A hybridization chain reaction-based electrochemical sensor for rapid detection of respiratory pathogens

Respiratory viral infections continue to pose a significant challenge to global public health. Electrochemical nucleic acid sensors, with their high sensitivity and ease of miniaturization, demonstrate great diagnostic potential. However, the development of high-performance sensors requires comprehensive consideration of factors such as capture probe density, steric hindrance effects, and electrostatic repulsion, which pose significant challenges for the direct detection of long-sequence nucleic acids and limit the application of electrochemical sensors in pathogen diagnosis. This work reports a novel sensor platform based on a concatenated DNA circuit and modified electrodes to achieve efficient and rapid detection of long-sequence nucleic acids. First, the pathogen genomic target sequence replaces the Trigger strand (Ts) through a toehold displacement (TD) reaction. After this, Ts initiates the HCR and the two biotin-modified hairpins hybridize to generate a long double-stranded product. Crowding agents were introduced into the system to enhance the hybridization efficiency of the toehold displacement-mediated hybridization chain reaction (TDHCR) by increasing the local nucleic acid concentration and reducing the free water content in the system, resulting in a significant reduction in reaction time from 120 min to 30 min. Subsequently, the biotin labeled on the TDHCR products rapidly binds to streptavidin immobilized on the electrode surface, enabling efficient and rapid product immobilization. This strategy overcomes the common limitation of low nucleic acid hybridization efficiency at the solid-liquid interface. Finally, the HRP-catalyzed redox reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and H₂O₂ converts the DNA hybridization event into a measurable electrochemical signal, generating a significant current response. The entire detection process can be completed in less than 50 min, with a detection limit as low as 4.876 fM. This platform demonstrates great potential for clinical pathogen detection and is well-suited for integration into microfluidic devices.