Detection of E.coli 23S rRNA by electrocatalytic ?off-on? DNA beacon assay with femtomolar sensitivity

Prevention of food spoilage, environmental bio-contamination, and pathogenic infections requires rapid and sensitive bacterial detection systems. Among microbial communities, the bacterial strain of Escherichia coli is most widespread, with pathogenic and non-pathogenic strains being biomarkers of bacterial contamination. Here, we have developed a fM-sensitive, simple, and robust electrocatalytically-amplified assay facilitating specific detection of E.coli 23S ribosomal rRNA, in the total RNA sample, after its site-specific cleavage by RNase H enzyme. Gold screen-printed electrodes (SPE) were electrochemically pre-treated to be productively modified with a methylene-blue (MB) - labelled hairpin DNA probes, which hybridization with the E. coli-specific DNA placed MB in the top region of the DNA duplex. The formed duplex acted as an electrical wire, mediating electron transfer from the gold electrode to the DNA-intercalated MB, and further to ferricyanide in solution, enabling its electrocatalytic reduction otherwise impeded on the hairpin-modified SPEs. The assay facilitated 20 min 1 fM detection of both synthetic E. coli DNA and 23S rRNA isolated from E.coli (equivalent to 15 CFU mL-1), and can be extended to fM analysis of nucleic acids isolated from any other bacteria.

Automated summary

In this study, a sensitive and simple electrocatalytic amplification method for detecting Escherichia coli 23S ribosomal rRNA has been developed. The RNA sample was cleaved by RNase H enzyme, and then hybridized with a methylene-blue (MB) - labelled DNA probe. The formed duplex acted as an electrical wire for electron transfer, enabling the electrocatalytic reduction on the modified electrodes. This assay allowed the detection of synthetic E. coli DNA and 23S rRNA with a limit of 1 fM.