Accurate and sensitive detection of the Pax-5a gene is of great importance in the early diagnosis and prognosis of acute leukaemia. Herein, a label-free electrochemical sensing system was proposed for the detection of the acute leukaemia Pax-5a gene based on enzyme-assisted signal amplification to generate abundant G-quadruplex/hemin DNAzyme. The presence of Pax-5a can open the hairpin probe (HP), which acts as a template. Under the action of the restriction enzymes Nt.BbvCI and Klenow fragment polymerase , the target gene Pax-5a is cycled to open the HP; On the other hand, a large number of G-quadruplex sequences are produced. The resulting G-quadruplex sequence is capable of forming the G-quadruplex/hemin complex on the surface of the electrode in the presence of hemin . The ultrasensitive label-free electrochemical detection of Pax-5a can be realized via the G-quadruplex/hemin complex-catalysed reduction of H 2 O 2 , and the detection limit was estimated to be as low as 4.6 fM. In addition, the biosensor has good specificity and stability, and also has excellent detection capabilities in a complex substrate environment. Therefore, the sensor shows great potential in bioanalysis and clinical diagnosis.
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 H 2 O 2 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.
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