Use of capillary electrophoresis with chemiluminescence detection for sensitive determination of homocysteine
Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education), College of Chemistry and Chemical Engineering, Guangxi Normal University, Guilin, P. R. China.Journal of Separation Science (Impact Factor: 2.74). 01/2012; 35(2). DOI: 10.1002/jssc.201100650
A sensitive capillary electrophoresis (CE) method with chemiluminescence (CL) detection was developed for the determination of homocysteine (HCys) in human plasma. In this work, N-(4-aminobutyl)-N-ethylisoluminol was used as tagging reagent to label the analyte for achieving high assay sensitivity. N-(4-Aminobutyl)-N-ethylisoluminol-tagged HCys after CE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase, producing CL emission. Experimental conditions for labeling analyte, CE separation, and CL detection were studied. The CL intensity was proportional to the concentration of HCys in the range of 2.5×10(-8) to 5.0×10(-6) M. Detection limit (S/N=3) was 7.6×10(-9) M. Human plasma samples from healthy donors were analyzed by the presented method. HCys levels were found to be in the range of 9.50-15.3 μM.
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ABSTRACT: A capillary electrophoresis method based on dual-enzyme co-immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co-immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. Enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed capillary electrophoresis method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screen system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two enzyme inhibition by the present method. This article is protected by copyright. All rights reserved.Journal of Separation Science 08/2013; 36(15). DOI:10.1002/jssc.201300315 · 2.74 Impact Factor
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