This work was supported with funding provided from the Office of Naval Research.&#160;J. Fontana acknowledges the National Research Council for a postdoctoral associateship.

1. Self-assembled Ligand-nanoparticle Monolayers
As an illustrative example of the self-assembly technique, macroscopic, thiol-alkane capped gold nanosphere monolayer films are produced as follows:
<ol>
	<li>Concentrate 15&#160;nm gold nanospheres (commercially available at a number density: 1012 particles/ml) to ~1013 particles/ml&#160;in water.
	<ol>
		<li>Place 15 ml of the dilute nanosphere water suspension into an ultra centrifugal filter (100&#160;K nominal molecular weight limit).</li>
		<li>Centrifuge the filter/vial at 4,500 x g for 2 min&#160;or until only a few ml remain in the filter chamber.</li>
	</ol>
	</li>
	<li>Resuspend the nanosphere in approximately 1 ml of deionized (DI) water such that the nanoparticle concentration is 1013 particles/ml. The suspension is stable for several hours once resuspended in DI water.
	<ol>
		<li>Verify the number density and confirm the nanoparticles have not aggregated. Dilute the concentrated nanoparticle suspension by a factor of 1:10 back to the original concentration by placing 0.150 ml of the suspension into a cuvette (1 cm path length), and to this add 1.35 ml of DI water.</li>
		<li>Place the cuvette into a spectrometer and measure the absorbance spectrum of the suspension and the original suspension. Compare the peak position and full width at half maximum to ensure aggregation has not occurred. The magnitude of the absorbance peaks for both samples should be approximately the same, thus ensuring the concentrated sample is denser by a factor of 10.</li>
	</ol>
	</li>
	<li>In a separate clean 20&#160;ml borosilicate glass vial add 1 ml of tetrahydrofuran (THF).</li>
	<li>Add the thiol-alkane ligands (e.g. 5 ml of 1,6-hexanedithiol and 5 ml of 1-dodecanethiol) to the THF and shake the solution to mix uniformly. Enough ligand should be added to cover at least the entire surface area of the suspended nanoparticles. Excess ligand increases the speed and efficiency of the reaction.</li>
	<li>In a fume hood, pour the contents of the vial containing the gold nanospheres into the vial of THF-ligands.</li>
	<li>Quickly screw on the lid and shake the vial vigorously for 15 sec.</li>
	<li>Remove the lid and set the vial down in the fume hood, Figure 1(a). Depending on the ligands used, domains of gold nanoparticle films quickly form at the air-liquid interface, Figure 1(c). The films will then begin to translate up the sides of the vial, Figure 1(d). Nearly all the nanoparticles are capped with thiol-ligand, removed from the suspension, and transported to the sides of the vial within 1&#160;hr, Figure 1(e).</li>
</ol>
2. Transferring the Monolayers onto Removable Substrates
<ol>
	<li>To transfer the films onto removable glass and silicon wafer substrates: cut the substrates into an area of 12.5 mm x 25.4 mm using a scribing pen/wheel.
	<ol>
		<li>Glass Substrates: clean using an acetone rinse, followed by an isopropyl alcohol rinse, and finally a DI water rinse. Allow the substrates to dry, proceed to section 2.2.</li>
		<li>Silicon Wafer Substrates: in a fume hood prepare Piranha solution (3 parts concentrated sulfuric acid to 1 part 30% hydrogen peroxide, CAUTION: oxidizer, corrosive). Place 15 ml of sulfuric acid into a 20&#160;ml borosilicate glass vial. To this slowly add 5 ml of 30% hydrogen peroxide. Do not cap the vial. Use caution; the mixture is highly exothermic. See reference for more safety information19.</li>
		<li>Carefully submerse the silicon wafer substrates into Piranha solution for 30 min, remove, rinse with DI water and dry with nitrogen.</li>
		<li>As an optional step, the vial used for the nanoparticle ligand exchange and self-assembly can be salinized to force all the nanoparticles onto the glass substrate or silicon wafer instead of the walls of the glass vial, otherwise proceed to section 2.2.</li>
		<li>Fill the glass vial with piranha solution (CAUTION: oxidizer, corrosive), refer to section 2.1.2.</li>
		<li>Allow the vial to soak for 30 min. After 30 min&#160;rinse the vial out with DI water.</li>
		<li>Fill vial with 1% v/v of hexamethyldisilazane in acetone and cap.</li>
		<li>Allow the sealed vial to soak for 24 hr, then rinse with DI water and dry with nitrogen.</li>
	</ol>
	</li>
	<li>Prior to shaking (section 1.6) insert the substrate into the vial. Screw on lid and shake.</li>
	<li>After shaking remove lid and, using tweezers, position the substrate nearly vertical against the vial wall.</li>
	<li>Use a pipette to coat the reaction mixture onto the substrate. The reaction stops when all the organic solvent has evaporated or all the nanoparticle have been removed from the suspension.</li>
</ol>
3. Monolayer Analysis
<ol>
	<li>Estimate the packing efficiency of the nanospheres in the monolayer quickly by observing the transmission and reflective properties of the film. Illuminate the monolayer on glass substrates from behind with a white-light source. With a white light source, a uniform colored film should be observed for high-density nanoparticle monolayer films in transmission and a gold-like reflection observed in reflection, Figure 2.</li>
	<li>Use a spectrometer (see section 1.2.2) to quantify the macroscopic absorbance spectrum from the monolayers, Figure 4. Normalize the absorbance spectrum with a clean glass slide. Mount the monolayer film, on a glass substrate, into the beam path of the spectrometer and collect the absorbance spectrum. 
	Note: The absorbance peak should be significantly red-shifted&#160;several hundred nanometers depending on the ligand used. The quality factor of the absorbance peak should be comparable to the dilute suspension value, but only slightly broadened (Figure 4). If the absorbance peak is very broad or not well defined then the monolayer films are probably of poor quality, proceed to section 3.3 for further characterization.</li>
	<li>Examine the nanoscopic organization of the nanospheres using scanning electron microscopy (SEM) of monolayers transferred onto silicon wafer substrates (see section 2.1.2) as shown in Figure 3. If the films are on glass substrates connect conductive tape to one corner of the film and ground it to the SEM pedestal to prevent charging and allow imaging.</li>
</ol>
4. Efficient Phase Transfer Technique for Organic Soluble Nanoparticles
<ol>
	<li>To use the technique as an efficient means to functionalize the nanoparticles with thiol-ligands, decant the remaining solution from the bottom of the vial after the reaction is complete, section 1.7, and dry the material in the vial under nitrogen.</li>
	<li>Add an organic solvent (e.g. chloroform, toluene) to re-suspend the nanoparticles with nearly 100% particle phase transfer and recovery.</li>
	<li>Repeat section 1.2.1 to ensure the nanoparticles have not aggregated upon re-suspension into the organic solvent. If the absorbance peak is board, relative to the original suspension, sonicate the sample for 15 min&#160;to help redisperse the nanoparticles, Figure 4.</li>
</ol>

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S., Sastry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+spontaneous+growth+of+colloidal+nanoparticle+superlattices.">A new technique for the spontaneous growth of colloidal nanoparticle superlattices.</a> Langmuir. 15, 1902-1904 (1999).</li><li>Spain, E. M., Johnson, D. D., Kang, B., Vigorita, J. 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The authors have nothing to disclose.

This protocol describes a single &#8216;one-pot&#8217; self-assembly technique to create macroscopic nanoparticle-ligand monolayer films using phase transfer, phase separation and surface tension gradients.&#160; The advantage of this technique is that it combines three self-assembly processes into a single, low-cost process; by quickly and efficiently phase transferring the nanoparticles, assembling the particles into monolayers at the air-fluid interface and transporting the monolayer films onto template-free substrates.
The most critical elements for creating high-density monolayers are using freshly synthesized citrate stabilized gold nanoparticles, choosing a suitable substrate/ligand/solvent, controlling the evaporation rate and temperature, and using materials free of contaminates.
The rate of the nanosphere phase transfer was observed to decrease with the age of the nanoparticles, presumably from changes in the nanosphere surface chemistry21. Typically the &#8216;life-time&#8217; of the gold nanoparticles was less than 3 months from when they were synthesized.&#160; If the gold nanoparticles are purchased, the phase transfer rate is also greatly reduced if the manufacturer stabilizes the nanoparticles in excessive amounts of &#8216;unknown&#8217; surfactants.&#160; The phase transfer rate was difficult to quantify for commercial nanospheres since the age and surfactant were unknowns. For larger nanosphere diameters (&#62;30 nm) the size of the nanospheres hinders large film formation and the film areas are decreased typically to square millimeters.&#160; Hydrophobic substrates, such as Teflon, did not form good films since the water-THF could not wet the surface and subsequently transport the film onto the substrate surface.
The amount of ligand used needs to be sufficient to cover the entire surface area of the nanoparticles in suspension to observe film formation at the air-liquid interface and to enable the films to translate up the side of the vial.&#160; Adding excess ligands greatly increased the speed and final height the films reached on the substrate1.&#160;The packing density of the nanoparticle into monolayer films also depends on the specific ligand selected; thiol-alkane/ene/phenol were tested and all worked reasonably well, singularly or in combination. The pH of the reaction mixture is also an important parameter and will be the subject of future studies.
This self-assembly technique, with further refinement, may enable the future development of high-throughput, tunable nanoparticle-ligand structures.

The self-assembly of macroscopic nanoparticle films has attracted great attention for their unique properties determined from the geometry and composition of the elements5 and may lead to of a wide range of optical, electronic and chemical applications6-14. To self-assemble such films metallic nanoparticles capped with ligands must be packed into high density, monolayers. However several assembly issues need to be addressed to advance the development of such materials.
First, surfactant stabilized metallic nanoparticles are typically synthesized by wet-chemistry methods in dilute suspensions15. To prevent aggregation and to control the interparticle spacing of the nanoparticles in the films, the nanoparticles need to be capped with ligand shells. After the nanoparticles have been functionalized with ligands the nanoparticles typically remain in relatively dilute suspensions.&#160;A technique is then needed to self-assemble the nanoparticles into macroscopic, high-density, monolayer films16,17.
Cheng et al.18 phase transferred gold nanorods using thiolated polystyrene in a water-tetrahydrofuran suspension.&#160; The nanorods where then re-suspended in chloroform and a drop was placed at an air-water interface and evaporated slowly, forming monolayer films. Bigioni et al.17 created macroscopic monolayers of dodecanethiol capped gold nanospheres using excess ligand and rapid solvent evaporation, but the nanospheres needed to be phase transferred prior to self-assembling.&#160;
Once the monolayer films are formed they typically need to be transported onto a substrate.&#160; Mayya et al.3 confined nanospheres at a water-toluene interface and transferred them onto template-free substrates using surface tension gradients. Similarly, Johnson et al.4&#160;suspended silver nanospheres in excess ligand and then translated the nanoparticles up the walls of the vial using surface tension gradients from two immiscible fluids. While assembly techniques exist to address each of these issues the need for more efficient techniques is needed to aid in the development of large-scale nanoparticle film production.
Here we demonstrate a straightforward and robust technique that combines the three self-assembly issues described above in to a single &#8216;one-pot&#8217; technique, shown in Figure 1. A water miscible organic solvent (e.g. tetrahydrofuran, dimeythl sulfoxide), is used to first quickly and efficiently functionalize thiol-ligands (e.g. thiol-alkane, thiol-ene, thiol-phenol) onto the nanoparticles (e.g. gold nanospheres, nanorods, etc.). The mixture then drives&#160;self-assembly of the nanoparticles into macroscopic, high-density, monolayer films at the air-fluid interface using phase separation.&#160; Finally, monolayer films of nanoparticles form onto template-free substrates using surface tension gradients from the water/organic solvent mixture, Figure 2 and Figure 3.

<table border="0" cellpadding="0" cellspacing="0" width="628">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">1-6 hexanedithiol</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:140px;">H12005-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">1-dodecanethiol</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:140px;">471364-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">20 ml liquid scintillation vials</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:140px;">Z253081-1PAK</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:224px;">acetone</td>
			<td style="width:97px;">Sigma</td>
			<td>650501-1L</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:224px;">amicon ultra-15 centrifugal filter&#160;</td>
			<td style="width:97px;">Millipore</td>
			<td style="width:140px;">100K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">centrifuge</td>
			<td style="width:97px;">Sorvall&#160;</td>
			<td style="width:140px;">RC5B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">centrifuge&#160;</td>
			<td style="width:97px;">Eppendorf</td>
			<td style="width:140px;">5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">deionized water&#160;</td>
			<td style="width:97px;">in-house&#39;</td>
			<td style="width:140px;">N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:224px;">glass slides</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:140px;">CLS294875X25-72EA</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:224px;">15 nm gold nanospheres</td>
			<td style="width:97px;">Ted Pella, Inc</td>
			<td style="width:140px;">15703-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">hexamethyldisilazane</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:140px;">52619-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">hydrogen peroxide (30%)</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:140px;">216763-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">scanning electron microscope</td>
			<td style="width:97px;">Carl Zeiss</td>
			<td style="width:140px;">Model 55</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">polished silicon wafer</td>
			<td style="width:97px;">Sun Edison</td>
			<td style="width:140px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">spectrometer</td>
			<td style="width:97px;">OceanOptics</td>
			<td style="width:140px;">USB4000-VIS-NIR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">sulfuric acid</td>
			<td style="width:97px;">Fisher</td>
			<td style="width:140px;">A300-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">tetrahydrofuran</td>
			<td style="width:97px;">Sigma</td>
			<td style="width:140px;">401757-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

Figure 1(a) shows a suspension of gold nanospheres, thiol-alkane ligands, tetrahydrofuran and water in a glass vial immediately after mixing. A schematic of the three main self-assembly stages, phase transfer, phase separation, and surface tension gradient mediated film transport&#160;is shown in Figure 1(b) as an expanded view at the air-fluid interface near the side of the vial.
The thiol groups on the ligands rapidly bond to the gold nanospheres after mixing, displacing the ionic surfactant, causing the nanospheres to become hydrophobic and more miscible in THF. The lower density of THF, relative to the water, aids to quickly transport the nanospheres to the air-fluid interface where they become confined by the reduction of free energy, Figure 1(c). Suspending the ligands in a water-miscible organic solvent also increases the available surface area between the thiol-ligands and nanospheres, increasing the rate the nanospheres phase transfer, compared to systems that use two immiscible fluids3,4.
Macroscopic monolayer domains of nanospheres typically begin to form at the air-fluid interface within several minutes after mixing, but this process is ligand dependent. The shaking of the vial also coats the sides of the vial with a thin film of the suspension. The THF in the thin film on the sides of the vial evaporates faster than the water, creating a surface tension gradient between the thin film and bulk suspension. The fluid then flows from the low to the high surface tension regions carrying the nanospheres domains from the air-fluid interface up the side of the vial, Figure 1(d)3. After all the organic solvent has evaporated or all the nanosphere have been removed from the suspension the reaction is complete, Figure 1(e).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51282/51282fig1highres.jpg" width="500" /> 
Figure 1. Self-assembly technique. (a) A suspension of 15&#160;nm gold nanospheres, thiol-alkane ligands, tetrahydrofuran and water in a glass vial. (b) Schematic of the three main self-assembly stages; phase transfer, phase separation, and film transport. (c) Phase transfer and separation of the nanoparticles at the air-fluid interface. (d) Surface tension gradient mediated transport of the nanoparticle monolayer domains. (e) Completed reaction.
Macroscopic nanoparticle-ligand monolayer films (~cm) are demonstrated using this technique without multilayers or large particle density gradients on a template-free substrate. Figure 2 is an image of a thiol-alkane capped 15&#160;nm gold nanosphere monolayer film on a glass substrate partially reflecting light (right side), signifying the high volume fraction of nanospheres and transmitting light (left side), demonstrating the preservation of the plasmon resonances, uniformity and optical clarity. If more than one layer is present it can easily be seen with the naked eye1. The right edge of the film has excess surfactant present on the top surface giving rise to the slight discoloration in the reflected light. Additional monolayer images can be found in references1,2.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51282/51282fig2highres.jpg" width="500" /> 
Figure 2. Macroscopic gold nanosphere monolayers. Alkane-thiol capped gold nanosphere monolayer film on a glass substrate is partially transmitting light (left side) and reflecting light (right side).
Figure 3&#160;presents false-colored SEM images of a thiol-alkane capped 15 nm gold nanosphere monolayer film on a silicon wafer substrate. Figure 3(a) shows the edge of the film, demonstrating the films are monolayers and the nanospheres pack into amorphous domains at microscopic length scales. At nanoscopic length scales the films contain hexagonally close packed domains as demonstrated by the Fourier transform of the image in Figure 3(b) (inset).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51282/51282fig3highres.jpg" width="500" /> 
Figure 3. False-colored SEM images. Thiol-alkane capped 15&#160;nm gold nanosphere monolayer films. The inset in upper-right corner of (b) is the Fourier transform of the image.
The normalized experimental absorbance from a monolayer film consisting of thiol-alkane capped 15&#160;nm gold nanospheres on a glass substrate (red line), and a suspension of 15&#160;nm gold nanospheres in water (blue line) and phase transferred into chloroform (green line) is shown in Figure 4. Although shifted and slightly broadened, relative to the aqueous suspension, due to particle-particle coupling20 and changes in the host medium, the plasmon resonances are preserved well for the monolayer film given the density of nanospheres.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51282/51282fig4highres.jpg" width="500" /> 
Figure 4. Normalized experimental absorbance spectra. Macroscopic absorbance from 15&#160;nm gold nanospheres in a water suspension (blue line), phase transferred into a chloroform suspension (green line) and as a monolayer film (red line).

Watch this Scientific Journal Video about A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates at JoVE.com

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

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Nanyang Technological University

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Chemistry

10.8K Views.  Naval Research Laboratory. A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

Video: A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

Atomically Defined Templates for Epitaxial Growth of Complex Oxide Thin Films

Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain such surfaces are described. The first approach is the preparation of single terminated perovskite SrTiO3 (001) and DyScO3 (110) substrates. Wet etching was used to selectively remove one of the two possible surface terminations, while an annealing step was used to increase the smoothness of the surface. The resulting single terminated surfaces allow for the heteroepitaxial growth of perovskite oxide thin films with high crystalline quality and well-defined interfaces between substrate and film. In the second approach, seed layers for epitaxial film growth on arbitrary substrates were created by Langmuir-Blodgett (LB) deposition of nanosheets. As model system Ca2Nb3O10- nanosheets were used, prepared by delamination of their layered parent compound HCa2Nb3O10. A key advantage of creating seed layers with nanosheets is that relatively expensive and size-limited single crystalline substrates can be replaced by virtually any substrate material.

Various procedures are outlined to prepare atomically defined templates for epitaxial growth of complex oxide thin films. Chemical treatments of single crystalline SrTiO3 (001) and DyScO3 (110) substrates were performed to obtain atomically smooth, single terminated surfaces. Ca2 Nb3 O10- nanosheets were used to create atomically defined templates on arbitrary substrates.

Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain ...

Fabrication of Large-area Free-standing Ultrathin Polymer Films

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films. Typically, ultrathin films are prepared using either sacrificial layers, which may damage the film or affect its mechanical properties, or they are made on freshly cleaved mica, a substrate that is difficult to scale. Further, the size of ultrathin film is typically limited to a few square millimeters. In this method, we modify a surface with a polyelectrolyte that alters the strength of adhesion between polymer and deposition substrate. The polyelectrolyte can be shown to remain on the wafer using spectroscopy, and a treated wafer can be used to produce multiple films, indicating that at best minimal amounts of the polyelectrolyte are added to the film. The process has thus far been shown to be limited in scalability only by the size of the coating equipment, and is expected to be readily scalable to industrial processes. In this study, the protocol for making the solutions, preparing the deposition surface, and producing the films is described.

We describe a method for the fabrication of large-area (up to 13 cm diameter) and ultrathin (as thin as 8 nm) polymer films. Instead of using a sacrificial interlayer to delaminate the film from its substrate, we use a self-limiting surface treatment suitable for arbitrarily large areas.

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films.  Typically, ultrathin films are prepared ...

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 ...

Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements are simple high-throughput characterization experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glassy states for a variety of glassy materials. This technique allows for these parameters to be measured in a single experiment, while other methods must combine a variety of different techniques to investigate all of these properties. Measurements of dynamics close to Tg are particularly challenging. The advantage of cooling rate dependent Tg measurements over other methods which directly probe bulk and surface relaxation dynamics is that they are relatively quick and simple experiments, which do not utilize fluorophores or other complicated experimental techniques. Furthermore, this technique probes the average dynamics of technologically relevant thin films in temperature and relaxation time (&#964;&#945;) regimes relevant to the glass transition (&#964;&#945; &#62; 100 sec). The limitation to using ellipsometry for cooling rate dependent Tg experiments is that it cannot probe relaxation times relevant to measurements of viscosity (&#964;&#945; &#60;&#60; 1 sec). Other cooling rate dependent Tg measurement techniques, however, can extend the CR-Tg method to faster relaxation times. Furthermore, this technique can be used for any glassy system so long as the integrity of the film remains throughout the experiment.

Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements ...

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Reactive Vapor Deposition of Conjugated Polymer Films on Arbitrary Substrates

We demonstrate a method of conformally coating conjugated polymers on arbitrary substrates using a custom-designed, low-pressure reaction chamber. Conductive polymers, poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-propylenedioxythiophene) (PProDOT), and a semiconducting polymer, poly(thieno[3,2-b]thiophene) (PTT), were deposited on unconventional highly-disordered and textured substrates with high surface areas, such as paper, towels and fabrics. This reported deposition chamber is an improvement of previous vapor reactors because our system can accommodate both volatile and nonvolatile monomers, such as 3,4-propylenedioxythiophene and thieno[3,2-b]thiophene. Utilization of both solid and liquid oxidants are also demonstrated. One limitation of this method is that it lacks sophisticated in situ thickness monitors. Polymer coatings made by the commonly used solution-based coating methods, such as spin-coating and surface grafting, are often not uniform or susceptible to mechanical degradation. This reported vapor phase deposition method overcomes those drawbacks and is a strong alternative to common solution-based coating methods. Notably, polymer films coated by the reported method are uniform and conformal on rough surfaces, even at a micrometer scale. This feature allows for future application of vapor deposited polymers in electronics devices on flexible and highly textured substrates.

This paper presents a protocol for reactive vapor deposition of poly(3,4-ethylenedioxythiophene), poly(3,4-propylenedioxythiophene), and poly(thieno[3,2-b]thiophene) films on glass slides and rough substrates, such as textiles and paper.

We demonstrate a method of conformally coating conjugated polymers on arbitrary substrates using a custom-designed, low-pressure reaction chamber. ...

Growth of Gold Dendritic Nanoforests on Titanium Nitride-coated Silicon Substrates

In this study, a high-power impulse magnetron sputtering system is used to coat a flat and firm titanium nitride (TiN) film on silicon (Si) wafers, and a fluoride-assisted galvanic replacement reaction (FAGRR) is employed for the rapid and easy deposition of gold dendritic nanoforests (Au DNFs) on the TiN/Si substrates. Scanning electron microscopy (SEM) images and energy-dispersive X-ray spectroscopy patterns of TiN/Si and Au DNFs/TiN/Si samples validate that the synthesis process is accurately controlled. Under the reaction conditions in this study, the thickness of the Au DNFs increases linearly to 5.10 &#177; 0.20 &#181;m within 15 min of the reaction. Therefore, the employed synthesis procedure is a simple and rapid approach for preparing Au DNFs/TiN/Si composites.

This study presents a feasible procedure for synthesizing gold dendritic nanoforests on titanium nitride/silicon substrates. The thickness of gold dendritic nanoforests increases linearly within 15 min of a synthesis reaction.

In this study, a high-power impulse magnetron sputtering system is used to coat a flat and firm titanium nitride (TiN) film on silicon (Si) wafers, and a ...