Chemiluminescently labeled aptamers as the affinity probe for interaction analysis by capillary electrophoresis

Aptamers are nucleic acid oligonucleotides, which can recognize targets with high affi-nity and specificity. Fluorescently labeled aptamers have been used as affinity probes in CE for interaction analysis. In this study, a method of labeling aptamers chemiluminescently with isoluminol isothiocyanate (ILITC) through covalent bonds was proposed and realized. The ILITC-labeled aptamers were characterized by HPLC-MS and purified by HPLC. After desalination, the ILITC-labeled aptamers were employed as the affinity probe for interaction analysis in CE coupled with chemiluminescence detection (CE-CL) by interface of end column reaction mode, the apparatus of which was home-designed and setup. CE-CL experiment conditions, including buffer pH, concentrations of horseradish peroxidase and H₂O₂, were optimized first. The system of thrombin and its 29-mer aptamer was chosen as the model. Binding parameters, namely the dissociation constant (K_d) and the binding site number (n), were calculated. The K_d obtained was 124.0±6.9 nM in agreement with the reported values. Thus, interaction analysis method based on chemiluminescently labeled aptamers as the affinity probe in CE-CL has been established. This method can be widely applied due to the ease and universality of the labeling method, simplicity of CE-CL apparatus and combination with aptamers for a wide range of targets.