The key factor to control the incidence rate of diseases caused by bacteria is rapid detection and early diagnosis. Herein, we proposed a new electrochemical bacterial sensor by coupling DNA walking and rolling circle amplification (RCA) reaction-assisted "silver-link" crossing electrode. Staphylococcus aureus (S. aureus) was detected using this proof-of concept strategy. Aptamer/DNA walker and auxiliary sequence (AS)/RCA reaction probe (RP) duplexes were modified on the electrode surface. The binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker and released DNA walker. With the help of Exo III, DNA walker moved along the electrode surface and AS in AS/RP duplex was continuously digested to release RP. By introducing phi29 DNA polymerase, RCA reaction was performed using RP as the reaction primer to form long single-strand RCA extension products between the electrodes. The "silver-link" crossing electrode was formed by metallization of "gene-link", significant conductivity was thus acquired for bacteria detection. The limit of detection (LOD) was 10 CFU/mL and detection time was 2 h. The proposed sensor has high efficiency, good stability and low background signal, human serum and milk samples were successfully detected, which emerged a promising potential in the food monitoring and clinical diagnosis.
Keywords: Bacteria detection; Conductivity; Crossing electrode; DNA walking; Electrochemical sensor; Rolling circle amplification.
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