The main objective of this project is to advance in the solution of the analytical problem associated with the still unsatisfactory hydrolytic stability of current organic films that coat the inner-wall of the fused-silica tubes used in capillary electrophoresis (CE). The purpose is to fully exploit the superb efficiency of CE by significantly decreasing the limitation due to the undesirable interactions between the analyte and the silica surface. The ultimate goal is to obtain clear-cut separation profiles that enable better characterization of the protein species present in complex samples from a variety of tissues or bodily fluids. The project will focus on the development of chemically modified capillaries through a three-step process that starts with the formation of an inorganic silicon hydride precursor on the silica surface. Next, attachment of benzyl chloride groups is accomplished by reacting the hydride intermediate with a terminal olefin, p-vinyl-benzyl chloride, in the presence of a Pt- catalyst. Finally, polymerization of an N-substituted acrylamide monomer (namely, N-acryloyl- aminopropanol), initiated at the benzyl chloride group in the presence of a Cu-catalyst, produces a hydrophilic layer chemically attached to the silica surface. Thus, our strategy brings together the best of three worlds: first, a very stable anchorage of the coating to the inner wall of the capillary tube by means of Si-C linkages;second, a similarly stable polymeric film whose strength arises from the N-substitution on acrylamide;and third, an in-situ "living" polymerization method that permits easy control of the coating thickness. The fundamentals of the various phases of the project have been set out in previous work of this researcher as well as that of others, and their experimental soundness has been separately demonstrated. The modified surfaces will be characterized by IR and solid-state NMR spectroscopies, atomic force microscopy, contact-angle measurements, etc. Naturally, the CE migration profile of a number of chemical probes will also be thoroughly examined and, eventually, analytical procedures will be developed for selected proteome samples. A thorough assessment of the stability of all new bonded materials will also be an essential part of this work. A successful completion of this project should have a significant impact on proteomics, the analytical characterization of protein species and the elucidation of their biological role in cell function.

Public Health Relevance

Analytical mapping of protein molecular species in human tissues and fluids is extremely important from a public health point of view because understanding protein composition and fate will provide valuable insights into the mechanisms of many diseases and important implications for drug development. This project will pursue the development of an improved CE coating to move toward the solution of the analytical problem associated with the less than satisfactory separation and quantification of proteins by current CE methodologies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM089759-03
Application #
8323809
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Edmonds, Charles G
Project Start
2010-09-15
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$29,164
Indirect Cost
Name
University of Valle
Department
Type
DUNS #
880188859
City
Cali
State
Country
Colombia
Zip Code
N/A
Espinal, Jose H; Gomez, Jorge E; Sandoval, Junior E (2013) Closer look at the operating definition of protein recovery in CE. Electrophoresis 34:1141-7