A Cascade Signal Amplification Strategy for the Ultrasensitive Fluorescence Detection of Cu<sup>2+</sup> via λ-Exonuclease-Assisted Target Recycling with Mismatched Catalytic Hairpin Assembly
Zhen Liu,
Chen Liu,
Liqiong He,
Jinquan Liu,
Le Li,
Shengyuan Yang,
Yan Tan,
Xing Liu,
Xilin Xiao
Affiliations
Zhen Liu
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Chen Liu
Hunan Province Key Laboratory for Typical Environmental Pollution and Health Hazards, School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China
Liqiong He
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Jinquan Liu
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Le Li
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Shengyuan Yang
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Yan Tan
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Xing Liu
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Xilin Xiao
Hunan Key Laboratory of Typical Environmental Pollution and Health Hazards, School of Public Health, Hengyang Medical School, University of South China, Hengyang 421001, China
Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu2+ was designed using the cascade signal amplification strategy, coupling λ-exonuclease-assisted target recycling and mismatched catalytic hairpin assembly (MCHA). In the designed detection system, the target, Cu2+, can activate the Cu2+-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (tDNA). Then, tDNA binds to hairpin DNA (H0) with an overhanging 5′-phosphorylated terminus to form dsDNA with a blunt 5′-phosphorylated terminus, which activates the dsDNA to be digested by λ-Exo and releases tDNA along with another ssDNA (iDNA). Subsequently, the iDNA initiates MCHA, which can restore the fluorescence of carboxyfluorescein (FAM) previously quenched by tetramethylrhodamine (TAMRA), resulting in a strong fluorescent signal. Furthermore, MCHA efficiently improves the signal-to-noise ratio of the detection system. More importantly, tDNA recycling can be achieved with the λ-Exo digestion reaction to release more iDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+ with a detection limit of 60 fM. The practical application of the developed biosensor was also demonstrated by detecting Cu2+ in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.
