Sensitive and label-free fluorescent microRNA detection via catalytic DNAzyme assembly initiated primer exchange reaction

MicroRNAs (miRNAs) have an intricate connection to the development of human diseases, such as malignancies, diabetes, and viral infections; therefore, their rapid and precise identification is critical in disease diagnosis and treatment. In this study, we have developed an innovative technique for detecting miRNA using a highly sensitive and label-free fluorescence approach. This method combines the target recognition-based catalytic hairpin assembly (CHA) driven DNAzyme system with the primer exchange reaction (PER). This method involves three signal amplification processes: CHA-based target recycling, DNAzyme-based signal amplification, and PER, which endow the method with a high sensitivity. Furthermore, a block sequence has been incorporated into the template probe for PER, resulting in a substantial decrease in interferences caused by non-specific targets. Finally, the created G-quadruplex can fix thioflavin T (ThT) to generate fluorescence signals in a label-free manner. Based on this, the approach demonstrates a high miRNA detection sensitivity with a low limit of detection of 0.32 fM. Furthermore, this approach effectively identified miRNA-21 in cellular extracts, thereby showcasing its exceptional resistance to interference. The suggested technology utilizing G-quadruplex/ThT offers intrinsic advantages that make it suitable for widespread application in bioanalysis and early identification of illnesses.