Investigation of the effects of metal ions in sample buffer on capillary electrophoresis coupled with laser-induced fluorescence analysis of thrombin using a 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 uses a fluorescently labeled aptamer probe and relies on the change of electrophoretic mobility of the 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 of thrombin. In this work, we made a further detailed investigation of the effects of metal ions (e.g. Na⁺, K⁺, and Mg²⁺) in sample buffer on the CE-LIF analysis of thrombin using a dye labeled 29-mer DNA aptamer probe and achieved sensitive detection of thrombin. A complex of thrombin and the 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 of thrombin using dye-labeled Apt29, and it caused significant decrease of complex peaks of thrombin–Apt29. The use of both Na⁺ and Mg²⁺ in sample buffer was favorable for the sensitive detection of thrombin because a larger peak area of complex peaks of thrombin and the aptamer probe was obtained. Under the optimized conditions, as low as 0.1 nM thrombin was successfully detected with good specificity. This work shows that metal ions in sample buffer have a large effect on aptamer affinity CE-LIF analysis of thrombin using an Apt29 probe.