Capillary electrophoresis instrumentation and applications for the analysis of neural systems

Capillary Electrophoresis;Tryptophan Metabolism;Serotonin;Mast Cell

Dailey, Christopher

关键词: Capillary Electrophoresis;    Tryptophan Metabolism;    Serotonin;    Mast Cell;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/42454/Christopher_Dailey.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Capillary electrophoresis (CE) separations for the analysis of the chemically-complex environment of the nervous system have been demonstrated to be an effective tool in neuroscience.The inherent scaling laws of CE allow for the analysis of low-volume samples at high separation efficiencies which provide a platform for the study of neural systems. Here, CE systems using laser-induced, native-fluorescence (LINF) detection are described along with its applications for neural analysis across a variety of metazoan life.Selective detection of analytes after separation allows for the expedited analysis of complex biological systems by substantially decreasing the electropherogram complexity and reducing background interference.Many tryptophan and tyrosine metabolites present in biological systems are amenable to LINF detection.An enhanced wavelength-resolved CE-LINF instrument was developed in an effort to take advantage of improvements in technology and electronics to increase sensitivity and versatility over prior, similar instruments our lab has developed.The enhanced instrument uses a frequency-doubled, argon-ion laser operating in the deep ultraviolet (UV) region (either 264 or 229 nm) for efficient excitation of indolamines and catecholamines as well as an UV enhanced charge-coupled device for wavelength-resolved detection.Using 264 nm excitation detection limits were 1 nM for serotonin and 36 nM for dopamine, approximately 5-fold improvement for both as compared to previous systems.Additionally, the use of 229 nm excitation improved limits of detection for tyrosine metabolites by a factor of 2-3 over excitation at 264 nm. Our enhanced, wavelength-resolved CE-LINF system has been used to investigate a variety of model systems. Using the mouse as a mammalian model, we have demonstrated a significant, non-neuronal source of serotonin to the hippocampus from nearby mast cells, a major component of the immune system.Also, using a genetically modified strain of Drosophila melanogaster, we have developed a single-neuron sampling method which stabilizes the neuron by adding a temperature-gated arrest of neurosecretory activity.This strain resulted in improved reliability for the detection of serotonin in well-characterized neuron as well as the detection of tryptamine which had not been previously detected. Additionally, we have investigated the absence of serotonin and many other classical neurotransmitters in the basal phyla Ctenophore which compliments genomic studies by our collaborators indicating a lack an enzymatic pathway.While serotonin is an important and well-studied tryptophan metabolite, we have begun expanding our system to detect metabolites in the kynurenine pathway. In fact, this pathway is responsible for the majority of tryptophan metabolism in mammalian physiology and results in the neuroactive compounds kynurenine and kynurenic acid which are natively fluorescent.An automated version of a CE-LINF instrument was optimized and evaluated for the quantitation of both tryptophan and tyrosine metabolites.This system uses a metal-vapor, He-Ag laser with 224 nm output for excitation and a tunable emission detection system using a spectrometer and photomultiplier tube, all relatively low-cost components.The system demonstrated excellent linearity over several orders of magnitude of concentration and intraday precision of 1–11% relative standard deviation.Limits of detection ranged from 4 to 30 nmol/L for serotonin and tyrosine, respectively.Extracts from mammalian brain stem were analyzed for serotonin and tryptophan with an intraday precision of less than 10%.