An Ultrasensitive Duplex Aptasensor Featuring the Target-Triggered Lanthanide Luminescence Antenna Effect for Wash-Free Detection of Epithelial Cancerous Exosomes

Fluorescence sensing is widely used in in vitro detection due to its high sensitivity and rapid result delivery. However, detection systems based on nanomaterials involving complex and cumbersome purification steps can lead to sample loss and significantly reduce the accuracy of the results. To address this issue, we proposed a lanthanide-based aptasensor featuring the target-triggered antenna effect to significantly enhance the time-resolved luminescence (TRL) of chelated Tb³⁺ combined with a wash-free strategy. This sensor enabled highly sensitive detection of the epithelial cell adhesion molecule (EpCAM) on exosomes from epithelial tumors, eliminating the necessity for purification steps and thereby delivering more stable and reliable results. Specifically, using computer-aided design, a sequence capable of self-folding into a stable hairpin structure (Antenna-Hairpin-Tb) was obtained. After hybridization with an EpCAM-specific aptamer (Apt_EpCAM), a duplex aptasensor (Probe-Tb) was constructed for detection. When the targeted binding occurred between the Apt_EpCAM and exosomes, the Antenna-Hairpin-Tb dehybridized from the Probe-Tb and underwent self-folding, leading to a significant sensitized TRL signal due to the antenna effect-induced energy transfer from the antenna ligand to the chelated Tb³⁺. Over a broad range of exosome concentrations (spanning from 6.19 × 10² to 6.19 × 10⁸ particles/mL), the emission intensity of chelated Tb³⁺ at 543 nm linearly increased with the exosome concentration (linear relationship: Y = 332.62 lg(N_exosomes) + 3690.5, R² = 0.9956), achieving a theoretical detection limit as low as 17 particles/mL in the buffer. Furthermore, Probe-Tb enabled a swift and effective differentiation of all epithelial cancer serum samples from nonepithelial ones within just 30 min of a wash-free operation, achieving 100% sensitivity, 100% specificity, and 100% accuracy.