Facile strategy to enhance the specificity and sensitivity of hairpin molecular devices for detecting pax-5a gene by an integration probe and the specific function of exonuclease III

A label-free DNA sensor is proposed to detect the acute leukemia gene pax-5a, in which the system only relies on a functional hairpin probe (HP) containing a G-quadruplex sequence and the cleavage of exonuclease III (Exo III). Non-single-stranded fragments in the HP structure can also be digested, including double-stranded stems and G-quadruplex fragments, by Exo III at a certain concentration. In the presence of pax-5a, the HP sequence can be cyclically opened through the shearing action of Exo III, causing a large number of G-quadruplex fragments to be released. In the absence of pax-5a, the G-quadruplex fragment is digested by Exo III. The G-quadruplex-hemin DNAzyme can catalyze the oxidation of 3, 3', 5, 5'-tetramethylbenzidine by H2O2 to generate a detectable visible absorption signal, thereby achieving facile and visual detection of acute leukemia gene pax-5a. The detection limit of the label-free DNA sensor that we proposed is as low as 25 fM in the pax-5a concentration range of 25 fM to 25 nM. In addition, the sensor has good discrimination ability of non-target genes and excellent detection ability in complex matrices. Therefore, the DNA sensor shows great potential in biological analysis and clinical diagnosis.