This work was supported by the Academy of Finland (projects 286845, 308578, 303804, 267497), the Jane and Aatos Erkko Foundation, and the Sigrid Jus&#233;lius Foundation. This work was carried out under the Academy of Finland Centers of Excellence Programme (2014&#8211;2019). We acknowledge the provision of facilities and technical support by Aalto University Bioeconomy Facilities and OtaNano &#8211; Nanomicroscopy Center (Aalto-NMC) and Micronova Nanofabrication Center.

The authors have nothing to disclose.

The protocol provides great freedom and accuracy in the shape of produced nanostructures. By changing the design of the DNA origami, the shape of the metal nanostructures can be controlled. The final, exact shape of the metal structures is additionally determined by the mask growth step (Step 9) and to a lesser degree by the mask etching (Step 10) should it not be anisotropic. If the mask growth time is extended enough, the holes in the mask will start to grow shut. This can be used to omit the thinnest features of some structures and control gap sizes, as demonstrated in Shen et al.34 with separated triangles of the bowtie origami (Figures 5B). Conversely, thinner shapes can be better preserved by shortening the oxide growth time. This means that it is possible to tune the optical properties displayed in Figure 6, not just by changing the used origami design, but also by tuning the SiO2 film growth.

If the mask thickness is changed significantly, that change must also be reflected in the SiO2 RIE step. Only a very thin layer of SiO2 should be etched (2-5 nm) to barely pierce through the mask holes. This is the most sensitive and crucial part of the whole process. Since the etching time is extremely short, only 10-20 s, exact settings must be experimentally determined when first attempted with new equipment. This is also true for Step 10.4 as some SiO2 is also etched during the a-Si etching. The extent of etched SiO2 is determined by the selectivity of the used a-Si etch parameters, equipment and even individual equipment calibrations. Care should be taken not to etch away the entire SiO2 layer during these two processes.

Another sensitive step is the SiO2 growth. The growth process is dependent on both the chamber humidity and the current activity of the used TEOS. TEOS degrades as it adsorbs water from the air, causing it to become less effective with age. This can manifest as a significantly slower, less controllable growth rate within months even with proper storage of the chemical.34 If the resulting SiO2 layer is thinner than intended, this can indicate a problem with TEOS rather than chamber humidity. While a lower humidity can also result in lower growth rate and thinner film, the resulting film should also be smoother than normal. Meanwhile a coarse grained and rough layer would conversely indicate a problem with high humidity.

It is also possible to perform this protocol on any other freely chosen substrate with two requirements: It must tolerate both HF etching (Step 12) and the 200-300 &#176;C temperatures of PECVD (Step 6). The temperature can be safely lowered to 100 &#176;C for the PECVD of a-Si if a more sensitive substrate is used, but HF cannot be avoided if the protocol is followed exactly as described. To circumvent HF, the application of an additional sacrificial layer would be required. If the requirement of the HF etching is removed, this protocol would become compatible with a wider selection of substrate materials and metals.

As this protocol consists of commonly used and robust micro- and nanofabrication processes, it could be combined with any number of other microfabrication protocols where small feature sizes and complex metal shapes are desired. In the near future, especially with the coming of low-cost DNA origami mass-production31, there is potential for this method to facilitate both general use and high-throughput nanopatterning for interface-based nanophotonics and plasmonics55.

Structural DNA nanotechnology has rapidly evolved during the recent decade1,2, and the most influential development in the field has arguably been the invention of DNA origami3,4. The DNA origami technique allows fabrication of virtually any nanoshape with accurate structural features3,4. This powerful method can be used in (sub)nanometer-precise spatial arrangement and anchoring of other nano-objects, such as carbon nanotubes5, metal nanoparticles6,7,8,9, enzymes/proteins10,11,12,13 and therapeutic materials14,15,16,17. Importantly, these structures are not merely static, but they can also be programmed to act in a dynamic manner18,19. The countless applications of DNA origami range from drug delivery20,21,22 to molecular electronics/plasmonics5,23,24,25 and from materials science26,27 to novel imaging and calibration techniques28.

Besides the applications mentioned above, the extreme spatial resolution of the DNA origami shapes could be harnessed in nanopatterning and delicate nanoscale lithography29,30. This protocol describes a lithography method for creating discrete and accurate inorganic nanostructures on substrates using DNA origami templates. These templates can be efficiently produced in various shapes and in large quantities31, and deposited effortlessly onto chosen substrates at large scales32. These properties allow a highly parallel fabrication of billions of nanostructures in one step as opposed to commonly used but rather slow electron beam lithography or other scanning-based nanofabrication techniques.

Herein, the fabrication process is demonstrated by creating gold bowtie-shaped structures on silicon nitride and sapphire substrates; in other words, the spatial information of DNA origami is transferred to entirely metallic nanostructures. As discussed here, the technique is not limited to the selected bowtie DNA origami structure since the method enables the use of virtually any DNA origami shape. Moreover, with methodical modifications, the technique can be extended to different metals and substrates paving the way towards fabrication of metasurfaces33.

The surfaces patterned with the DNA origami-mediated fabrication may serve as versatile sensors; for example, they can be used in surface-enhanced Raman spectroscopy (SERS). As a result of the small dimensions of the individual nanoshapes, the created surfaces may find uses in optical and plasmonic applications at the visible wavelength range.

<table><tbody><tr><td>Acetone</td><td>Honeywell</td><td>40289H</td><td>Semiconductor grade ULSI, &amp;ge; 99.5 %</td></tr><tr><td>Agarose</td><td>Fisher Bioreagents</td><td>1036603</td><td>Low-EEO, multi-purpose and molecular biology grade</td></tr><tr><td>Ammonium hydroxide</td><td>Fisher Chemical</td><td>10652251</td><td>25 % ammonia solution, Certified AR for Analysis, d = 0.91</td></tr><tr><td>BRANSON 5510</td><td>Branson</td><td></td><td>Ultrasonic bath</td></tr><tr><td>Dimension Icon</td><td>Bruker</td><td></td><td>Atomic force microscope</td></tr><tr><td>Electron-beam evaporator IM-9912</td><td>Instrumentti Mattila</td><td></td><td>Evaporator for PVD</td></tr><tr><td>Ethidium bromide</td><td>Sigma Aldrich</td><td>E8751</td><td>Fluorescent dye for DNA staining</td></tr><tr><td>Eon Microplate spectrophotometer</td><td>BioTek</td><td></td><td>UV/Vis spectrophotometer used for DNA origami concentration measurements</td></tr><tr><td>Gel Doc XR+ Documentation System</td><td>BioRad</td><td></td><td>Gel imaging system</td></tr><tr><td>Gel Loading Dye, Blue (6&amp;times;)</td><td>New England Biolabs</td><td>B7021S</td><td>Bromophenol blue-based loading dye for agarose gel electrophoresis</td></tr><tr><td>G-storm GS1 Thermal cycler</td><td>Gene Technologies</td><td></td><td></td></tr><tr><td>HBR 4</td><td>IKA</td><td></td><td>Heating bath</td></tr><tr><td>Hydrofluoric acid</td><td>Honeywell</td><td>40213H</td><td>Semiconductor grade, 49.5-50.5 %</td></tr><tr><td>Isopropanol</td><td>Honeywell</td><td>40301H</td><td>Semiconductor grade VLSI, &amp;ge; 99.8 %</td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M8266</td><td>Anhydrous, &amp;ge; 98 %</td></tr><tr><td>Mini-Sub Cell GT Horizontal Electrophoresis System</td><td>BioRad</td><td></td><td></td></tr><tr><td>Plasmalab 80+ PECVD</td><td>Oxford Instruments</td><td></td><td>PECVD system</td></tr><tr><td>Plasmalab 80+ RIE</td><td>Oxford Instruments</td><td></td><td>RIE system</td></tr><tr><td>Poly(ethylene glycol)</td><td>Sigma Aldrich</td><td>89510</td><td>BioUltra, 8,000</td></tr><tr><td>PowerPac HC High-Current Power Supply</td><td>BioRad</td><td></td><td></td></tr><tr><td>Sapphire substrate (Al2O3)</td><td>University Wafer</td><td></td><td>Thickness: 430 &amp;mu;m, Polish: DSP, Size: 50.8 mm</td></tr><tr><td>Sigma VP</td><td>Zeiss</td><td></td><td>Scanning electron microscope</td></tr><tr><td>Silica gel</td><td>Merck</td><td>1019691000</td><td>With indicator (orange gel), granulate ~1-3 mm</td></tr><tr><td>Single-stranded Scaffold DNA, type p7249</td><td>Tilibit Nanosystems</td><td></td><td>At 100 nM concentration</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S9888</td><td>ACS reagent, &amp;ge; 99.0 %</td></tr><tr><td>Staple strands (oligonucleotides)</td><td>Integrated DNA Technologies</td><td></td><td>Sequences can be ordered e.g. at 100 micromolar in Rnase-free water</td></tr><tr><td>TAE buffer (50&amp;times;) pH 8.0</td><td>VWR Chemicals</td><td>444125D</td><td>Electran Electrophoresis grade</td></tr><tr><td>Take3 micro-volume plate</td><td>BioTek</td><td></td><td>Used for DNA origami concentration measurements</td></tr><tr><td>Tetraethyl orthosilicate</td><td>Sigma Aldrich</td><td>86578</td><td>&amp;ge; 99.0 % (GC)</td></tr></tbody></table>

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.

A schematic figure of the bowtie DNA origami design and its structural details are shown in Figure 1. Agarose gel electrophoresis and AFM are used to analyze the DNA origami folding and the quality of PEG purification (Figure 2). The process flow of the nanolithography steps is displayed in Figure 3. Representative AFM images after SiO2 mask growth are shown in Figure 4 (this step is depicted in Figure 3D), while SEM images of the final metal nanostructures can be seen in Figure 5 (this step is depicted in Figure 3H). Figure 6 demonstrates the optical functionality of the metallic nanostructures templated by the bowtie DNA origami.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="5">Folding buffer (FOB) component concentrations [mM]</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Tris</td>
			<td>Acetic acid</td>
			<td>EDTA</td>
			<td>Magnesium chloride</td>
			<td>pH</td>
		</tr>
		<tr>
			<td>2.5x FOB</td>
			<td>100</td>
			<td>47.5</td>
			<td>2.5</td>
			<td>31.25</td>
			<td>~8,3</td>
		</tr>
		<tr>
			<td>1x FOB</td>
			<td>40</td>
			<td>19</td>
			<td>1</td>
			<td>12.5</td>
			<td>~8,3</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of the folding buffer (FOB).
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Temperature range [oC]</td>
			<td>Cooling rate</td>
		</tr>
		<tr>
			<td>90-70</td>
			<td>-0.2 &#176;C / 8 s</td>
		</tr>
		<tr>
			<td>70-60</td>
			<td>-0.1 &#176;C / 8 s</td>
		</tr>
		<tr>
			<td>60-27</td>
			<td>-0.1 &#176;C / 2 s</td>
		</tr>
		<tr>
			<td>12</td>
			<td>Hold until stopped</td>
		</tr>
	</tbody>
</table>
Table 2: Thermal ramp for the bowtie origami folding. After annealing, the origami will be stored at 12 oC until the program is manually stopped.
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>PECVD and RIE parameters</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Gas</td>
			<td>Gas flow [sccm]</td>
			<td>Chamber pressure [mTorr]</td>
			<td>RF power [W]</td>
			<td>Temperature [oC]</td>
			<td>Duration [s]</td>
		</tr>
		<tr>
			<td>PECVD of a-Si</td>
			<td>5% SiH4 in N2</td>
			<td>500</td>
			<td>1000</td>
			<td>15</td>
			<td>250</td>
			<td>90</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>O2 plasma treatment</td>
			<td>O2</td>
			<td>50</td>
			<td>40</td>
			<td>200</td>
			<td>30</td>
			<td>1200</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RIE of SiO2</td>
			<td>CHF3</td>
			<td>25</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Ar</td>
			<td>25</td>
			<td>30</td>
			<td>100</td>
			<td>25</td>
			<td>10-22</td>
		</tr>
		<tr>
			<td>RIE of a-Si</td>
			<td>O2</td>
			<td>8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>SF6</td>
			<td>100</td>
			<td>90</td>
			<td>50</td>
			<td>30</td>
			<td>35</td>
		</tr>
		<tr>
			<td>RIE of remaining a-Si</td>
			<td>O2</td>
			<td>8</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>SF6</td>
			<td>100</td>
			<td>90</td>
			<td>50</td>
			<td>30</td>
			<td>35-40</td>
		</tr>
	</tbody>
</table>
Table 3: Process parameters for plasma-enhanced chemical vapor deposition (PECVD) and reactive ion etching (RIE). The process parameters for these devices are specific to individual instruments and they may need to be adapted when used.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60313/60313fig1.jpg" /> 
Figure 1: Design of the bowtie DNA origami. (A) Schematic representation of the bowtie origami design in which the core structure is shown as double helices and the polyT-overhangs are depicted as wavy lines. (B) Screenshot of a part of the bowtie origami design in the caDNAno software. The red crosses denote the base pair skipping for the twist correction, and the T8-overhangs are added to prevent blunt-end base-stacking. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/60313fig1large.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/60313/60313fig2.jpg" /> 
Figure 2: Characterization of the bowtie DNA origami structure. (A) Agarose gel electrophoresis of the bowtie structure before and after poly(ethylene glycol) (PEG) purification. The 7249 nucleotides long scaffold is used as reference. (B) Atomic force microscopy (AFM) image of the bowtie structures before the purification. (C) AFM image of the bowtie structures after PEG purification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/60313fig2large.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/60313/60313fig3.jpg" /> 
Figure 3: Scheme of fabrication process flow (the dimensions are not in scale). (A) Dice and clean the substrate. (B) Deposit an a-Si layer by plasma-enhanced chemical vapor deposition (PECVD). *It is possible to employ an additional sacrificial layer under the a-Si to enable lift-off with etchant other than HF. (C) Treat the sample surface with O2 plasma and deposit DNA origami onto it. (D) Grow the SiO2 mask in desiccator. (E) Etch a thin layer of SiO2 and through the a-Si underneath it by reactive ion etching (RIE). (F) Deposit metal through the mask by physical vapor deposition (PVD). (G) Lift-off with HF. (H) remove the remaining a-Si by RIE. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/60313fig3large.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/60313/60313fig4.jpg" />
Figure 4: Representative AFM images of SiO2 film with the DNA origami shaped pattern. (A) 10 &#956;m x 10 &#956;m scanning area demonstrates the high yield of the pattern formation. (B) A closer 3 &#956;m x 3 &#956;m scan shows the accurate individual patterns in the SiO2 film. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/60313fig4large.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/60313/60313fig5.jpg" /> 
Figure 5: Representative scanning electron microscopy (SEM) images of metallic nanostructures templated with structurally different DNA origami. (A) Cross-shaped DNA origami, i.e., so-called Seeman tile origami54. (B) Bowtie antennas. (C) Chiral double-L (CDL) structures. Insets show individual structures with box sizes of 150 nm x 150 nm. The fabrication yield of exact structures is up to 76% for the bowtie origami and ~ 50% for the other structures displayed here34. This figure has been adapted and modified from Shen et al.34. The figure is reproduced with permission of the authors and published by The American Association for the Advancement of Science, 2018. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/60313fig5large.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/60313/60313fig6.jpg" /> 
Figure 6: Representative optical/functional properties of resulting nanostructures. (A) Localized surface plasmon resonance (LSPR) measurements of an individual gold bowtie structure with different polarization (color coded as orange and blue). The solid lines are measured spectra and the dashed lines are simulation results. Insets show the SEM image of the measured particle (left) and the model used for simulation (right). (B) Surface enhanced Raman spectroscopy (SERS) of rhodamine 6G and 2,2-bipyridine measured on a surface covered with bowtie nanostructures. The baseline of each sample shows the signal level when the nanostructures were absent. This figure has been adapted and modified from Shen et al.34. The figure is reproduced with permission of the authors and published by The American Association for the Advancement of Science, 2018. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/60313fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental File 1" src="/files/ftp_upload/60313/60313sup1.jpg" /> 
Supplemental File 1: CaDNAno file <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/bowtie_poly-T.json" target="_blank">Please click here to download this file.</a>
<img alt="Supplemental File 2" src="/files/ftp_upload/60313/60313sup2.jpg" /> 
Supplemental File 2: m13mp18 sequence <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/m13mp18_sequence.txt" target="_blank">Please click here to download this file.</a>
<img alt="Supplemental File 3" src="/files/ftp_upload/60313/60313sup3.jpg" /> 
Supplemental File 3: Staple strand sequence <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60313/bowtie_staple_sequences.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications at JoVE.com

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

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 ...

dna-origami-mediated-substrate-nanopatterning-inorganic-structures

Nanyang Technological University

Research

JoVE Journal

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11.5K Views.  Aalto University. Dit protocol combineert de bottom-up gebaseerde DNA origami techniek met top-down nanofabricage methoden waardoor de parallelle fabricage van anorganische nanostructuren met sub-10 nanometer functie maten op verschillende substraten. Deze techniek kan worden gebruikt om miljarden nauwkeurige nanostructuren in één keer te creëren, vrijwel in elke vorm zonder de noodzaak van dure patronenmethoden. Deze methode heeft het potentieel om te worden benut in het ontdekken van toepassingen zoals op...

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Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates

In recent years &pi;-conjugated organic semiconductors have emerged as the active material in a number of diverse applications including large-area, low-cost displays, photovoltaics, printable and flexible electronics and organic spin valves. Organics allow (a) low-cost, low-temperature processing and (b) molecular-level design of electronic, optical and spin transport characteristics. Such features are not readily available for mainstream inorganic semiconductors, which have enabled organics to carve a niche in the silicon-dominated electronics market. The first generation of organic-based devices has focused on thin film geometries, grown by physical vapor deposition or solution processing. However, it has been realized that organic nanostructures can be used to enhance performance of above-mentioned applications and significant effort has been invested in exploring methods for organic nanostructure fabrication.	
A particularly interesting class of organic nanostructures is the one in which vertically oriented organic nanowires, nanorods or nanotubes are organized in a well-regimented, high-density array. Such structures are highly versatile and are ideal morphological architectures for various applications such as chemical sensors, split-dipole nanoantennas, photovoltaic devices with radially heterostructured "core-shell" nanowires, and memory devices with a cross-point geometry. Such architecture is generally realized by a template-directed approach. In the past this method has been used to grow metal and inorganic semiconductor nanowire arrays. More recently &pi;-conjugated polymer nanowires have been grown within nanoporous templates. However, these approaches have had limited success in growing nanowires of technologically important &pi;-conjugated small molecular weight organics, such as tris-8-hydroxyquinoline aluminum (Alq3), rubrene and methanofullerenes, which are commonly used in diverse areas including organic displays, photovoltaics, thin film transistors and spintronics.	
Recently we have been able to address the above-mentioned issue by employing a novel "centrifugation-assisted" approach. This method therefore broadens the spectrum of organic materials that can be patterned in a vertically ordered nanowire array. Due to the technological importance of Alq3, rubrene and methanofullerenes, our method can be used to explore how the nanostructuring of these materials affects the performance of aforementioned organic devices. The purpose of this article is to describe the technical details of the above-mentioned protocol, demonstrate how this process can be extended to grow small-molecular organic nanowires on arbitrary substrates and finally, to discuss the critical steps, limitations, possible modifications, trouble-shooting and future applications.

We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and spintronics.

In  recent years &pi;-conjugated  organic semiconductors have emerged as the active material in a number of  diverse applications including large-area, ...

Engineering

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Bioengineering

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 ...

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 ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

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.

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.

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

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

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