Ultra-efficient amplification-free biosensor based on electric field and crowding agent-promoted hybridization for the detection of pathogenic bacteria

Pathogenic bacteria represent a significant threat to public health and the global economy due to their high infectiousness and lethality. Consequently, the rapid and accurate identification of these bacteria remains a major challenge. Electrochemical amplification-free detection methods have shown several advantages, including rapidity, simplicity and high sensitivity. However, these methods also possess certain drawbacks, such as the inefficient solid-phase hybridization of long-chain nucleic acids on the electrode surface, it is challenging to directly detect long-chain nucleic acids. In response, we have established a novel sensor platform for efficient and rapid detection of long chain nucleic acids based on the principle of HRP signal amplification. This platform incorporates macromolecular crowding agents and electric field assistance to enhance pathogenic bacteria detection efficiency in clinical samples without amplification. The detection process begins by introducing the target into a strand displacement reaction system containing a crowding agent. The crowding effect of the crowding agent is such that the long chain target rapidly replaces the biotinylated probe. Subsequently, an electric field is applied to the surface of the screen-printed electrodes for rapid hybridization, which has been modified with a capture probe. This assay is straightforward and expeditious, reducing hybridization time from 150 min to 90 s. The sensor platform showed a detection linear range of 10²–10⁷ CFU mL⁻¹ with the limit of detection of 10 CFU mL⁻¹ within 25min. This platform showcases the significant potential for on-site detection of pathogenic bacteria in clinical settings and is expected to be integrated into existing diagnostic equipment.