The authors describe a fluorometric strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. This strategy relies on the combination of target-modulated photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA (labeled with silver nanoclusters) along with hairpin probe-based circular exponential amplification. The reaction system involves three hairpin probes (H1, H2 and H3). Probe H1 contains an aptamer against S. Typhimurium and the recognition sequence for nicking endonuclease. It is used to recognize S. Typhimurium and participates in polymerase-catalyzed target recycle amplification and secondary-target recycle amplification. Probe H2 contains an aptamer against hemin and is used to form the G-quadruplex DNAzyme in the presence of hemin and potassium ion. It acts as the electron acceptor and quenches the fluorescence of the labeled DNA. Fluorescence is best measured at excitation/emission wavelengths of 567/650 nm. Probe H3 contains the template sequence for the synthesis of AgNCs and the H2-annealing sequence. Both H2 and H3 are utilized to perform a strand displacement reaction and to achieve PET between G-quadruplex DNAzyme and DNA/AgNCs. To the best of our knowledge, this is the first example of a PET between G-quadruplex DNAzyme and DNA/AgNCs coupled with circular exponential amplification. The assay has an ultra-low detection limit 8 cfu·mL-1 of S. Typhimurium. The assay is rapid, accurate, inexpensive and simple. Hence, the strategy may represent a useful platform for ultrasensitive and highly specific detection of pathogenic bacteria as encountered in food analysis and clinical diagnosis. Graphical abstract The reaction system includes three hairpin probes (H1, H2 and H3), primer probe (P), Phi 29 DNA ploymerase (Phi 29) and nicking endonuclease Nt.AlwI (Nt.AlwI). Phi 29 and Nt.AlwI -assisted signal amplification leads to the recycling of target and produces numerous single stranded-DNAs (S). Strand displacement amplification leads to photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA/AgNCs. HAP-based circular exponential amplification of PET results in an ultrasensitive fluorometric assay.
Keywords: Bioanalysis; Biosensor; Gel electrophoresis; Hairpin; Nicking endonuclease; Polymerase; S. Typhimurium; Signal amplification; Strand displacement amplification.