Catalytic DNA/RNA, such as DNAzyme, has been widely adopted to construct biosensors, especially for metal ion analysis. However, traditional DNAzyme biosensors still suffer from fluctuating and relatively high background. Herein, we proposed a temperature-robust DNAzyme, conferring ultralow background in various temperatures, thus leading to highly sensitive and robust detection of metal ions. Instead of labeling substrate to directly output fluorescence signal, our proposed DNAzyme biosensor utilized a sequential detection process with a couple of proximity fluorescent probes, confirming very low background regardless of the conditions of cleavage reaction. This sequential DNAzyme biosensor conferred a signal to background ratio over 20 when the temperature of the catalytic reaction ranged from 20 to 41 °C. Benefitting from its ultralow background, it could confer a detection limit of 0.22 nM, which ranked as one of the highest sensitivity levels among DNAzyme-based fluorescent biosensors. This DNAzyme biosensor was over 6000 times more selective for Pb2+ against the most active interfering metal ions, Zn2+. Further, it has been successfully applied for analyzing lead pollution in tap water and eggs, with total recoveries ranging from 87% to 114%. This facile, simple, and effective design strategy would significantly improve the detection performance of DNAzyme biosensors, thus facilitating its practical applications for both food safety analysis and environment monitoring.
Keywords: DNA nanotechnology; DNAzyme; fluorescent biosensor; functional nucleic acids; metal ions.