Circular exponential amplification of photoinduced electron transfer using hairpin probes, G-quadruplex DNAzyme and silver nanocluster-labeled DNA for ultrasensitive fluorometric determination of pathogenic bacteria

Abstract

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 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.

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

“…Thus, alternatively, one can develop an intrinsic signal amplification of reporters' signal by either cationic and fluorescent conjugated polymers or direct inhibition of fluorescent loss using fluorophore encapsulation. For signal amplification, the CRISPR system might be promoted by using other fluorescent-intensity-based nanostructures (e.g., quantum dots and silver nanostructures) and up conversion nanomaterials alongside with robust quenchers such as gold nanoparticles [105] and carbon nanomaterials (graphene oxides and single-walled carbon nanotubes) [106]. Additionally, the use of G-quadruplex-mediated catalysis for colorimetric detection of isothermal amplified amplicons or cleaved-CRISPR reporters could eliminate the need of more enzymes and thus allow conveniently the one-pot reaction toward simplified, cost-effective, specific, and sensitive on-site detection of nucleic acids.…”

The Fluorescent Palette of DNA-Templated Silver Nanoclusters for Biological Applications

“…(D-F) The fluorescence signal is based directly on DNA-AgNCs (Huang et al, 2018;Li et al, 2018a;Dang et al, 2020). (G) The fluorescence signal is based on PET (Leng et al, 2018). (H) The fluorescence signal is based on reactive quenchers (Peng et al, 2015).…”

“…This strategy was dependent on a combination of target modulated photoinduced electron transfer (PET) and cyclic exponential amplification of hairpin probes. The biosensor system has high selectivity to S. Typhimurium with a detection limit of 8 cfu•mL −1 (Leng et al, 2018) (Figure 2G).…”

Recent Innovations in Bacterial Infection Detection and Treatment

“…As with colorimetric biosensors, fluorescence-based sensors also produce light changes; however, these sensors require specific wavelengths of light to excite fluorescent probes or labels to produce a signal. − These biosensors typically have higher sensitivity than most colorimetric biosensors because of the specificity of the emitted light and the required use of external instrumentation to detect this light. Various materials and recognition elements have been investigated in fluorescence-based biosensors for sensitive bacteria detection.…”