Two-layer three-dimensional DNA walker with highly integrated entropy-driven and enzyme-powered reactions for HIV detection.

Here, we propose a new two-layer three-dimensional (3-D) DNA walker sensor with highly integrated entropy-driven and enzyme-powered reactions for the first time. The 3-D DNA walker sensor is constructed by assembling densely carboxyfluorescein-labeled single strand oligonucleotides (inner-layer tracks) and nucleic acid complex S (outer-layer tracks) on a microparticle. In the presence of the target, outer and inner tracks are activated in turn, thereby releasing a great deal of the signal reporters for signal reading. As a result, our 3-D DNA walker sensor can realize the target detection in the range from 2 pM to 5 nM within one hour. Besides, the specific walker sensor can clearly distinguish even one-base mismatched target analogue. More importantly, our walker sensor can also test the target in human serum samples in the concentrations as low as 0.1 nM, which provides a bridge between real sample detection and clinical application. Certainly, this smart strategy could also be generalized to any target of interest by proper design.