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Investigation of Effects of Metal Ions in Sample Buffer on Capillary Electrophoresis Coupled with Laser-Induced Fluorescence Analysis of Thrombin Using Dye-Labeled 29-mer DNA Aptamer

Abstract

Thrombin is an important serine protease in blood and a therapeutic biomarker. The aptamer-based assays for thrombin take advantage of unique features of nucleic acid aptamers in selection, preparation, stability, and modification of functional groups. Aptamer affinity capillary electrophoresis coupled with laser induced fluorescence (CE-LIF) analysis for thrombin applies fluorescently labeled aptamer probe and relies on the change of electrophoretic mobility of aptamer probe caused by protein-aptamer binding. A high-affinity 29-nt DNA aptamer (Apt29) has been used as an affinity probe in CE-LIF analysis for thrombin. In this work, we made a further detailed investigation about the effects of metal ions (e.g. Na+, K+, and Mg2+) in sample buffer on the CE-LIF analysis of thrombin using the dye labeled 29-mer DNA aptamer probe and achieved sensitive detection of thrombin. The complex of thrombin and Apt29 probe was well separated from the unbound probe in CE separation. We found that the addition of K+ in sample buffer was not preferred in CE-LIF analysis for thrombin using dye-labeled Apt29, and it caused significant decrease of complex peak of thrombin-Apt29. The use of both Na+ and Mg2+ in sample buffer was favorable for sensitive detection of thrombin because larger peak area of complex peaks of thrombin and aptamer probe was obtained. Under the optimized conditions, as low as 0.1 nM thrombin was successfully detected with good specificity. This work shows the metal ions in sample buffer have large effect in aptamer affinity CE-LIF analysis of thrombin using Apt29 probe.

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Publication details

The article was received on 23 Jul 2017, accepted on 02 Sep 2017 and first published on 04 Sep 2017


Article type: Paper
DOI: 10.1039/C7AY01791J
Citation: Anal. Methods, 2017, Accepted Manuscript
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    Investigation of Effects of Metal Ions in Sample Buffer on Capillary Electrophoresis Coupled with Laser-Induced Fluorescence Analysis of Thrombin Using Dye-Labeled 29-mer DNA Aptamer

    Y. Bai and Q. Zhao, Anal. Methods, 2017, Accepted Manuscript , DOI: 10.1039/C7AY01791J

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