Publicação
Development of a genosensing platform for antimicrobial resistant bacteria based on graphene’s nearfield effects
| Resumo: | Deoxyribonucleic Acid (DNA) hairpin probes are single-stranded DNA sequences that foldin to a stem-loop structure. These molecules, combined with fluorophores and quencher agents, are widely used in the biosensing field to detect specific DNA sequences. Currently, sensing platforms as such are of major importance to address solutions for global health threats. As na example, they can be used to study Antimicrobial Resistance (AMR), a complex process in which bactéria adapt to the continuous exposure to antibiotics, making infections harder to treat. In this work, a graphene-based genosensing platform was developed on a glass cover slip to individually detect and monitor the interaction between a DNA hair pin probe and four different DNA target sequences: one that is fully complementary, and three that include10-base pair mismatch located at the top, middle, and bottom of the probe. The graphene surface allows for Resonance Energy Transfer (RET) from fluorescent dyes to this 2D material, which leads to a decrease in both the fluorescence intensity and lifetime near its surface. During DNA hybridization, the DNA hairpins are expected to unfold into a double-stranded configuration, displacing the dye and resulting in a detectable acquired optical signal. A Total Internal Reflection Fluorescence (TIRF) microscope was used to track this unfolding kinetic phase at the single molecule level. Using a super-resolution approach, it was possible to localize individual hairpin molecules and perform kinetic assessments of their unfolding behaviour in the presence of different DNA target strands. The study of unfolding times revealed that the DNA probe exhibited a longer unfolding time in the presence of the middle mismatch DNA strand compared to the other three sequences. Fluorescence LifetimeImaging Microscopy (FLIM) was used to characterize the dye lifetimes after the hybridization process, that were then converted into dye-to-graphene distances using a mathematical function. For the fully complementary case, the dye was displaced by a distance consistente with the expected DNA molecular length. The top and bottom mismatches showed similar distances to each other, while the middle mismatch resulted in a distinctly lower dye-to-graphene distance. This genosensing platform, based on graphene near field effects combined with the aforementioned optical detection methods, can be adopted for biomolecular research and biorecognition studies. |
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| Autores principais: | Soares, Maria Miguel de Oliveira |
| Assunto: | DNA sensing FLIM Fluorescencequenching Graphene TIRF Deteçãode ADN Extinção de fluorescência Grafeno Microscopia de Fluorescência de Reflexão Interna Total Microscopia de Imagem de Tempos de Vida de Fluorescência |
| Ano: | 2025 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Deoxyribonucleic Acid (DNA) hairpin probes are single-stranded DNA sequences that foldin to a stem-loop structure. These molecules, combined with fluorophores and quencher agents, are widely used in the biosensing field to detect specific DNA sequences. Currently, sensing platforms as such are of major importance to address solutions for global health threats. As na example, they can be used to study Antimicrobial Resistance (AMR), a complex process in which bactéria adapt to the continuous exposure to antibiotics, making infections harder to treat. In this work, a graphene-based genosensing platform was developed on a glass cover slip to individually detect and monitor the interaction between a DNA hair pin probe and four different DNA target sequences: one that is fully complementary, and three that include10-base pair mismatch located at the top, middle, and bottom of the probe. The graphene surface allows for Resonance Energy Transfer (RET) from fluorescent dyes to this 2D material, which leads to a decrease in both the fluorescence intensity and lifetime near its surface. During DNA hybridization, the DNA hairpins are expected to unfold into a double-stranded configuration, displacing the dye and resulting in a detectable acquired optical signal. A Total Internal Reflection Fluorescence (TIRF) microscope was used to track this unfolding kinetic phase at the single molecule level. Using a super-resolution approach, it was possible to localize individual hairpin molecules and perform kinetic assessments of their unfolding behaviour in the presence of different DNA target strands. The study of unfolding times revealed that the DNA probe exhibited a longer unfolding time in the presence of the middle mismatch DNA strand compared to the other three sequences. Fluorescence LifetimeImaging Microscopy (FLIM) was used to characterize the dye lifetimes after the hybridization process, that were then converted into dye-to-graphene distances using a mathematical function. For the fully complementary case, the dye was displaced by a distance consistente with the expected DNA molecular length. The top and bottom mismatches showed similar distances to each other, while the middle mismatch resulted in a distinctly lower dye-to-graphene distance. This genosensing platform, based on graphene near field effects combined with the aforementioned optical detection methods, can be adopted for biomolecular research and biorecognition studies. |
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