| Summary: | Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu<sup>2+</sup> 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<sup>2+</sup>, can activate the Cu<sup>2+</sup>-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (<i>t</i>DNA). Then, <i>t</i>DNA 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 <i>t</i>DNA along with another ssDNA (<i>i</i>DNA). Subsequently, the <i>i</i>DNA 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, <i>t</i>DNA recycling can be achieved with the λ-Exo digestion reaction to release more <i>i</i>DNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu<sup>2+</sup> with a detection limit of 60 <i>f</i>M. The practical application of the developed biosensor was also demonstrated by detecting Cu<sup>2+</sup> 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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