A fluorescent sensor based on strand displacement amplification and primer exchange reaction coupling for label-free detection of miRNA

MicroRNAs (miRNAs) are closely associated with human disease occurrence, including cancers, diabetes, inflammation, heart diseases, and viral infections, and their rapid and accurate detection is vital for the diagnosis and treatment of these diseases. Based on one-step reaction of strand displacement amplification (SDA) and primer exchange reaction (PER), a label-free and highly sensitive miRNA-21 detection strategy was developed. In this strategy, the target miRNA-21 binds directly to the hairpin template, triggering the SDA reaction and generating a large number of single strand DNAs as primers for PER amplification. With the help of polymerase, plenty of G-quadruplex fragments of different lengths were accumulated, and the organic dye thioflavin T selectively binds to these G-quadruplex fragments to produce a strong fluorescent signal. There is a wide detection range in this method, miRNA-21 can be detected in the range of 10 fM - 1 nM, the detection limit is low (1.25 fM). This method has good specificity and can effectively distinguish single-base mismatches of miRNA. In addition, the versatility of the method was validated by changing the target recognition site of SDA template.