Label-free homogeneous electrochemical biosensor for HPV DNA based on entropy-driven target recycling and hyperbranched rolling circle amplification

Development of rapid and low-cost detection strategies to screen HPV16 E6 and E7 oncogenes are urgently needed for cervical cancer screening. A highly sensitive, selective and label-free homogeneous electrochemical biosensor for these oncogenes was developed in this study. The assay combines the superior recognition ability of entropy-driven amplification with the excellent signal amplification capability of hyperbranched rolling circle amplification (HRCA). In the absence of a target, most of the electrochemical indicator, methylene blue (MB), can diffuse freely onto the surface of the negatively charged indium tin oxide (ITO) electrode, resulting in the production of a distinct signal. Conversely, the target DNA can trigger entropy-driven target recycling, causing the release of multiple assistant probe 1, which can generate numerous long strand ssDNA and dsDNA through HRCA. Because MB can embed into DNA fragments and the strong electrostatic repulsion between long strand DNAs and the negative charged electrode causes a minimal amount of MB to arrive at the electrode surface, a signal drop associated with the target concentration can be observed. The electrochemical response has a linear relationship with the target concentration in the range of 0.1 fM to 10 pM with a low detection limit of 18.6 attomole (S/N = 3). The biosensor was applied to detect the target in clinical samples and shows good consistency with the classical method.