A Cascade Signal Amplification Strategy for the Ultrasensitive Fluorescence Detection of Cu2+ via λ-Exonuclease-Assisted Target Recycling with Mismatched Catalytic Hairpin Assembly

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 releasestDNA along with another ssDNA (iDNA). Subsequently, theiDNA 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 moreiDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+with a detection limit of 60fM. 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.Keywords:Cu2+detection;λ-exonuclease-assisted target recycling;mismatched catalytic hairpin assembly;cascade signal amplification;environmental pollutant