Highly sensitive fluorometric determination of thrombin by on-chip signal amplification initiated by terminal deoxynucleotidyl transferase

The article describes an on-chip amplification scheme initiated by a terminal deoxynucleotidyl transferase (TdT) for highly sensitive fluorometric determination of protein. Two thrombin-binding aptamers were designed to capture thrombin as they can form a sandwich structure for improved specificity. An amino-modified aptamer (TBA29) was first immobilized on a silicon chip. After capture of thrombin, a second aptamer (TBA15) was conjugated to the second binding site of thrombin. The 3’-terminal of aptamer TBA15 is exposed on the chip surface, and then fluorescein-labeled 12-dATP associates to the 3’-terminal with the help of TdT. This results in signal amplification, and eventually leads to highly sensitive detection. Under optimal conditions, fluorescence intensity is linearly related to the logarithm of thrombin concentration in the range of 100 fM - 0.1 μM, and the detection limit is as low as 2.0 fM. The assay is sensitive and selective even over potentially interfering proteins and in the presence of human serum. Schematic strategy for thrombin detection. Two thrombin-binding aptamers were designed to capture thrombin to form a sandwich structure for improved specificity. The protein detection is based on TdT initiated on-chip fluorescent amplification.