Rolling Circle Amplification/G‐Quadruplex‐Based Dual‐Signal Ratiometric Electrochemical Aptasensor for Ultrasensitive Detection of Pathogenic Bacteria

Abstract Accurate and efficient detection of pathogenic bacteria plays a crucial role in the diagnosis of infectious diseases. However, rapid and highly sensitive detection of specific bacteria in clinical samples remains challenging. In this study, we developed a ratiometric dual‐signal electrochemical biosensor for ultrasensitive detection of pathogenic bacteria, based on an aptamer recognition‐induced rolling circle amplification (RCA)/G‐quadruplex strategy. On the surface of a gold electrode chip, we used an aptamer sequence (P1) conjugated to ferrocene (Fc) to capture the target bacterium. This capture released a different, previously bound sequence (P2) sequence that initiated the RCA/G‐quadruplex cascade, which, in the presence of potassium ions, was able to bind the electrochemical indicator methylene blue (MB). We integrated the signals from the loss of Fc and the appearance of MB (I MB /I FC ratio) to quantify the target bacteria concentration. This biosensor showed excellent detection performance and specificity, with a detection limit of 10 CFU/mL. Notably, it showed great diagnostic potential for clinical infectious diseases caused by pathogenic bacteria in precise/personalized medicine applications.