Periodic Reporting for period 1 - Plas_OpMap (Towards faster super-resolution DNA optical mapping using plasmon-enhanced fluorescence)
Periodo di rendicontazione: 2023-07-01 al 2025-06-30
The human gut microbiome is increasingly recognized as a key player in health and disease, with influence over digestion, immunity, and even neurological conditions. However, studying how this complex microbial community evolves over time remains a major challenge. Existing DNA sequencing technologies, although powerful, often fall short when it comes to capturing large-scale genome structures or delivering results quickly enough for real-time or longitudinal studies.
This project, Plas_OpMap, tackles this gap by advancing an emerging technology known as optical DNA mapping. Unlike conventional sequencing, which fragments DNA into short pieces, optical mapping visualises entire DNA molecules, allowing scientists to observe large-scale structural variations. This approach is particularly useful for understanding microbiome composition and dynamics. But despite its promise, optical mapping is currently limited by slow imaging speeds and the need for lengthy data acquisition, making it impractical for high-throughput applications.
A major innovation proposed in Plas_OpMap is the use of plasmon-enhanced fluorescence to significantly boost the speed and precision of DNA imaging. This involves placing fluorescently labelled DNA on specially engineered plasmonic substrates-surfaces coated with gold nanostructures that amplify the emission of light from fluorescent tags. By enhancing photon output, these substrates reduce the time needed to capture each image, allowing faster data collection without sacrificing resolution.
The project focuses on developing a reliable and scalable plasmonic substrate using wet-chemically synthesized gold nanotriangles. These nanoparticles are deposited uniformly across a glass surface, creating a dense, stable, and optically active layer. Their unique geometry and material properties support strong plasmon resonances that enhance fluorescence signals from DNA molecules positioned nearby. This design avoids the complexity of conventional nanofabrication and can be adapted to existing imaging setups.
Three key objectives guide the project:
1.Develop and optimize plasmonic substrates with high nanoparticle coverage.
2.Validate the enhancement effect on fluorescence imaging of labelled DNA molecules.
3.Demonstrate optical mapping of DNA extracted from human gut microbiota using the new substrate.
By combining nanomaterials, microscopy, molecular biology, and bioinformatics, Plas_OpMap brings together expertise across several scientific domains. The long-term goal is to enable faster, more scalable genomic analysis tools that support real-time monitoring of microbial populations that are critical for personalized medicine, diagnostics, and environmental health.
While the project is focused on gut microbiome research, the underlying platform has broader potential across biomedical fields. Faster optical mapping could improve how we track infections, study disease progression, and even respond to public health challenges. In this way, Plas_OpMap contributes to the EU’s broader goals in health innovation, research infrastructure, and digital health technologies.
This project, Plas_OpMap, tackles this gap by advancing an emerging technology known as optical DNA mapping. Unlike conventional sequencing, which fragments DNA into short pieces, optical mapping visualises entire DNA molecules, allowing scientists to observe large-scale structural variations. This approach is particularly useful for understanding microbiome composition and dynamics. But despite its promise, optical mapping is currently limited by slow imaging speeds and the need for lengthy data acquisition, making it impractical for high-throughput applications.
A major innovation proposed in Plas_OpMap is the use of plasmon-enhanced fluorescence to significantly boost the speed and precision of DNA imaging. This involves placing fluorescently labelled DNA on specially engineered plasmonic substrates-surfaces coated with gold nanostructures that amplify the emission of light from fluorescent tags. By enhancing photon output, these substrates reduce the time needed to capture each image, allowing faster data collection without sacrificing resolution.
The project focuses on developing a reliable and scalable plasmonic substrate using wet-chemically synthesized gold nanotriangles. These nanoparticles are deposited uniformly across a glass surface, creating a dense, stable, and optically active layer. Their unique geometry and material properties support strong plasmon resonances that enhance fluorescence signals from DNA molecules positioned nearby. This design avoids the complexity of conventional nanofabrication and can be adapted to existing imaging setups.
Three key objectives guide the project:
1.Develop and optimize plasmonic substrates with high nanoparticle coverage.
2.Validate the enhancement effect on fluorescence imaging of labelled DNA molecules.
3.Demonstrate optical mapping of DNA extracted from human gut microbiota using the new substrate.
By combining nanomaterials, microscopy, molecular biology, and bioinformatics, Plas_OpMap brings together expertise across several scientific domains. The long-term goal is to enable faster, more scalable genomic analysis tools that support real-time monitoring of microbial populations that are critical for personalized medicine, diagnostics, and environmental health.
While the project is focused on gut microbiome research, the underlying platform has broader potential across biomedical fields. Faster optical mapping could improve how we track infections, study disease progression, and even respond to public health challenges. In this way, Plas_OpMap contributes to the EU’s broader goals in health innovation, research infrastructure, and digital health technologies.
This project set out to accelerate super-resolution DNA optical mapping by using plasmon-enhanced fluorescence to increase signal brightness and reduce imaging time. The core achievement was the development of a novel plasmonic substrate composed of uniformly deposited gold nanotriangles (AuNTs), synthesized through a wet-chemical method. These substrates offer tunable plasmonic properties, adjusted by controlling synthesis conditions, making them adaptable for specific fluorescent dyes.
Over 95 percent surface coverage was achieved, and the substrate’s quality and structure were confirmed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). UV-visible spectroscopy was used to characterize the plasmon resonance. To verify that enhancement was occurring, tests were carried out using weakly emitting dye (crystal violet). Increased fluorescence intensity confirmed the substrate’s ability to enhance emission.
The protocol for substrate fabrication was refined to ensure consistent coverage and reproducibility. Next, efforts focused on stretching DNA molecules over the plasmonic surface. A spacer layer made of Zeonex was introduced to control the distance between the DNA and the nanoparticles. The thickness of this layer, ranging from 5 to 25 nm, was measured using ellipsometry. This step is crucial to place the DNA close enough to benefit from enhancement without causing signal quenching.
Initial experiments used Lambda DNA that was site-specifically labelled with fluorescent dyes through enzymatic reactions. Training and support for this labelling method were provided by the host group. It was found that DNA could not be reliably stretched on substrates with spacer layers thinner than 10 nm, but stretching worked well above that. However, despite good sample preparation, enhancement effects seen with freely diffusing dyes were not observed with labelled DNA.
To understand this, a theoretical investigation using Boundary Element Method (BEM) simulations was performed. These simulations offered insights into how factors like dye orientation, local field distribution, and distance from the surface might be affecting enhancement in more complex systems like labelled DNA.
The developed plasmonic substrate represents a valuable outcome of the project. It offers a versatile and scalable platform for enhancing fluorescence signals and could be applied beyond DNA mapping, including in single-molecule sensing, imaging, and other nanophotonic applications. Although full optical mapping of labelled microbiome DNA could not be completed within the project timeframe, the scientific groundwork including substrate fabrication, fluorescence enhancement validation, DNA stretching, and theoretical modeling provides a strong foundation for further development. These results contribute to broader efforts to create faster and more precise tools for molecular-scale analysis.
Over 95 percent surface coverage was achieved, and the substrate’s quality and structure were confirmed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). UV-visible spectroscopy was used to characterize the plasmon resonance. To verify that enhancement was occurring, tests were carried out using weakly emitting dye (crystal violet). Increased fluorescence intensity confirmed the substrate’s ability to enhance emission.
The protocol for substrate fabrication was refined to ensure consistent coverage and reproducibility. Next, efforts focused on stretching DNA molecules over the plasmonic surface. A spacer layer made of Zeonex was introduced to control the distance between the DNA and the nanoparticles. The thickness of this layer, ranging from 5 to 25 nm, was measured using ellipsometry. This step is crucial to place the DNA close enough to benefit from enhancement without causing signal quenching.
Initial experiments used Lambda DNA that was site-specifically labelled with fluorescent dyes through enzymatic reactions. Training and support for this labelling method were provided by the host group. It was found that DNA could not be reliably stretched on substrates with spacer layers thinner than 10 nm, but stretching worked well above that. However, despite good sample preparation, enhancement effects seen with freely diffusing dyes were not observed with labelled DNA.
To understand this, a theoretical investigation using Boundary Element Method (BEM) simulations was performed. These simulations offered insights into how factors like dye orientation, local field distribution, and distance from the surface might be affecting enhancement in more complex systems like labelled DNA.
The developed plasmonic substrate represents a valuable outcome of the project. It offers a versatile and scalable platform for enhancing fluorescence signals and could be applied beyond DNA mapping, including in single-molecule sensing, imaging, and other nanophotonic applications. Although full optical mapping of labelled microbiome DNA could not be completed within the project timeframe, the scientific groundwork including substrate fabrication, fluorescence enhancement validation, DNA stretching, and theoretical modeling provides a strong foundation for further development. These results contribute to broader efforts to create faster and more precise tools for molecular-scale analysis.
The project delivered a scalable plasmonic substrate with tunable plasmon response, providing a cost-effective alternative to lithographic nanostructures. Fluorescence enhancement was confirmed using weak emitters, and the platform shows broader potential in single-molecule sensing, imaging, and diagnostics. To improve performance for DNA mapping, future research could focus on tuning plasmonic modes through control of nanoparticle shape and spacing, or by integrating the wet-chemically prepared plasmonic substrates into photonic chips. Such hybrid systems could enable more efficient excitation of plasmonic fields and enhance signal strength in biologically relevant settings.