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On‐line chemiluminescence detection for isoelectric focusing of heme proteins on microchips
Author(s) -
Huang Xiangyi,
Ren Jicun
Publication year - 2005
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/elps.200500076
Subject(s) - horseradish peroxidase , luminol , myoglobin , chemistry , chemiluminescence , isoelectric focusing , hemeprotein , chromatography , heme , detection limit , peroxidase , cytochrome c , analytical chemistry (journal) , biochemistry , enzyme , mitochondrion
In this paper we present a sensitive chemiluminescence (CL) detection of heme proteins coupled with microchip IEF. The detection principle was based on the catalytic effects of the heme proteins on the CL reaction of luminol‐H 2 O 2 enhanced by para ‐iodophenol. The glass microchip and poly(dimethylsiloxane) (PDMS)/glass microchip for IEF were fabricated using micromachining technology in the laboratory. The modes of CL detection were investigated and two microchips (glass, PDMS/glass) were compared. Certain proteins, such as cytochrome c , myoglobin, and horseradish peroxidase, were focused by use of Pharmalyte pH 3–10 as ampholytes. Hydroxypropylmethylcellulose was added to the sample solution in order to easily reduce protein interactions with the channel wall as well as the EOF. The focused proteins were transported by salt mobilization to the CL detection window. Cytochrome c , myoglobin, and horseradish peroxidase were well separated within 10 min on a glass chip and the detection limits (S/N = 3) were 1.2×10 −7 , 1.6×10 −7 , and 1.0×10 −10 M , respectively.