Highly sensitive detection of human thrombin in serum by affinity capillary electrophoresis/laser-induced fluorescence polarization using aptamers as probes

The detection and quantification of disease-related proteins play critical roles in clinical practice and diagnostic assays. We present an affinity probe capillary electrophoresis/laser-induced fluorescence polarization (APCE/LIFP) assay for detection of human thrombin using a specific aptamer as probe. In the APCE/LIFP assay, the mobility and fluorescence polarization of complex are measured simultaneously during CE analysis. The affinity complex of human thrombin can be well separated from unbound aptamer on CE and clearly identified on the basis of its fluorescence polarization and migration. Because of the binding favorable G-quartet conformation potentially involved in the specific aptamer, it was assumed that monovalent and bivalent cations promoting the formation of a stable G quadruplex conformation in the aptamer may enhance the binding of the aptamer and thrombin. Therefore, we investigated the effects of various metal cations on the binding of human thrombin and the aptamer. Our results show that cations like K⁺ and Mg²⁺ could not stabilize the affinity complex. Without the use of typical cations, a highly sensitive assay of human thrombin was developed with the corresponding detection limits of 4.38 × 10⁻¹⁹ and 2.94 × 10⁻¹⁹ mol in mass for standard solution and human serum, respectively.