This research was supported by the European Research Council (ERC; consolidator Grant No. 772752).

1. DNA origami fork assembly
<ol>
	<li>Self-assemble the DNA origami structure in one pot. First, add 2.5 nM circular scaffold strand M13mp18 (7,249 nucleotides, see Table of Materials) and 100 nM of 201 short oligonucleotides (Table 1) in 1x TAE (tris[hydroxymethyl]aminomethane [tris], acetic acid, ethylenediamine acetic acid [EDTA]) supplemented with 15 mM MgCl2 buffer. Then, adjust the total volume to 100 &#181;L using ultrapure water.</li>
	<li>Anneal the solution subsequently by a temperature gradient in a thermocycler, first by rapidly heating to 80 &#176;C, then by cooling down from 80 &#176;C to 20 &#176;C at 1 &#176;C/12 min, then from 20 &#176;C to 16 &#176;C at 1 &#176;C/6 min, followed by rapidly cooling down from 16 &#176;C to 8 &#176;C.</li>
	<li>Use 100 kDa molecular weight cut-off (MWCO) Amicon filters (see Table of Materials) to purify the mixture from excess staples.
	<ol>
		<li>Add 100 &#181;L of the DNA origami solution to the Amicon filters, as well as 400 &#181;L of ultrapure water, then centrifuge at 6,000 x g for 8 min at room temperature (RT).</li>
		<li>Discard the filtrate by removing the filter and flipping the tube to remove the wash-out in the sink, then add 400 &#181;L of ultrapure water to the filter and centrifuge again. Repeat this step one more time.</li>
		<li>To collect the purified nanostructure solution, flip the filter upside down in a new tube and centrifuge (1,000 x g, 2 min, RT) following the manufacturer&#39;s instructions (see Table of Materials). This solution can be stored in a fridge at 8 &#176;C for up to 2 weeks. 
		&#8203;NOTE: Use 1x TAE buffer or 1x TAE supplemented with 15 mM MgCl2 buffer instead of ultrapure water for the Amicon filter purification step, since water decreases the stability and lowers the concentration of the DNA origami forks obtained. For a single dye molecule measurement, the DNA staple strand mixture is replaced with a modified strand mixture containing a TAMRA dye on the 5&#39; end. This modified strand is in the middle of the nanofork bridge (Table 2).</li>
	</ol>
	</li>
</ol>
2. Gold nanoparticle (AuNP) coating
NOTE: A modified version of the Liu et al.23&#160;protocol was used to coat the AuNPs, and the coating process involved freezing the AuNP-DNA solution.
<ol>
	<li>Centrifuge (3,500 x g, 5 min, RT) 400 &#181;L of 60 nm diameter AuNP solution (commercially obtained; see Table of Materials), and using a pipette, remove the supernatant. Then, resolubilize the pellet in 25 &#181;L of ultrapure water. The final concentration of the AuNPs is ~0.3 nM.</li>
	<li>Add 1 &#181;L of 100 mM tris-(2-carboxyethyl)phosphine (TCEP) solution to 4 &#181;L of thiol-modified DNA (100 &#181;M as supplied by the manufacturer; see Table of Materials) and incubate for 10 min at RT.</li>
	<li>Following incubation, add the 5 &#181;L mixture to the concentrated AuNP solution (step 2.1), vortex for 5 s, and freeze for at least 2 h at -20 &#176;C.</li>
	<li>After thawing at RT, centrifuge (3,500 x g, 5 min, RT) the mixture to remove the excessively added coating DNA. Using a pipette, remove the supernatant, and resolublize the pellet in 10 &#181;L of water. 
	NOTE: There are two coating strands for each of the AuNPs on the nanofork (Table 2). The steps are the same except for the sequence of the DNA coating strand. Once the particles are coated, they are very stable; they can stay as they are for months in the fridge and can even be frozen. For fully coated dye AuNPs, the coating DNA strands have an internal TAMRA dye.</li>
</ol>
3. DONA assembly
<ol>
	<li>Add the coated AuNP solution to the nanofork solution, with a concentration molar ratio of 1.5:1.</li>
	<li>Add MgCl2 to a final concentration of 4 mM using a 50 mM MgCl2 stock solution. Adjust the final volume to 20 &#181;L using ultrapure water.</li>
	<li>Hybridize the DONAs using a temperature gradient in a thermocycler. First, rapidly heat to 40 &#176;C, then cool from 40 &#176;C to 20 &#176;C at 1 &#176;C/10 min, and then rapidly cool from 20 &#176;C to 8 &#176;C.</li>
</ol>
4. Gel electrophoresis
NOTE: The unbound nanoparticles in the DONA solution are removed by agarose gel electrophoresis.
<ol>
	<li>Prepare 1% agarose gel. For this, dissolve 0.8 g of agarose in 80 mL of 1x TAE supplemented with 5 mM MgCl2.</li>
	<li>Add 2.25 &#181;L of loading buffer (30% glycerol, 13 mM MgCl2;&#160;see Table of Materials) to 18 &#181;L of DONA solution to reach a final concentration of 5 mM MgCl2 from 4 mM in step 3.2. The loading buffer also ensures that the sample stays inside the pocket of the gel.</li>
	<li>Run the gel for 60 min at 70 V in an ice water bath. The running buffer is 1x TAE supplemented with 5 mM MgCl2.</li>
	<li>Cut out the band of interest and place it on a paraffin plastic film-wrapped microscopy slide, and then squeeze out the solution using a second paraffin plastic film-wrapped microscopy slide. Using a pipette, collect the squeezed liquid into a 500 &#181;L tube.</li>
</ol>
5. Colocalization of AFM and Raman measurements
<ol>
	<li>Plasma treat a silicon chip (see Table of Materials) for 10 min, then incubate 10 &#181;L of the purified DONA solution plus 10 &#181;L of 100 mM MgCl2 on the chip for 3 h. Then, wash the chip two times with a 1:1 mixture (by volume) of ethanol and water and blow-dry with compressed air. Then, tape the chip on a magnetic disc and insert it into the instrument for imaging. 
	NOTE: Before starting the measurements, the position of the Raman excitation laser is adjusted to be on top of the AFM probe tip.&#160;In the present work a HORIBA LabRAM HR Evolution Raman microscope coupled to a HORIBA Omegascope AFM is used. The instrument is controlled using the software LabSpec and AIST.</li>
	<li>For the AFM measurement, use tapping mode (AC mode) with ACCESS-NC-A (resonance frequency: 300 kHz; spring constant: 45 N/m) tips24. The AC mode automatically controls all the parameters except for the scan rate, which is set to 1 Hz. 
	NOTE: Following AFM imaging, the AFM tip is withdrawn so that it is not in the path of the Raman laser. This is done using a macro function, &#34;Probe away&#34;, programmed in the system. In this way, for any chosen point in the AFM image, the laser will be in the same position with no offset compared to AFM.</li>
	<li>Choose the desired laser wavelength, power, and accumulation time for the Raman measurements, as all these parameters depend on the measured sample.
	<ol>
		<li>Use the mentioned parameters for a fully coated TAMRA AuNP measurement: 633 nm laser, 100 &#181;W power, and 1 s integration time.</li>
		<li>Use the mentioned parameters for a single TAMRA measurement: 633 nm laser, 400 &#181;W power, and 4 s integration time.</li>
	</ol>
	</li>
	<li>After adjusting the parameters, place the cursor on top of the desired DONA in the AFM image; the &#34;move cursor&#34; function in the software does this. Then, start the Raman measurement. 
	NOTE: There are different modes for acquiring the spectra: single point measurement-one-dimension time mapping-where a single point is measured over time25; and two-dimension XY area mapping, where a motorized stage is moved under the laser to acquire spectra from an array of points in an XY grid26.</li>
</ol>

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placement+and+orientation+of+individual+DNA+shapes+on+lithographically+patterned+surfaces.">Placement and orientation of individual DNA shapes on lithographically patterned surfaces.</a> Nature Nanotechnology. 4 (9), 557-561 (2009).</li><li>Dutta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+states+and+spin+crossover+of+hemin+studied+by+DNA+origami+enabled+single-molecule+surface-enhanced+Raman+scattering.">Molecular states and spin crossover of hemin studied by DNA origami enabled single-molecule surface-enhanced Raman scattering.</a> Nanoscale. 14 (44), 16467-16478 (2022).</li><li>Langer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+of+surface-enhanced+Raman+scattering.">Present and future of surface-enhanced Raman scattering.</a> ACS Nano. 14 (1), 28-117 (2020).</li><li>Jones, R. 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The authors have no competing financial interests or other conflicts of interest.

SM SERS is a powerful tool that allows researchers to study the behavior and interactions of individual molecules within a sample1. Such a technique allows the analysis of systems at an unprecedented level of sensitivity, providing new insights into the fundamental behavior of single molecules and the distribution of chemical or physical properties over an ensemble of molecules, and helps to identify relevant intermediates in chemical processes. However, placing a single molecule in the hot spot while also making sure that the hot spot has enough surface enhancement can be quite challenging27. The described DONAs in this protocol can precisely place a single molecule in the hot spot between two gold nanoparticles while ensuring a 1011-fold surface enhancement is reached.
The gap between the nanoparticles is critical for the DONA assembly to reach the required surface enhancement for SM SERS studies. The DONAs are optimized for spherical nanoparticles with sizes between 60 and 80 nm. Furthermore, the quality of the nanoparticles can significantly impact the hybridization of the nanoparticles with the DNA origami forks; when the nanoparticles that are used in the coating step are older than 6 months, the efficiency of hybridization begins to decline.
Another critical aspect of the protocol is that the steps that require an exact ratio between components must be followed precisely, or the DONAs will not be formed correctly. The DNA origami fork is extremely sensitive to the temperature ramping protocol, with changes affecting the structure&#39;s integrity or preventing forks from forming.
Amorphous carbon generation is a significant issue during SERS measurements because its peaks are typically in the same range as the fingerprint area for many molecules (1,200-1,700 cm-1). While the formation is not yet fully understood, it is usually associated with high laser power or long integration times28. As a precaution, the lowest laser power and the shortest integration time possible should be used. However, this is not easily accomplished because a balance must be achieved between obtaining the desired SERS signal and avoiding the generation of amorphous carbon.
The DONAs are very versatile as an SM system regarding the different types and shapes of nanoparticles that could be used, such as&#160;silver spheres, gold flowers, or stars. Additionally, the molecule under investigation can be easily replaced by changing only the DNA strand in the middle of the bridge, with no changes to the procedure. Switching to proteins like cytochrome C could be done by having a pyridine-modified DNA capture strand in the bridge, which would bind the cytochrome C and ensure that it is in the hot spot for SM SERS measurements22. This also translates to flexibly choosing the laser for irradiation, potentially using a laser that gives maximum enhancement.
In summary, this method is reliable for assembling DONA structures and using them for single-molecule surface-enhanced Raman spectroscopy measurements.

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).

<table><tbody><tr><td>100 kDa MWCO Amicon filters, 0.5 mL</td><td>Merck</td><td>UFC5100BK</td><td></td></tr><tr><td>10x TAE buffer (0.4 M Tris, 0.2 M acetic acid, 0.01 M EDTA)</td><td>SIGMA Aldrich</td><td>T9650</td><td></td></tr><tr><td>60 nm goldspheres, bare (citrate)</td><td>NanoComposix</td><td>AUCN60</td><td></td></tr><tr><td>ACCESS-NC-A AFM probes</td><td>SCHAEFER-TEC</td><td></td><td></td></tr><tr><td>Agarose powder</td><td>SIGMA Aldrich</td><td>9012-36-6</td><td></td></tr><tr><td>AuNP DNA coating strands</td><td>IDT</td><td></td><td></td></tr><tr><td>AuNP DNA coating strands (TAMRA)</td><td>SIGMA Aldrich</td><td></td><td></td></tr><tr><td>Glycerol</td><td>SIGMA Aldrich</td><td>56-81-5</td><td></td></tr><tr><td>Heraeus Fresco 17 centrifuge</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>HORIBA OmegaScope with a LabRAM HR evolution</td><td>HORIBA</td><td></td><td></td></tr><tr><td>Magnesium chloride</td><td>SIGMA Aldrich</td><td>7786-30-3</td><td></td></tr><tr><td>Nanofork DNA bridge strand (TAMRA)</td><td>Metabion</td><td></td><td></td></tr><tr><td>Nanofork DNA staple strands</td><td>SIGMA Aldrich</td><td></td><td></td></tr><tr><td>ParafilmM</td><td>Carl Roth</td><td>CNP8.1</td><td></td></tr><tr><td>Primus 25 Thermocycler</td><td>Peqlab/VWR</td><td></td><td></td></tr><tr><td>Silicon wafer</td><td>Siegert wafer</td><td>BW14076</td><td></td></tr><tr><td>Single-stranded scaffold DNA, type p7249 (M13mp18)</td><td>Tilibit nanosystems</td><td></td><td></td></tr><tr><td>TCEP solution</td><td>SIGMA Aldrich</td><td>51805-45-9</td><td></td></tr></tbody></table>

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.

Following the protocol, one should ensure that the DNA origami fork is correctly assembled; the preferred method for examining the structure of the forks is AFM imaging. Most forks are expected to be solid, with no broken arms. On the other hand, the bridge between the arms is difficult to image because of its small diameter and high flexibility; it also requires a very sharp AFM tip (Figure 2).
The solution color change at each step of the AuNP coating process indicates that everything works correctly. The color starts deep red with just the AuNPs, but as soon as the DNA is added, it changes to dark-purplish red. Freezing changes the color to purple and, after thawing, returns it to deep red (Figure 3A). Figure 3B depicts the absorbance spectra of bare AuNPs and DNA-coated AuNPs.
After that, in the DONA assembly steps, the color stays deep red throughout the whole process. During the agarose gel purification, a dimer band appears above the free AuNP band, the fastest-running band in the sample. This dimer band corresponds to the DONAs and is subsequently cut out and squeezed to extract the sample (Figure 4).
Finally, for colocalization measurements, AFM imaging of the sample is performed in search of DONAs (Figure 5). Following that, Raman spectra from single DONAs are collected and compared to ensure that the spectra obtained are from TAMRA molecules (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65310/65310fig01.jpg" /> 
Figure 1: Schematic representation of the DNA origami fork and the fully assembled DONA. (A) Dimensions of the DNA origami fork with a bridge 90 nucleotides long. (B) Schematic side-view of an assembled DONA, with two AuNPs and the DNA origami fork in between. (C) Schematic top-view of the assembled DONA showing the placement position of the SM in the middle of the bridge. <a href="https://www.jove.com/files/ftp_upload/65310/65310fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65310/65310fig02.jpg" /> 
Figure 2: AFM image of the DNA origami forks after assembly. The forks are well formed, with the bridge visible in some of the forks. The forks have a height between 1.5-2 nm. Scale bar = 500 nm. <a href="https://www.jove.com/files/ftp_upload/65310/65310fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65310/65310fig03.jpg" /> 
Figure 3: Color development and absorbance spectra of AuNPs. (A) Tubes showing the color of the AuNP solution in different steps. (1) Deep red color of the bare AuNP solution. (2) Dark-purplish red after the addition of the coating DNA to the AuNPs. (3) Purple color after freezing the coating DNA-AuNP mixture. (4) The color returns to deep red after thawing of the mixture. (B) Absorbance spectra of the 60 nm AuNPs, showing the shift in absorbance peak from bare AuNPs (tube 1) to DNA-coated AuNPs (tube 4). <a href="https://www.jove.com/files/ftp_upload/65310/65310fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65310/65310fig04.jpg" /> 
Figure 4: Agarose gel of the DONA solution. Both lanes have the same sample, and both the dimer band (DONA) and the free AuNP band are clearly visible. <a href="https://www.jove.com/files/ftp_upload/65310/65310fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65310/65310fig05.jpg" /> 
Figure 5: AFM image of DONAs. (A) The image shows multiple DONA structures; this AFM image is used for the colocalization measurements. (B) Zoomed-in image of the circled DONA and vertical cross-section of the DONA. <a href="https://www.jove.com/files/ftp_upload/65310/65310fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65310/65310fig06.jpg" /> 
Figure 6: SERS spectra of DONAs equipped with fully coated TAMRA AuNPs and with a single TAMRA molecule. The vertical gridlines indicate the main TAMRA peaks. The main TAMRA peaks are visible in the fully coated TAMRA AuNP. Although the signal-to-noise ratio for the SM TAMRA spectra is lower, the main peaks are identifiable: 1,360 cm-1: C-C stretching; 1,509 cm-1: C=C stretching; 1,536 cm-1: C=C stretching; 1,654 cm-1: C=O stretching. <a href="https://www.jove.com/files/ftp_upload/65310/65310fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: List of DNA origami fork staples. <a href="https://www.jove.com/files/ftp_upload/65310/65310_Table 1_Nanofork staple strands list.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: List of modified DNA strands. <a href="https://www.jove.com/files/ftp_upload/65310/65310_Table 2_DNA modified strands.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas at JoVE.com

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

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.

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) ...

single-molecule-surface-enhanced-raman-scattering-measurements

Research

JoVE Journal

Chemistry

3.1K Views.  University of Potsdam. 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.

Video: Author Spotlight: Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas  

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Engineering

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.

A procedure for fabrication and performing the filter-based surface enhanced Raman spectroscopic (SERS) assay for the detection of chemical contaminants (i.e., pesticide ferbam and antibiotic ampicillin) is presented.

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic nanostructures. The high spatial precision of a DNA origami-templated assembly allows controlling the coupling between plasmonic resonances of individual particles and enables tailoring optical properties of the constructed nanostructures. Recently, chiral plasmonic systems attracted a lot of attention due to the strong correlation between the spatial configuration of plasmonic assemblies and their optical responses (e.g., circular dichroism [CD]). In this protocol, we describe the whole workflow for the generation of DNA origami-based chiral assemblies of gold nanorods (AuNRs). The protocol includes a detailed description of the design principles and experimental procedures for the fabrication of DNA origami templates, the synthesis of AuNRs, and the assembly of origami-AuNR structures. In addition, the characterization of structures using transmission electron microscopy (TEM) and CD spectroscopy is included. The described protocol is not limited to chiral configurations and can be adapted for the construction of various plasmonic architectures.

We describe the detailed protocol for the DNA origami-based assembly of gold nanorods into chiral plasmonic metamolecules with strong chiroptical responses. The protocol is not limited to chiral configurations and can be easily adapted for the fabrication of various plasmonic architectures.

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic ...

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

﻿Analyzing the Heterogeneous Performance of Single Nanoparticles with Electrochemical Methods

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This nanoscale heterogeneity remains hidden during the ensemble-averaged characterization of nanoparticles. Electrochemical techniques have been developed to measure currents from single nanoparticles but do not provide information about the structure and identity of the molecules that undergo reactions at the electrode surface. Optical techniques such as surface-enhanced Raman scattering (SERS) microscopy and spectroscopy can detect electrochemical events on individual nanoparticles while simultaneously providing information on the vibrational modes of electrode surface species. In this paper, a protocol to track the electrochemical oxidation-reduction of Nile Blue (NB) on single Ag nanoparticles using SERS microscopy and spectroscopy is demonstrated. First, a detailed protocol for fabricating Ag nanoparticles on a smooth and semi-transparent Ag film is described. A dipolar plasmon mode aligned along the optical axis is formed between a single Ag nanoparticle and Ag film. The SERS emission from NB fixed between the nanoparticle and the film is coupled into the plasmon mode, and the high-angle emission is collected by a microscope objective to form a donut-shaped emission pattern. These donut-shaped SERS emission patterns allow for the unambiguous identification of single nanoparticles on the substrate, from which the SERS spectra can be collected. In this work, a method for employing the SERS substrate as a working electrode in an electrochemical cell compatible with an inverted optical microscope is provided. Finally, tracking the electrochemical oxidation-reduction of NB molecules on an individual Ag nanoparticle is shown. The setup and the protocol described here can be modified to study various electrochemical reactions on individual nanoparticles.

Author Spotlight: Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy  

The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging.

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This ...

Label-Free SERS Analysis: Au@Carbon Dot Nanoprobes for Molecular Fingerprinting

Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular fingerprint information of biological samples, as well as its potential in single-cell analysis. This work aims to establish a simple strategy for label-free SERS bioanalysis based on Au@carbon dot nanoprobes (Au@CDs). Here, polyphenol-derived CDs are utilized as the reductant to rapidly synthesize core-shell Au@CD nanostructures, which allows powerful SERS performance even when the concentration of methylene blue (MB) is as low as 10-9 M, due to the cooperative Raman enhancement mechanism. For bioanalysis, Au@CDs can serve as a unique SERS nanosensor to identify the cellular components of biosamples (e.g., cancer cells and bacteria). The molecular fingerprints from different species can be further distinguished after combination with the principal component analysis. In addition, Au@CDs also enable label-free SERS imaging to analyze intracellular composition profiles. This strategy offers a feasible, label-free SERS bioanalysis, opening up a new prospect for nanodiagnosis.

Author Spotlight: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging 

In this study, we developed a low-cost surface-enhanced Raman scattering (SERS)-based fingerprint nanoprobe with favorable biocompatibility to show label-free live cell bioimaging and detect two bacterial strains, showing in detail how to get SERS spectra of living cells in a non-destructive method.

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular ...