Development of radionuclide detectors for use in capillary electrophoresis
Tracht, Scott
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Permalink
https://hdl.handle.net/2142/21145
Description
Title
Development of radionuclide detectors for use in capillary electrophoresis
Author(s)
Tracht, Scott
Issue Date
1996
Doctoral Committee Chair(s)
Sweedler, Jonathan V.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Chemistry, Biochemistry
Language
eng
Abstract
The development of two radionuclide detection methods for capillary electrophoresis (CE) is described. The methods are used to determine the neuropeptide content of single neurons. The application of CE to single cell studies by several research groups is also reviewed with an emphasis on the variety of detection schemes and sampling protocols developed for assaying these complex micro-environments.
In the first postcolumn radionuclide detection system for capillary electrophoresis, eluant from an electrophoresis capillary is directed onto a peptide binding membrane previously coated with a solid scintillator. The membrane is moved in a preselected pattern relative to the fixed capillary outlet during electrophoresis. Light emission from the scintillation is imaged onto a charge-coupled device (CCD) using a series of 35 mm camera lenses. Detection of two low energy beta emitters (35S and 3H) not previously reported for capillary electrophoresis is demonstrated. The separation efficiencies are similar to those obtained with on-line UV detection. The response for 35S labeled methionine is linear from 66 amol to 11 fmol. Detection limits are: 88 zmol (0.03 Bq) for 32P labeled analytes, 17 amol (0.94 Bq) for 35S labeled analytes, and 8 fmol (8.5 Bq) for 3H labeled analytes.
The second detection scheme uses a commercial phosphor-imaging detector, and has been optimized for low energy beta emitters. Eluant from the separation capillary is deposited on a membrane. Emission from radioactive analytes on the membrane is integrated using the phosphor-imaging system for 10 to 72 hours. Results from the phosphor-imaging system are converted to conventional electropherograms. Modifications to a prior postcolumn CE deposition system have been accomplished by adding a buffer make-up capillary; this broadens the electrolyte pH range and improves reproducibility. The limit of detection (LOD) for 35S labeled analytes is 0.13 amol (8.7 pM or 0.007 Bq), while the LOD for 32P labeled analytes is 4.9 zmol (0.33 pM or 0.002 Bq), with a linear range for 35S-met of 1.5 amol to 1.5 fmol. Two neuropeptides in a single Aplysia californica neuron are tentatively identified using 35S-met labeling followed by analysis by CE with radiochemical detection.
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