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>

<ol>
	<li>Fontana, J., Naciri, J., Rendell, R., Ratna, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+self-assembly+and+optical+characterization+of+nanoparticle&#8211;ligand+metamaterials.">Macroscopic self-assembly and optical characterization of nanoparticle&#8211;ligand metamaterials.</a> Advanced Optical Materials. 1, 100-106 (2013).</li><li>Fontana, 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=Large+surface-enhanced+Raman+scattering+from+self-assembled+gold+nanosphere+monolayers.">Large surface-enhanced Raman scattering from self-assembled gold nanosphere monolayers.</a> Applied Physics Letters. 102, (2013).</li><li>Mayya, K. 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. L., Amram, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marangoni+flow+of+Ag+nanoparticles+from+the+fluid-fluid+interface.">Marangoni flow of Ag nanoparticles from the fluid-fluid interface.</a> J Phys Chem A. 112, 9318-9323 (2008).</li><li>Sihvola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metamaterials:+A+Personal+View.">Metamaterials: A Personal View.</a> Radioengineering. 18, 90-94 (2009).</li><li>Valentine, J., Li, J. S., Zentgraf, T., Bartal, G., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+cloak+made+of+dielectrics.">An optical cloak made of dielectrics.</a> Nature Materials. 8, 568-571 (2009).</li><li>Seo, E., 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=Double+hydrophilic+block+copolymer+templated+Au+nanoparticles+with+enhanced+catalytic+activity+toward+nitroarene+reduction.">Double hydrophilic block copolymer templated Au nanoparticles with enhanced catalytic activity toward nitroarene reduction.</a> The Journal of Physical Chemistry C. , (2013).</li><li>Ward, D. R., 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=Simultaneous+measurements+of+electronic+conduction+and+Raman+response+in+molecular+junctions.">Simultaneous measurements of electronic conduction and Raman response in molecular junctions.</a> Nano Letters. 8, 919-924 (2008).</li><li>Perez-Gonzalez, O., 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=Optical+Spectroscopy+of+Conductive+Junctions+in+Plasmonic+Cavities.">Optical Spectroscopy of Conductive Junctions in Plasmonic Cavities.</a> Nano Letters. 10, 3090-3095 (2010).</li><li>Xiao, S. M., Chettiar, U. K., Kildishev, A. V., Drachev, V. P., Shalaev, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yellow-light+negative-index+metamaterials.">Yellow-light negative-index metamaterials.</a> Optics Letters. 34, 3478-3480 (2009).</li><li>Fang, N., Lee, H., Sun, C., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limited+optical+imaging+with+a+silver+superlens.">Sub-diffraction-limited optical imaging with a silver superlens.</a> Science. 308, (2005).</li><li>Liu, Z. W., Lee, H., Xiong, Y., Sun, C., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Far-field+optical+hyperlens+magnifying+sub-diffraction-limited+objects.">Far-field optical hyperlens magnifying sub-diffraction-limited objects.</a> Science. 315, 1686-1686 (2007).</li><li>Valentine, 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=Three-dimensional+optical+metamaterial+with+a+negative+refractive+index.">Three-dimensional optical metamaterial with a negative refractive index.</a> Nature. 455, (2008).</li><li>Law, M., Greene, L. E., Johnson, J. C., Saykally, R., Yang, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanowire+dye-sensitized+solar+cells.">Nanowire dye-sensitized solar cells.</a> Nature Materials. 4, 455-459 (2005).</li><li>Frens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Nucleation+for+Regulation+of+Particle-Size+in+Monodisperse+Gold+Suspensions.">Controlled Nucleation for Regulation of Particle-Size in Monodisperse Gold Suspensions.</a> Nature-Phys Sci. 241, 20-22 (1973).</li><li>Ye, X., Chen, J., Diroll, B. T., Murray, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+Plasmonic+Coupling+in+Self-Assembled+Binary+Nanocrystal+Superlattices+Studied+by+Correlated+Optical+Microspectrophotometry+and+Electron+Microscopy.">Tunable Plasmonic Coupling in Self-Assembled Binary Nanocrystal Superlattices Studied by Correlated Optical Microspectrophotometry and Electron Microscopy.</a> Nano Letters. 13, 1291-1297 (2013).</li><li>Bigioni, T. P., 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=Kinetically+driven+self-assembly+of+highly+ordered+nanoparticle+monolayers.">Kinetically driven self-assembly of highly ordered nanoparticle monolayers.</a> Nature Materials. 5, (2006).</li><li>Ng, K. C., 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=Free-Standing+Plasmonic-Nanorod+Super+lattice+Sheets.">Free-Standing Plasmonic-Nanorod Super lattice Sheets.</a> Acs Nano. 6, 925-934 (2012).</li><li>Romero, I., Aizpurua, J., Bryant, G. W., de Abajo, F. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmons+in+nearly+touching+metallic+nanoparticles:+singular+response+in+the+limit+of+touching+dimers.">Plasmons in nearly touching metallic nanoparticles: singular response in the limit of touching dimers.</a> Optics Express. 14, 9988-9999 (2006).</li><li>Caragheorgheopol, A., Chechik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+aspects+of+ligand+exchange+in+Au+nanoparticles.">Mechanistic aspects of ligand exchange in Au nanoparticles.</a> Physical Chemistry Chemical Physics. 10, 5029-5041 (2008).</li></ol>

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><tbody><tr><td>1,6-hexanedithiol</td><td>Sigma</td><td>H12005-5G</td><td></td></tr><tr><td>1-dodecanethiol</td><td>Sigma</td><td>471364-100ML</td><td></td></tr><tr><td>20 ml liquid scintillation vials</td><td>Sigma</td><td>Z253081-1PAK</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501-1L</td><td></td></tr><tr><td>Amicon ultra-15 centrifugal filter</td><td>Millipore</td><td>100K</td><td></td></tr><tr><td>Centrifuge</td><td>Sorvall</td><td>RC5B</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Deionized water</td><td>In-house</td><td>N/A</td><td></td></tr><tr><td>Glass slides</td><td>Sigma</td><td>CLS294875X25-72EA</td><td></td></tr><tr><td>15 nm gold nanospheres</td><td>Ted Pella, Inc</td><td>15703-1</td><td></td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma</td><td>52619-50ML</td><td></td></tr><tr><td>Hydrogen peroxide (30%)</td><td>Sigma</td><td>216763-100ML</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Carl Zeiss</td><td>Model 55</td><td></td></tr><tr><td>Polished silicon wafer</td><td>Sun Edison</td><td>N/A</td><td></td></tr><tr><td>spectrometer</td><td>OceanOptics</td><td>USB4000-VIS-NIR</td><td></td></tr><tr><td>Sulfuric acid</td><td>Fisher</td><td>A300-212</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Sigma</td><td>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 ...

a-technique-to-functionalize-self-assemble-macroscopic-nanoparticle

Nanyang Technological University

Research

JoVE Journal

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

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 ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Bioengineering

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 ...

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.
Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.
The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.

In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates.

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in ...

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

Engineering

Large Area Substrate-Based Nanofabrication of Controllable and Customizable Gold Nanoparticles Via Capped Dewetting

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and high-density data storage have demonstrated the potential benefit of their use in industrial applications. These applications require precise control over nanoparticle size, spacing, and sometimes shape. These requirements have resulted in the use of time and cost intensive processing steps to produce nanoparticles, thus making the transition to industrial application unrealistic. This protocol will resolve this issue by providing a scalable and affordable method for the large-area production of nanoparticle films with improved nanoparticle control compared to the current techniques. In this article, the process will be demonstrated with gold, but other metals can also be used.

This protocol details a novel nano-manufacturing technique that can be used to make controllable and customizable nanoparticle films over large areas based on the self-assembly of dewetting of capped metal films.

Recent scientific advances in the utilization of metallic nanoparticle for enhanced energy conversion efficiency, improved optical device performance, and ...

Procedure for the Transfer of Polymer Films Onto Porous Substrates with Minimized Defects

The fabrication of devices containing thin film composite membranes necessitates the transfer of these films onto the surfaces of arbitrary support substrates. Accomplishing this transfer in a highly controlled, mechanized, and reproducible manner can eliminate the creation of macroscale defect structures (e.g., tears, cracks, and wrinkles) within the thin film that compromise device performance and the usable area per sample. Here, we describe a general protocol for the highly controlled and mechanized transfer of a polymeric thin film onto an arbitrary porous support substrate for eventual use as a water filtration membrane device. Specifically, we fabricate a block copolymer (BCP) thin film on top of a sacrificial, water-soluble poly(acrylic acid) (PAA) layer and silicon wafer substrate. We then utilize a custom-designed, 3D-printed transfer tool and drain chamber system to deposit, lift-off, and transfer the BCP thin film onto the center of a porous anodized aluminum oxide (AAO) support disc. The transferred BCP thin film is shown to be consistently placed onto the center of the support surface due to the guidance of the meniscus formed between the water and the 3D-printed plastic drain chamber. We also compare our mechanized transfer-processed thin films to those that have been transferred by hand with the use of tweezers. Optical inspection and image analysis of the transferred thin films from the mechanized process confirm that little-to-no macroscale inhomogeneities or plastic deformations are produced, as compared to the multitude of tears and wrinkles produced from manual transfer by hand. Our results suggest that the proposed strategy for thin film transfer can reduce defects when compared to other methods across many systems and applications.

We present a procedure for highly controlled and wrinkle-free transfer of block copolymer thin films onto porous support substrates using a 3D-printed drain chamber. The drain chamber design is of general relevance to all procedures involving transfer of macromolecular films onto porous substrates, which is normally done by hand in an irreproducible fashion.

The fabrication of devices containing thin film composite membranes necessitates the transfer of these films onto the surfaces of arbitrary support ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

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 ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.