Jason B. Shear
Department of Chemistry and Biochemistry
The University of Texas at Austin
Austin, TX 78712
1998 Searle Scholar
Research in the Shear lab is directed toward bioanalytical technique development with the goal of characterizing fundamental chemical properties of individual neurons. An ideal analysis approach for studying neurotransmission at individual synapses would provide chemical profiles of many neurotransmitter species with millisecond time resolution and with a sensitivity capable of quantitatively assaying the contents of the smallest packets (or "vesicles") of neurotransmitter -- approximately 1000 molecules. Toward these goals, we are actively involved in development of rapid microcolumn chemical separation procedures and novel detection approaches that draw on recent advances from both the biology and physics communities.
The development of microcolumn separation procedures in the 1980s has opened new possibilities in single cell analysis. Capillary electrophoresis (CE), the most common microcolumn technique, can accomodate sample volumes as small as 100 femtoliters and can separate components in complex mixtures with efficiencies in excess of 10 E6 theoretical plates. Although CE separations typically require 5 to 20 minutes, recent work has demonstrated the feasibility of performing repetitive CE separations at rates greater than one per second (Moore and Jorgensen (1993) Anal. Chem. 65, 3550). We are examining possibilities for expanding the utility and efficiency of this technique, with the goal of studying changes in the secretory activity of cultured neurons during different stimulation patterns. The results of such studies should help clarify cellular and chemical mechanisms of learned responses.
Detection of minute quantities of biomolecules is as challenging a task as fractionation, and we are pursuing two main strategies to achieve the necessary sensitivities. Laser-induced fluorescence detection is currently the most sensitive detection approach for CE, but the sensitivity is ultimately limited by interference from scatter. For fluorophores that are ordinarily excited using UV or blue light, background caused by scatter can be virtually eliminated when analytes are excited via absorption of two or three photons of near-IR light from a mode-locked laser source. Because the excitation wavelength is significantly longer than the fluorescence wavelengths, discrimination of fluorescence from scatter is easily accomplished, and detection sensitivities can be improved by up to several orders of magnitude. We are currently devising approaches for coupling multiphoton excited (MPE) fluorescence measurements with capillary separations for measurement of both natively fluorescent neurotransmitters (e.g. catecholamines, serotonin, and neuroactive peptides) and of neurotransmitters tagged with fluorogenic reagents.
We are also developing measurement strategies that rely on the highly sensitive and selective detectors that cells have already developed -- neurotransmitter receptors. Use of entire cultured neuronal-type cells as postcolumn detectors for CE has been demonstrated previously (Shear et al. (1995) Science 267, 74), but improvements in sensitivity, reproducibility, and quantitation are possible in some cases by using detectors that rely on isolated neurotransmitter receptors. Such "biosensors" offer the possibility for measurement of neurotransmitters at the single vesicle level, even for species such as acetylcholine that are difficult to detect with traditional measurement approaches.
|SITE MAP CONTACT US||© COPYRIGHT 2017 KINSHIP FOUNDATION. ALL RIGHTS RESERVED.|