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Poly‐ N ‐hydroxyethylacrylamide (polyDuramide™): A novel, hydrophilic, self‐coating polymer matrix for DNA sequencing by capillary electrophoresis
Author(s) -
Albarghouthi Methal N.,
Buchholz Brett A.,
Huiberts Piet J.,
Stein Thomas M.,
Barron Annelise E.
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200205)23:10<1429::aid-elps1429>3.0.co;2-a
Subject(s) - capillary electrophoresis , polymer , monomer , aqueous solution , microchannel , chemistry , chromatography , radical polymerization , polyacrylamide , molar mass , chemical engineering , analytical chemistry (journal) , polymer chemistry , materials science , organic chemistry , nanotechnology , engineering
A replaceable polymer matrix, based on the novel monomer N ‐hydroxyethylacrylamide (HEA), has been synthesized for application in DNA separation by microchannel electrophoresis. The monomer was found by micellar electrokinetic chromatography analysis of monomer partitioning between water and 1‐octanol to be more hydrophilic than acrylamide and N,N ‐dimethylacrylamide. Polymers were synthesized by free radical polymerization in aqueous solution. The weight‐average molar mass of purified polymer was characterized by tandem gel permeation chromatography‐multiangle laser light scattering. The steady‐shear rheological behavior of the novel DNA sequencing matrix was also characterized, and it was found that the viscosity of the novel matrix decreases by more than 2 orders of magnitude as the shear rate is increased from 0.1 to 1000 s –1 . Moreover, in the shear‐thinning region, the rate of change of matrix viscosity with shear rate increases with increasing polymer concentration. Poly‐ N ‐hydroxyethylacrylamide (PHEA) exhibits good capillary‐coating ability, via adsorption from aqueous solution, efficiently suppressing electroosmotic flow (EOF) in a manner comparable to that of poly‐ N,N ‐dimethylacrylamide. Under DNA sequencing conditions, adsorptive PHEA coatings proved to be stable and to maintain negligible EOF for over 600 h of electrophoresis. Resolution of DNA sequencing fragments, particularly fragments > 500 bases, in PHEA matrices generally improves with increasing polymer concentration and decreasing electric field strength. When PHEA is used both as a separation matrix and as a dynamic coating in bare silica capillaries, the matrix can resolve over 620 bases of contiguous DNA sequence within 3 h. These results demonstrate the good potential of PHEA matrices for high‐throughput DNA analysis by microchannel electrophoresis.