Label-free, background-free detection of nucleic acid with immobilization-free heterogeneous biosensor and one-pot hybridization chain reaction amplification

Although immobilization-free and label-free electrochemical DNA (E-DNA) biosensors have engaged tremendous interest due to their superior properties, such as easy operation, time-saving and cost-saving, most of them are fabricated in homogeneous modes and usually produce high background current. In the present work, we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction (HCR) for one-pot nucleic acid detection with zero background. The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins, resulting in the generation of HCR products with multibranched arms, which can be captured onto the electrode via π-π stacking interactions between multibranched arms and reduced graphene oxide (rGO). Prior to the incubation process with an electrode, two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes. Thus, an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized. This strategy also presents additional merits of simplicity and cheap cost, since probe immobilization, signal tag labeling, and multiple incubation processes are avoided. Therefore, the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.