Enzyme-free ultrasensitive fluorescence detection of epithelial cell adhesion molecules based on a toehold-aided DNA recycling amplification strategy

Epithelial cell adhesion molecules (EpCAMs) play a significant role in tumorigenesis and tumor development. EpCAMs are considered to be tumor signaling molecules for cancer diagnosis, prognosis and therapy. Herein, an enzyme-free and highly sensitive fluorescent biosensor, with a combined aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy, was developed for sensitive and specific fluorescence detection of EpCAMs. Due to highly specific binding between EpCAMs and corresponding aptamers, strand a, which is released from the complex of aptamer/strand a in the presence of EpCAMs which is bound to the corresponding aptamer, triggered the toehold-mediated strand displacement process. An amplified fluorescent signal was achieved by recycling strand a for ultrasensitive EpCAM detection with a detection limit as low as 0.1 ng mL⁻¹, which was comparable or superior to that of reported immunoassays and biosensor strategies. In addition, high selectivity towards EpCAMs was exhibited when other proteins were selected as control proteins. Finally, this strategy was successfully used for the ultrasensitive fluorescence detection of EpCAMs in human serum samples with satisfactory results. Importantly, the present strategy may be also expanded for the detection of other targets using the corresponding aptamers.

A fluorescent biosensor with a combined aptamer-based EpCAM recognition and toehold-aided DNA recycling amplification strategy was developed.