Ultrasensitive detection of carcinoembryonic antigen based on exonuclease Ⅲ-assisted recycling and hybridization chain reaction strategies

In this work, we developed a novel ratiometric electrochemical aptasensor for the sensitive detection of carcinoembryonic antigen (CEA). Upon the addition of CEA, the specific binding between CEA and hairpin probe 1 (HP1) could trigger the Exo Ⅲ cleavage reaction and release large amounts of single stranded DNA (S1), which then hybridized with thiolated ferrocene-labeled hairpin probe 3 (Fc-HP3) on the electrode surface. Exo Ⅲ then cyclically digested the resulting duplexes to generate Fc-HP3 fragments on the electrode, accompanying the Fc molecules far away from the electrode surface and the Fc oxidation peak current signal (I Fc ) decreased. The HCR process was triggered by the Fc-HP3 fragment as an initiator, producing many long DNA sequences for electroactive probe methylene blue (MB) intercalation; thus, the MB oxidation peak current signal (I MB ) increased. The I MB /I Fc ratio was proportional to the logarithm value of CEA concentration from 100 fg mL −1 to 50 ng mL −1 , with a lower detection limit of 30.5 fg mL −1 . Furthermore, the aptasensor exhibited good selectivity and stability, with satisfactory reproducibility. Additionally, the developed aptasensor effectively measured CEA in real serum samples, verifying its practical application in biological samples.