Presentation Information
[P01-106]Detection of target mRNA in bacteria on a surface by spraying DNA-based biosensing molecules
○Hirotaka Nakamura1, Takeshi Ikeda1, Takenori Ishida1, Ryuichi Hirota1, Akio Kuroda1, Hisakage Funabashi1 (1. Hiroshima University (Japan))
Keywords:
Spray-based detection,Biosensing molecule,DNA nano-tweezers,Surface detection
[Purpose]
Detecting targets on solid surfaces involves sampling, dissolving the samples into a solution, and analyzing them using conventional methods. However, these procedures are often complex and therefore not suitable for simple use by non-specialists. To address these issues, we propose a novel method for direct imaging-based detection of targets on solid surfaces by spraying biosensing molecules. Biosensing molecules are biomaterial-based molecular tools that generate signals only upon recognizing their targets, enabling the design of homogeneous assays that do not require washing steps. In this study, we aimed to develop a simple imaging method for detecting intracellular mRNA in bacteria on a solid surface using a nanotweezer-shaped DNA-based biosensing molecule (DNA-NT) developed in our laboratory.
[Methods]
The DNA-NTs form a tweezer-like nanostructure through the self-assembly of three single-stranded DNAs (O1, O2, and O3). O1 and O2 contain sequences complementary to a target nucleic acid, serving as recognition sites. O3 contains split DNAzyme sequences that exhibit peroxidase-like activity in the presence of hemin as a cofactor. The DNA-NTs exist in an open conformation, but upon target recognition, they undergo a structural change to a closed form. This conformational change brings the split DNAzyme fragments on O3 into proximity, activating peroxidase-like activity. When the DNA-NTs are sprayed onto a solid surface, followed by spraying a signal-generating substrate, signals are produced at locations where the target nucleic acid is present. These signals can be captured using a smartphone, enabling direct and convenient detection of targets on the solid surface.
[Results]
Escherichia coli cells were spotted onto filter paper, and β-galactosidase mRNA was selected as the target. Cells were successfully disrupted by spraying enzymes and surfactants to lyse the cell wall and membrane, along with proteases to degrade endogenous bacterial peroxidases. A solution containing the cell-disrupting reagents and DNA-NTs for β-galactosidase mRNA was then sprayed, followed by the application of luminol, a substrate for peroxidase-catalyzed chemiluminescence. Imaging in a dark environment using a smartphone revealed luminescence only at locations where E. coli were spotted. Furthermore, when cells cultured with isopropyl β-D-1-thiogalactopyranoside to induce β-galactosidase expression were used, increased luminescence corresponding to elevated mRNA levels was observed.
[Conclusion]
These results demonstrate that simple spray-based operations enable bacterial cell lysis on solid surfaces and direct imaging-based detection of intracellular target mRNA. This approach provides a convenient and wash-free platform for on-site detection, including point-of-care testing (POCT).
Detecting targets on solid surfaces involves sampling, dissolving the samples into a solution, and analyzing them using conventional methods. However, these procedures are often complex and therefore not suitable for simple use by non-specialists. To address these issues, we propose a novel method for direct imaging-based detection of targets on solid surfaces by spraying biosensing molecules. Biosensing molecules are biomaterial-based molecular tools that generate signals only upon recognizing their targets, enabling the design of homogeneous assays that do not require washing steps. In this study, we aimed to develop a simple imaging method for detecting intracellular mRNA in bacteria on a solid surface using a nanotweezer-shaped DNA-based biosensing molecule (DNA-NT) developed in our laboratory.
[Methods]
The DNA-NTs form a tweezer-like nanostructure through the self-assembly of three single-stranded DNAs (O1, O2, and O3). O1 and O2 contain sequences complementary to a target nucleic acid, serving as recognition sites. O3 contains split DNAzyme sequences that exhibit peroxidase-like activity in the presence of hemin as a cofactor. The DNA-NTs exist in an open conformation, but upon target recognition, they undergo a structural change to a closed form. This conformational change brings the split DNAzyme fragments on O3 into proximity, activating peroxidase-like activity. When the DNA-NTs are sprayed onto a solid surface, followed by spraying a signal-generating substrate, signals are produced at locations where the target nucleic acid is present. These signals can be captured using a smartphone, enabling direct and convenient detection of targets on the solid surface.
[Results]
Escherichia coli cells were spotted onto filter paper, and β-galactosidase mRNA was selected as the target. Cells were successfully disrupted by spraying enzymes and surfactants to lyse the cell wall and membrane, along with proteases to degrade endogenous bacterial peroxidases. A solution containing the cell-disrupting reagents and DNA-NTs for β-galactosidase mRNA was then sprayed, followed by the application of luminol, a substrate for peroxidase-catalyzed chemiluminescence. Imaging in a dark environment using a smartphone revealed luminescence only at locations where E. coli were spotted. Furthermore, when cells cultured with isopropyl β-D-1-thiogalactopyranoside to induce β-galactosidase expression were used, increased luminescence corresponding to elevated mRNA levels was observed.
[Conclusion]
These results demonstrate that simple spray-based operations enable bacterial cell lysis on solid surfaces and direct imaging-based detection of intracellular target mRNA. This approach provides a convenient and wash-free platform for on-site detection, including point-of-care testing (POCT).
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