Molecular recognition of a novel molecularly imprinted polymer on oligopeptides.
Date Issued
2006
Date
2006
Author(s)
Sung, Kuo-Ning
DOI
zh-TW
Abstract
In this work, I used peptide as template to make a MIP monolithic capillary and use this column to separate peptide samples in CEC. While trying to fabricate this column, the synthesis proceed encountered two limitation. The first one is that peptides are insoluble in organic solvents while the most often used MIP synthesis proceed were taken under organic environment. The second limitation is the size of template molecule. Using large molecule will give rise to steric effect and thermal disfavor. In order to overcome the first limitation, I tried to change the porogen and functional monomer to synthesize the MIP under water environment rather than to do derivation of peptide template. The second limitation was overcome by introducing the 『epitope approach』to MIP monolithic column formation that means we can use short-chain peptides as template instead of long-chain peptide. In the synthetic procedure, Tyr-Gly-Gly, methacrylic acid (as function monomer), N,N’-methylene bisacrylamide (as cross-linker) and α,α’-azobis(isobutyronitrile) (as initiator) were dissolved in porogen ( mixture of water and DMF [1:1, v/v] ) under 50 ℃water bath for 20 minutes. The solution was stirred for 10 minutes under room temperature, then it was filled in a fused silanized capillary. The mole ratio of template、functional monomer、cross-linker is 1：16.2：32.2. The filled capillary was polymerized by thermal initiated under 65.5℃ for 13.5 minutes to obtained a monolithic MIP column called Column-epitope . Column-epitope was washed by water / DMF(1:1)、MeOH / HAc(10:1) for two hours. The molecular recognition of Column-epitope due to epitope approach could be improved by separation 3 similar peptide, [Met5]-enkephalin, [Leu5]- enkephalin andβ-casomorphin Bovine. Other separation mechanisms such as relative mobility, hydrophobic force, electrostatic force…etc were also contributed to CEC separation. Column-epitope of 75 μm x 60 (35) cm with hydrostatic injection (10 cm x 10 sec), mobile phase of 5 % acetonitrile in 30 mM phosphate ( pH 4 ), applied voltage of voltage gradient 20 kV to 30 kV and detection UV 210 nm, eleven peptides, FRMF amide, β-casomorphin Human, β-casomorphin bovine, oxytocin, tocinoic acid, Angiotension I, Angiotension II, [Sar1,Thr8] Angiotension II, [Met5]-enkephalin, [Leu5]-enkephalin and template could be baseline separated within 40 minutes.
Subjects
烙印分子
毛細管電層析
MIP
epitope approach
CEC
Type
thesis