Proximity ligation assay induced hairpin to DNAzyme structure switching for entropy-driven amplified detection of thrombin

Highlights: • Entropy-driven amplified reaction was induced by hairpin to DNAzyme structure switching strategy. • This method provides high reliability and sensitivity under enzyme- and hairpin-free conditions. • This method has been successfully applied to thrombin detection in human serum. -- Abstract: A proximity ligation assay (PLA) induced hairpin to DNAzyme structure switching strategy has been described for entropy-driven amplified detection of thrombin. The enzyme-strand (E-DNA) and substrate-strand (S-DNA) of DNAzyme are locked in hairpins structure, and the catalytic activity of DNAzyme is inhibited simultaneously. However, in the presence of thrombin, the PLA can induce the unlocking of hairpin, and then the forming of active DNAzyme. Subsequently, the cleavage of DNAzyme can release DNA fragment to induce the entropy-driven amplification reaction, resulting significant recovery of fluorescent intensity by the separation of FAM from quencher. There was a good linear relationship in the range of 5 pM - 1 nM. This method provides high reliability and sensitivity under enzyme- and hairpin-free conditions.