Extended GR-5 DNAzyme-based Autonomous isothermal Cascade machine: An efficient and sensitive one-tube colorimetric platform for Pb2+ detection

Development of simple, rapid and highly sensitive platforms for heavy metal ions on-site visual monitoring is extremely challenging and appealing for addressing environmental pollution and public health issues. Herein, we present an extended GR-5 DNAzyme-based autonomous isothermal cascade machine (EGD-AICM) for visible detection of trace Pb2+ in a single tube system. A home-designed GR-5 complex is assembled from an extended GR-5 DNAzyme (GR-5E, integrating GR-5 DNAzyme with a DNA track) and its corresponding extended substrate (GR-5S). Once Pb2+ triggers GR-5S cleavage, the machine automatically initiates isothermal strand displacement amplification to synthesize numerous EAD2 DNA copies. The released copies further integrate with hemin to form horseradish peroxidase (HRP)-mimicking DNAzymes, which efficiently promotes H2O2-mediated oxidation of ABTS and generates amplified colorimetric signals. EGD-AICM enables label-free and one-tube determination of Pb2+ with a short assay time (similar to 50 min), excellent selectivity and high sensitivity (LOD = 35.25 pM). In addition, the proposed EGD-AICM has been employed to measure Pb2+ in real water and shrimp samples with satisfactory accuracy and precision. Such a straightforward and economical EGD-AICM platform demonstrates great potential for routinely screening metal ions in environmental monitoring, food safety assessment, and public safety fields.