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|Title:||Capillary Electrophoretic Technologies for Single Cell Metabolomics|
|Author(s):||Lapainis, Theodore E.|
|Doctoral Committee Chair(s):||Sweedler, Jonathan V.|
|Department / Program:||Chemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Understanding the functioning of the brain is hindered by a lack of knowledge of the full complement of neurotransmitters and neuromodulatory compounds. Single cell measurements aid in the discovery of neurotransmitters used by small subsets of neurons that would be diluted below detection limits or masked by ubiquitous compounds when working with larger brain regions. Also, as neurochemistry can be different even in adjacent neurons, single cell measurements allow a unique perspective on cell-cell signaling in the brain. Here several instrument platforms have been created that combine capillary electrophoresis (CE) with information-rich detection methods in order to perform single cell measurements. CE is an appropriate separation technique because of its compatibility with the small volumes of the cellular samples, as well as its ability to carry out online sample enrichment.
A multichannel laser-induced native fluorescence detection system was designed, constructed, and coupled to CE. The system uses a unique laser that provides efficient excitation for the catecholamines, resulting low nanomolar limits of detection (LODs), e.g., 40 nM LODs for dopamine. In addition, the multichannel detector provides a spectral "fingerprint" by which metabolites can be characterized. This instrument was used to detect the neurotransmitters present in single Lymnaea stagnalis neurons, and to differentiate the detected neurotransmitters based on spectral characteristics.
Electrospray ionization mass spectrometry was also coupled to CE in order to provide a more comprehensive view of the cellular metabolome. A nebulizer-free sheath flow interface was developed that can provide low nanomolar (attomole) LODs for a variety of small molecule neurotransmitters. The utility of this platform for metabolomic profiling of individual neurons was demonstrated by analyzing cells from Aplysia californica.
One strategy for reducing detection limits is to concentrate metabolites prior to detection. To this end, the utility of dynamic field gradient focusing (DFGF) for use in metabolomics was evaluated. DFGF uses an electric field gradient and a buffer counterflow to focus charged analytes. A prototype DFGF system was installed, and online coupling of DFGF to mass spectrometry was then demonstrated. Several design modifications were then identified and investigated to adapt small molecule DFGF to the microfluidic regime.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.
|Date Available in IDEALS:||2014-12-17|