Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu
2+ 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, Cu
2+, can activate the Cu
2+
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Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu
2+ 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, Cu
2+, can activate the Cu
2+-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 Cu
2+ with a detection limit of 60
fM. The practical application of the developed biosensor was also demonstrated by detecting Cu
2+ in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.
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