Serum-compatible screening and truncation optimization of copeptin aptamers for TdT-amplified fluorescent detection

Copeptin is a clinically relevant biomarker for early diagnosis and prognostic assessment of acute myocardial infarction (AMI). Traditional antibody-based assays suffer from long testing time, high cost and complicated operation. Herein, we first screened high-affinity copeptin aptamers under serum-mimicking conditions and built a sensitive fluorescent aptasensor using terminal deoxynucleotidyl transferase (TdT)-assisted signal amplification. After three rounds of capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX), six candidate aptamer sequences were obtained. Among them, Apt31, showed the highest binding affinity and was further truncated and optimized to generate Apt31-1, which had a dissociation constant of 19.8 nM. This represented an approximately 2.5-fold improvement in affinity compared with the original full-length aptamer. Using Apt31-1 as the recognition element, a fluorescent aptasensor was constructed by combining TdT-mediated poly(thymine) extension with copper nanoparticle generation. For copeptin detection in buffer and human serum, the sensor showed a linear detection range of 10-200 pg/mL and a low limit of detection of 2.9 pg/mL. The results also showed strong agreement with those obtained by ELISA, with a correlation coefficient of 0.9920. Overall, this study presents a sensitive aptamer-based strategy for copeptin detection and demonstrates the potential of combining serum-compatible aptamer screening with isothermal signal amplification for clinical biomarker analysis.