Dual DNAzyme amplification-based colorimetric sensing assay for the identification and quantification of tumor-associated miRNAs

Herein, we present a colorimetric sensing strategy for the identification and quantification of tumor-associated miRNAs based on dual DNAzyme amplification. In this sensing ensemble, the substrate portion of the Pb 2+ -dependent 8−17 DNAzyme combines with the G-quadruplex portion to form a hairpin substrate strand. The two split 8−17 DNAzyme strands are partially complementary to the substrate strand and serve as a recognition unit for binding the target miRNA. In the presence of the target miRNA, the activated DNAzyme cleaves the substrate strand, releasing the G-quadruplex. This G-quadruplex binds to hemin to form a G-quadruplex/hemin complex with horseradish peroxidase (HRP)-like properties, which catalyzes the oxidation of ABTS 2− by H 2 O 2 . This oxidation reaction produces a colorimetric signal output, enabling the detection of the target miRNA. Under the optimal reaction conditions explored in this study, the constructed sensing ensembles tailored for each of the specific target miRNAs successfully identified and quantified the four target miRNAs—miR-122, miR-21, miR-335, and miR-155—in both buffer solutions and cell extracts. This colorimetric sensing strategy offers significant advantages in terms of simplicity, cost, and versatility and holds great potential for wide application in biomedical research and clinical diagnostics.