular response to the presence of ROS and the detection of Rh-123 allows for a direct measurement of the exogenous ROS that have come in contact with the cell.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Ehsan, Mohammad

Contributors dc:contributor

• Sweedler, Jonathan V.

• Fluorescence Detection
• Capillary Electrophoresis
• Optics
• Optical Trap
• Optical Tweezers
• Wavelength Resolved

Statement dc:rights

• Copyright 2014 Mohammad Ehsan

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/50501

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F50501