Self-paired dumbbell DNA -assisted simple preparation of stable circular DNAzyme and its application in Pb²⁺ sensor

During its development in recent decades, DNAzyme has become a promising candidate for application in biosensor field. However, it still suffers from the problem of thermodynamic and biological instability such as nuclease digestion, which limits its applications in complex samples. Here we have presented a simple and common strategy to resolve this problem by engineering the linear DNAzyme into a circular shape DNAzyme based on the integration of substrate and enzyme parts into one single-stranded sequence. This circular DNAzyme system is indeed endowed with excellent stability due to the stable intramolecular double-stranded formation and extraordinary resistance to nuclease digestion due to the closed structure. We demonstrated that this circular DNAzyme system gained excellent stability and could active under conditions across a broader range of temperature, salt concentrations, and pH. Depending on this circular DNAzyme, combing with Terminal deoxynucleotidyl transferase (TdT)-generated G-quadruplexes, a label free colorimetric sensing platform for Pb²⁺ quantitation was developed, and a detection limit of 0.085 nM was achieved. Then the enzyme digestion cycle amplification was introduced to further improve the sensitivity of the sensing system, an ultralow detection limit of 0.0015 nM for this fluorescence method was achieved. Based on the two sensing platforms, ultrasensitive analysis of Pb²⁺ in environmental water and food samples was successfully realized. It is anticipated that this stable circular DNAzyme design will be helpful for trace detection in complex samples.