Immobilization-free electrochemical homogeneous aptasensor for highly sensitive detection of carcinoembryonic antigen by dual amplification strategy

Electrochemical aptasensor has been widely studied, while its practical application is limited by the unavoidable variations of aptamer loading densities and low signal amplification efficiency. To overcome these restrictions, an immobilization-free and label-free electrochemical homogeneous aptasensor was constructed for carci- noembryonic antigen (CEA) assay by combining RecJf exonuclease-mediated target cycling strategy and rolling circle amplification technology. In this system, the pre-immobilization of aptamers or other relevant signal el- ements on the electrode substrate is no longer necessary, thus the electrochemical homogeneous aptasensor shows good versatility on different transducers. Moreover, the whole recognition and signal amplification pro- cess are activated instantaneously by a non-professional operation of the solution mixture. This strategy can not only increase the stability (95.1% after 30 days of storage) and reproducibility (2.12% among five independent electrodes), but also further improve the sensitivity (detection limit of fg mL-1 level) due to the free target recognition and dual signal amplification in the homogeneous solution phase. The proposed immobilization-free electrochemical homogeneous aptasensors on different electrode substrates both achieve satisfactory results in actual sample tests, which has the potential for commercial applications and the establishment of other target platforms in the future.