In this paper, a "turn-off" biosensor for detecting copper (II) ions based on Cu2+-dependent DNAzyme and a "blocker" beacon were developed. Upon the copper ion being added, the Cu2+-dependent DNAzyme substrate strand was irreversibly cleaved, thereby blocking the occurrence of the ligation reaction and PCR, which inhibited the G-rich sequence from forming the G-quadruplex structure, efficiently reducing the detection signal. This method had the characteristics of strong specificity and high sensitivity compared with the existing method due to the application of ligation-dependent probe signal recognition and amplification procedures. Under the optimized conditions, this method proved to be highly sensitive. The signal decreased as the concentration of copper ions increased, exhibiting a linear calibration from 0.03125 μM to 0.5 μM and a limit of detection of 18.25 nM. Subsequently, the selectivity of this biosensor was verified to be excellent by testing different relevant metal ions. Furthermore, this detection system of copper (II) ions was successfully applied to monitor Cu2+ contained in actual water samples, which demonstrated the feasibility of the biosensor.
Keywords: Cu2+; DNAzyme; G-quadruplex; PCR; biosensor; ligation.