Quadruple CHA-assisted assembly of a three-way six-footed DNA walker enabled a waste-free electrochemical and colorimetric dual-mode biosensing

DNA walking machines have been used as important signal amplification tools for biosensors; however, how to improve their signal amplification ability and avoid the typical DNA waste problem still needs to be urgently addressed. Herein, we designed a quadruple catalytic hairpin assembly (CHA)-assisted three-way six-footed DNA walker assembly strategy and utilized its signal amplification to develop a novel dual-mode biosensing method. Two CHA cycles were triggered by aptamer recognition reaction, and the other two were triggered by DNA walking-cleavage to a methylene blue (MB)-labeled DNA hairpin and another DNA hairpin immobilized at an electrode. This realized the amplified assembly of the DNA walker and also caused high capture of MB onto electrode to achieve electrochemical signal transduction. In addition, four CHA-triggers were also designed for highly assembling a G-quadruplex-decorated DNA long chain to achieve colorimetric signal transduction. Due to DNA walking-based multiple signal amplification, two wide linear ranges with detection limits down to 1.5 fg mL^–1 and 0.73 pg mL^–1 were endowed to the method for assaying kanamycin antibiotic. The combination of this analytical performance with the simple assay manipulation and full nucleic acid utilization in the method determine its promising value for practical applications.