Periodic Reporting for period 4 - SMART-DNA (Single Molecule Analytical Raman Tools based on DNA nanostructures)
Reporting period: 2022-10-01 to 2023-03-31
The monitoring of single molecule reactions promises unrivalled insight into chemical reaction mechanisms, but represents one of the most challenging tasks in chemistry. Surface-enhanced Raman scattering (SERS) is a particularly attractive single molecule (SM) technique due to its high chemical specificity, which allows to directly detect relevant intermediates and molecular subpopulations. However, SM-SERS is still at a premature state due to the highly challenging task to place single molecules precisely in nanoscale gaps of plasmonic nanostructures. These are required to provide sufficiently high electromagnetic field enhancement to reach SM sensitivity. The aim of SMART-DNA was to exploit artificial DNA nanostructures to provide sufficient structural control to assemble both, nanoparticles and target molecules, with nanometer precision. By means of novel DNA origami nanostructures the distance between two nanoparticles is controlled, and at the same time target molecules are placed at the positions of highest Raman enhancement through DNA aptamers.
Apart from Raman enhancement the excitation of the localized surface plasmon resonance of the metallic nanostructures results in other plasmonic effects such as heating and possibly the transfer of hot electrons. This can lead to diffusion, conformational changes or even dissociation of the target molecules. These issues do not only concern SM-SERS, but also make quantitative SERS and the SERS analysis of complex (bio)molecules very challenging. By the improved structural control achieved by SMART-DNA, nanoscale heating and hot electron transfer and their effect on SERS spectra was studied on an ensemble and a SM level. Finally, reactions induced by plasmonically generated electrons in DNA and DNA modified with electrophilic molecules was studied by SERS with the aim to develop novel strategies to improve cancer radiation therapies such as photothermal therapy.
Objectives of SMART-DNA:
I. Provide control over multiple structural parameters using bionanotechnology. SMART-DNA will create and apply advanced DNA origami-based SERS substrates with aptamer binding sites.
II. Establish reliable, continuous SM-SERS detection for a broad range of molecules.
III. Explore nanoscale interactions between NPs, molecules and excitation light.
IV. Explore electron-transfer induced reactions in DNA and modified DNA.
Conclusions: The main goals of the project have been reached, i.e. a DNA origami nanofork has been designed, characterised and optimized for SM-SERS measurements. SM-SERS measurements have been performed for a range of dye molecules, different proteins and small molecules such as hemin under both, dry and liquid conditions. Chemical modifications induced by ligands present in solution have been characterized on the SM level and SMs can now be monitored over several minutes to monitor their behaviour in the SERS hot spot and possible chemical transformations. Furthermore, hot electron reactions have been studied in great detail using small aromatic molecules as well as modified DNA. Furthermore, strategies have been explored to improve cancer radiation therapy.
Apart from Raman enhancement the excitation of the localized surface plasmon resonance of the metallic nanostructures results in other plasmonic effects such as heating and possibly the transfer of hot electrons. This can lead to diffusion, conformational changes or even dissociation of the target molecules. These issues do not only concern SM-SERS, but also make quantitative SERS and the SERS analysis of complex (bio)molecules very challenging. By the improved structural control achieved by SMART-DNA, nanoscale heating and hot electron transfer and their effect on SERS spectra was studied on an ensemble and a SM level. Finally, reactions induced by plasmonically generated electrons in DNA and DNA modified with electrophilic molecules was studied by SERS with the aim to develop novel strategies to improve cancer radiation therapies such as photothermal therapy.
Objectives of SMART-DNA:
I. Provide control over multiple structural parameters using bionanotechnology. SMART-DNA will create and apply advanced DNA origami-based SERS substrates with aptamer binding sites.
II. Establish reliable, continuous SM-SERS detection for a broad range of molecules.
III. Explore nanoscale interactions between NPs, molecules and excitation light.
IV. Explore electron-transfer induced reactions in DNA and modified DNA.
Conclusions: The main goals of the project have been reached, i.e. a DNA origami nanofork has been designed, characterised and optimized for SM-SERS measurements. SM-SERS measurements have been performed for a range of dye molecules, different proteins and small molecules such as hemin under both, dry and liquid conditions. Chemical modifications induced by ligands present in solution have been characterized on the SM level and SMs can now be monitored over several minutes to monitor their behaviour in the SERS hot spot and possible chemical transformations. Furthermore, hot electron reactions have been studied in great detail using small aromatic molecules as well as modified DNA. Furthermore, strategies have been explored to improve cancer radiation therapy.
The project started with the design of a novel DNA origami nanofork to create plasmonic Dna Origami Nanofork Antennas (DONAs) for single-molecule SERS. The DONA structures have been characterised and optimized for SM-SERS measurements and a broad range of nanoparticles have been used (e.g. Au and Ag nanoparticle spheres and Au nanoflowers). SM-SERS measurements have been performed for a range of dye molecules (e.g. TAMRA, Cy5, Cy7), different proteins (e.g. cytochrome C and horseradish peroxidase) and small molecules such as hemin under both, dry and liquid conditions. The placements and orientations of the molecules of interest in the SERS hot spots has been analyzed in detail and strategies have been tested to orient molecules in a predetermined way in the SERS hot spot. Chemical modifications induced by ligands present in solution have been characterized on the SM level and SMs can now be monitored over several minutes to monitor their behaviour in the SERS hot spot and possible chemical transformations. One example is the change of the spin state of the iron ion in single hemin molecules.
Upon the irradiation of plasmonic nanoparticles, not only the Raman scattering is enhanced, but also hot charge carriers can be generated that interact with surrounding molecules. The kinetics of hot electron induced reactions on gold nanoparticles have been studied by SERS using a range of molecules and a detailed kinetic model is currently developed. Furthermore, we have studied electron transfer from nanoparticles to modified DNA indicating that DNA can serve as a conductive wire that is able to separate the source of the hot electrons (the nanoparticle) from the reaction centre. These reactions induced in DNA modified with electrophilic compounds such as brominated adenines could be used for cancer radiation therapy and strategies have been explored to improve such therapies. Furthermore, it turned out that hot electron chemistry could be used in the future to control chemical reaction pathways and for sustainable light-induced synthesis.
Upon the irradiation of plasmonic nanoparticles, not only the Raman scattering is enhanced, but also hot charge carriers can be generated that interact with surrounding molecules. The kinetics of hot electron induced reactions on gold nanoparticles have been studied by SERS using a range of molecules and a detailed kinetic model is currently developed. Furthermore, we have studied electron transfer from nanoparticles to modified DNA indicating that DNA can serve as a conductive wire that is able to separate the source of the hot electrons (the nanoparticle) from the reaction centre. These reactions induced in DNA modified with electrophilic compounds such as brominated adenines could be used for cancer radiation therapy and strategies have been explored to improve such therapies. Furthermore, it turned out that hot electron chemistry could be used in the future to control chemical reaction pathways and for sustainable light-induced synthesis.
The SMART-DNA project represents a fundamentally different approach to SM- and single nanostructure SERS spectroscopy compared to previous studies and enables the control of a large number of parameters that are critical for significant advancement of this field. The unprecedented degree of structural control on the nanometer scale enabled by the DNA origami technology was exploited to establish a versatile tool for spectroscopic investigations with significantly improved control over the target molecules. SMART-DNA represents frontier research of high risk, but the interdisciplinary approach (DNA nanofabrication combined with SERS spectroscopy) promises high gain by advancement of novel fields of application for SERS that showed only slow progress during the last years or even decades due to the general complexity of SERS substrates. It has been shown that the interparticle gaps have been reduced down to 1.2 nm to provide sufficient Raman enhancement, but at the same time the gap distance can be modified by variation of DNA strands to accommodate larger molecules. SMART-DNA addressed a broad range of fundamental scientific challenges such as the origin of SERS blinking in SM and few-molecule SERS and its role for ensemble measurements, the SERS signal distribution and a fundamental mechanistic understanding of electron transfer induced reactions in modified DNA. The latter mechanisms contribute to the development of a complete picture of physical and chemical radiosensitization in cancer radiation therapy, which will enable the improvement of currently applied therapeutics and will enable the development of novel treatment strategies. Furthermore, the tools developed within SMART-DNA are not only restricted to SERS, but can also be exploited by other surface-enhanced spectroscopy methods such as surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced fluorescence (SEF). Additionally, significant contributions to the young field of plasmonic chemistry have been made, which lay the ground for future coordinated research projects.