TY - JOUR
T1 - Detection of E.coli 23S rRNA by electrocatalytic “off-on” DNA beacon assay with femtomolar sensitivity
AU - Jamal, Rimsha B.
AU - Vitasovic, Toni
AU - Gosewinkel, Ulrich
AU - Ferapontova, Elena E.
PY - 2023/5
Y1 - 2023/5
N2 - 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.
AB - 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.
KW - Chronocoulometry
KW - DNA hairpin probes
KW - Electrocatalysis
KW - Genosensor
KW - Gold Screen-printeed electrodes
KW - Hybridization
KW - Methylene blue
KW - Ribosomal RNA
KW - Single nucleotide polymorphism
UR - http://www.scopus.com/inward/record.url?scp=85150821298&partnerID=8YFLogxK
U2 - 10.1016/j.bios.2023.115214
DO - 10.1016/j.bios.2023.115214
M3 - Journal article
C2 - 36906990
AN - SCOPUS:85150821298
SN - 0956-5663
VL - 228
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
M1 - 115214
ER -