The video outlines the process of self-assembling DNA origami structures using a specific mixture of scaffold and oligonucleotide staples. It details preparation, annealing procedures, purification using filtration, and collection steps, concluding with atomic force microscopy imaging to verify proper assembly, noting challenges in visualizing certain structural features.

dna origami fork assembly for dna origami nanoantenna (dona)

Mesures SERS d’une seule molécule rendues possibles par des nanoantennes d’origami d’ADN plasmonique

DNA origami is a nanotechnology technique that involves folding DNA strands into specific shapes and patterns. The ability to create structures with precise control at the nanoscale is one of the key advantages of DNA origami1. The ability to manipulate matter at such a small scale has the potential to revolutionize a wide range of fields, including medicine, electronics, and materials science2. For example, DNA origami structures have been used to deliver drugs directly to cancer cells3,4, create nanoscale sensors for detecting diseases5,6, and create intricate patterns on the surfaces of materials6,7. Furthermore, the ability to use DNA origami to create complex nanoscale structures has created new opportunities for studying fundamental biological processes at the nanoscale8.
Surface enhanced Raman scattering (SERS) is a robust analytical technique that detects and identifies molecules at extremely low concentrations9. It is based on the Raman effect, which is a change in the wavelength of scattered light10. SERS requires a plasmonic metal substrate to enhance the Raman signal of molecules adsorbed onto it. This enhancement can be up to 1011 times greater than the signal obtained from the same molecule measured by conventional Raman spectroscopy, making SERS a highly sensitive method for analyzing trace amounts of substances11.
The Raman signal enhancement of nanoparticles is mostly due to electromagnetic enhancement based on the excitation of localized surface plasmon resonance (LSPR)12. In this phenomenon, electrons within the metal nanoparticle oscillate collectively around the surface of the metal nanoparticle during light incidence. This results in the creation of a standing wave of electrons known as a surface plasmon, which can resonate with the incident light. The LSPR greatly enhances the electric field near the particle&#39;s surface, and the optical absorption of the particle is maximum at the plasmon resonant frequency. The surface plasmon&#39;s energy depends on the shape and size of the metal nanoparticle, as well as the properties of the surrounding medium13. A higher enhancement could be further achieved by plasmonic couplings, such as when two nanoparticles are close to each other at a distance of approximately 2.5 times the diameter length or less14. The proximity causes the LSPR of both nanoparticles to interact with each other, enhancing the electric field in the gap between the particles by several orders of magnitude, far exceeding the enhancement of a single nanoparticle15,16,17. The magnitude of the enhancement is inversely proportional to the distance between the nanoparticles; as the distance decreases, a critical point appears where the enhancement is sufficient to detect single molecules (SMs)18.
DNA origami represents a key technology that can efficiently arrange plasmonic nanoparticles to exploit and optimize the plasmonic coupling19. At the same time, molecules of interest can be placed precisely at the place where optical detection is most efficient. This has been demonstrated with DNA origami-based plasmonic nanoantennas for fluorescence detection20. In contrast to the detection of fluorescent labels, SERS provides the possibility to detect a direct chemical fingerprint of a molecule, making the single-molecule SERS very attractive for sensing as well as monitoring chemical reactions and mechanistic studies. DNA origami can also be used as a mask to fabricate plasmonic nanostructures with well-defined shapes21, although the possibility to position target molecules precisely into the hot spot is then lost.
Using colocalized atomic force microscopy (AFM) and Raman measurements, we can obtain the Raman spectra from a single DNA origami nanoantenna (DONA) and potentially a single molecule if placed at the position of highest signal enhancement. The DONA comprises a DNA origami fork and two precisely positioned nanoparticles, fully coated with DNA complementary to extended staple strands attached to the DNA origami. Upon DNA hybridization, the nanoparticles are bound to the DNA origami fork with a 1.2-2.0 nm gap between the nanoparticle surfaces22. Such assembly creates a hot spot in between the nanoparticles with a signal enhancement of up to 1011-fold, as calculated from finite difference time domain (FDTD) simulations22, thus permitting SM SERS measurements. However, the volume of the highest enhancement is small (in the 1-10 nm3 range), and consequently the target molecules need to be positioned precisely into this hot spot. The DNA origami fork allows the positioning of a single molecule between the two nanoparticles using a DNA fork bridge and suitable coupling chemistry. Nevertheless, observing such SMs in Raman spectra is very challenging22. Alternatively, the nanoparticles can be fully coated with the target molecule, such as a TAMRA dye, to allow for single DONA measurements with a higher SERS intensity21. In this case, the TAMRA is covalently attached to the DNA coating strand (Figure 1).

Pleins feux sur l’auteur : Mesures de diffusion Raman améliorées en surface d’une seule molécule rendues possibles par des nanoantennes d’origami d’ADN plasmonique  

Mesures de diffusion Raman améliorées en surface à molécule unique rendues possibles par des nanoantennes d’origami d’ADN plasmonique

Watch this Scientific Journal Video about DNA Origami Fork Assembly for DNA Origami Nanoantenna DONA at JoVE.com

DNA Origami Fork Assembly for DNA Origami Nanoantenna (DONA)  

Ensemble fourche en origami d’ADN pour nanoantenne d’origami d’ADN (DONA)

Pour auto-assembler la structure de l’origami d’ADN dans un pot, commencez par préparer un mélange de 2,5 nanomolaires du brin d’échafaudage circulaire M13mp18 et de 100 nanomolaires des 201 courtes agrafes oligonucléotidiques dans un tampon TAE complété par 15 millimolaires de chlorure de magnésium. Ajustez ensuite le volume total de la solution à 100 microlitres en utilisant de l’eau ultrapure. Recuit la solution dans un thermocycleur à l’aide d’un gradient de température.Commencez par chauffer rapidement à 80 degrés Celsius et suivez le programme de gradient de température affiché étape par étape. Pour purifier le mélange de l’excès d’agrafes, ajoutez 100 microlitres de la solution d’origami d’ADN à un filtre amicon de coupure de poids moléculaire de 100 kilodaltons. Suivi de l’ajout de 400 microlitres d’eau ultrapure.Puis centrifuger à 6 000G pendant huit minutes à température ambiante. Après la centrifugation, retirez le filtre et retournez le tube pour jeter le filtrat. Ajoutez 400 microlitres d’eau ultrapure au filtre et à la centrifugeuse comme démontré précédemment.Recueillir la solution de nanostructure purifiée. Retournez le filtre dans un nouveau tube et centrifugez à 1 000 G pendant deux minutes à température ambiante en suivant les instructions du fabricant. L’assemblage correct de la fourche d’origami d’ADN a été assuré à l’aide de la microscopie à force atomique ou de l’imagerie AFM.Comme prévu, la plupart des fourches étaient solides et ne présentaient pas de bras cassés. Cependant, le pont entre les bras était difficile à imager en raison de son petit diamètre et de sa grande flexibilité.

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Research

JoVE Journal

Chemistry

DNA Origami Fork Assembly for DNA Origami Nanoantenna (DONA)

503 vues. Pour auto-assembler la structure de l’origami d’ADN dans un pot, commencez par préparer un mélange de 2,5 nanomolaires du brin d’échafaudage circulaire M13mp18 et de 100 nanomolaires des 201 courtes agrafes oligonucléotidiques dans un tampon TAE complété par 15 millimolaires de chlorure de magnésium. Ajustez ensuite le volume total de la solution à 100 microlitres en utilisant de l’eau ultrapure. Recuit la solution dans un thermocycleur à l’aide d’un gradient de températ...

Vidéo: DNA Origami Fork Assembly for DNA Origami Nanoantenna (DONA)  

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...