Characterization of the Specific Binding Between Aptamers and Cytochrome <i>c</i> With Pressure‐Assisted Capillary Electrophoresis Frontal Analysis

Cytochrome c (cyt c) is a heme protein located in the mitochondrial intermembrane space. Because the release of cyt c is a highly specific event in apoptotic signaling, it can serve as an apoptosis-related marker. To date, three frequently used aptamers for cyt c (Apt40, Apt61, and Apt76) have been selected and applied in the field of sensing. The response of these aptamers is not clear, partly because of their weak affinity and nonspecific binding inherent to the system. In this study, pressure-assisted capillary electrophoresis frontal analysis (PACE-FA) was used to characterize the interactions between the aptamers and cyt c, and an electrophoretic mobility-based correction was introduced to obtain accurate binding constants. A nonlinear curve-fitting approach was used for evaluating specific binding interactions in the presence of nonspecific binding. Apt76 was found to bind specifically to cyt c, exhibiting the highest binding constant (1.53 × 10⁶ M^-1), and all three aptamers interacted with cyt c at 1:1 stoichiometry. Fluorescence titrations were performed to verify the effectiveness of the reference-free PACE-FA method. This study demonstrates that specific binding between biomolecules has different characteristics compared to nonspecific binding and that the PACE-FA method can be widely used in the evaluation of biological macromolecular interactions.