Figure 2.

Formation of the aptamer–thrombin complex. (A) Optimization of aptamer hybridization length. F, Q, and T represent the fluorophore-modified aptamer (TBA15-F), the quencher-modified aptamer (TBA29-Q), and thrombin, respectively. (F + Q)/F is the fluorescence intensity ratio between F + Q and F only, and (F + Q + T)/F is the fluorescence intensity ratio between the complex F + Q + T and F only. The concentrations of aptamers and thrombin were 200 nM. (B) Optimization of aptamer concentration. Different concentrations of aptamers and thrombin with identical molar ratios were tested. (C) Buffer selection. Assay performance was tested in different buffers, including Tris-HCl buffer with K⁺ and Na⁺ (20 mM Tris-HCl, 5 mM KCl, 140 mM NaCl, pH 7.5), Tris-HCl buffer with K⁺, Na⁺, Mg²⁺, and Ca²⁺ (20 mM Tris-HCl, 5 mM KCl, 140 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, pH 7.5), PBS buffer, and PBS buffer with 1 mM MgCl₂ and 1 mM CaCl₂. All intensities were normalized by F. (D) Molar ratio optimization between aptamers and thrombin. Concentration was 200 nM each for the two aptamers. All intensities were normalized by F. (E) Comparison between FQT ratio 1:1:1 and 1:1:1.25 with the addition of AT. (F) Kinetics of aptamer–thrombin complex formation. All data were normalized by the fluorescence intensity at 0 min.