G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection

• We report a novel G-quadruplex based two-stage isothermal amplification reaction.
• The assay achieves dual amplification of recognition event and signal readout.
• The strategy realizes simple, rapid, sensitive and label-free DNA colorimetry.
• The assay demonstrates good discrimination of mismatched sequences.

A novel G-quadruplex based two-stage isothermal exponential amplification reaction (GQ-EXPAR) was developed for label-free DNA colorimetric detection in this work. The exponential amplified trigger DNA in the first stage can convert into G-quadruplex sequence EAD2 by a linear amplification circuit in the second stage. Created EAD2 can form G-quadruplex/hemin DNAzyme to act as a direct signal readout element. The GQ-EXPAR combines the exponential amplification of DNA sequence and the peroxidase-mimicking DNAzyme induced signal amplification, which achieves tandem dual-amplification. Taking advantages of isothermal incubation, this label-free homogeneous assay obviates the need of thermal cycling . As no complex synthesis or extra downstream operation is needed, the whole easy handling procedure can be finished in no more than 1 h. This assay allows the sensing of the model DNA with the limit of detection to be 2.5 pM. Moreover, it demonstrates good discrimination of mismatched sequences. The strategy has also been successfully implemented to sensitively detect Tay–Sachs genetic disorder mutant.