Engineering aptamer nanoarchitectures for electrochemical detection of Exosomal CD63

Electrochemical aptamer-based biosensors (E-ABs) have emerged as powerful tools for biomolecular detection, yet limited insight into binding-induced conformational dynamics has constrained their development to more efficient E-ABs. Here, we present a strategy that integrates in-silico docking with electrochemical nanoarchitecture engineering to identify optimal aptamer-target binding sites, thereby guiding the rational placement of redox labels to enhance analytical performance. Three E-AB electrodes were fabricated, each with a methylene blue placed at distinct sites within the aptamer. Electrochemical analysis revealed that the spatial positioning of redox labels critically governs the biosensing performance by modulating both binding affinity and signal transduction efficiency. Circular dichroism (CD) spectroscopy was further employed to investigate conformational changes in the engineered aptamers upon interaction with CD63, an exosomal biomarker. Notably, the configuration with a redox label positioned at the distal end achieved the lowest detection limit, the highest binding affinity, and robust performance in proof-of-concept biological models.