This work was supported by JCO A*STAR Investigatorship grant (Project no. 1437C00135), A*STAR Science &amp; Engineering Research Council Public Sector Funding (Project no. 1421200076), and a JSPS-NUS Joint Research Projects grant (WBS R143-000-611-133).

1. Preparation of Precursor Stock Solutions
<ol>
	<li>TOP-Se stock solution.
	<ol>
		<li>In a nitrogen atmosphere glovebox, weigh out 11.84 g of selenium pellets into a 150-mL conical flask and place a magnetic stir bar in the flask.</li>
		<li>Add 100 mL of tri-n-octylphosphine (TOP) to the conical flask and seal the flask with a rubber septum.</li>
		<li>Stir the mixture overnight at 800 RPM. 
		NOTE: Once the selenium pellets have dissolved fully into the TOP, the TOP-Se stock solution (1.5 M) is ready to be used in further reactions.</li>
	</ol>
	</li>
	<li>TOP-S stock solution.
	<ol>
		<li>Weigh out 0.611 g of sulfur powder into a 20-mL single-necked round-bottom flask (RBF) and place a magnetic stir bar in the flask.</li>
		<li>Add 10 mL of TOP to the sulfur powder and seal the RBF with a rubber septum. Place it under a nitrogen atmosphere and stir at 800 RPM.</li>
		<li>Once the sulfur powder has dissolved and the solution is clear, use the TOP-S stock solution in further reactions.</li>
	</ol>
	</li>
</ol>
2. Synthesis of CdSe Quantum Dot (QD) Stock Solution
<ol>
	<li>Wurtzite CdSe QDs. 
	Note: See5.
	<ol>
		<li>Mix 9 g of tri-n-octylphosphine oxide (TOPO), 6 g of hexadecyl amine (HDA), and 0.25 mL of diisooctylphosphonic acid (DIPA) in a three-necked RBF. Add a magnetic stir bar to the mixture and insert a temperature probe into the RBF through a punctured rubber septum.
		<ol>
			<li>Mount a reflux condenser over the RBF and connect it to a Schlenk line via an adapter. Apply high vacuum grease at all glass-to-glass joints.</li>
		</ol>
		</li>
		<li>Degas the solution at 100 &#176;C for 2 h under constant stirring at 800 RPM.</li>
		<li>Separately, mix 0.320 g of cadmium acetylacetonate (Cd(acac)2), 0.570 g of hexadecanediol (HDDO), and 6 mL of 1-octadecene (ODE) in a single-necked RBF. Add a magnetic stir bar and degas the mixture under constant stirring over an oil bath for 2 h at 120 &#176;C and 800 RPM.</li>
		<li>After degassing for 2 h, increase the temperature of the three-necked RBF to 345 &#176;C. Concurrently, let the cadmium precursor mixture cool to room temperature and add 4 mL of TOP-Se stock solution to the single-necked RBF.</li>
		<li>Swiftly inject the entire cadmium and selenium precursor solution into the three-necked RBF once its temperature reaches 345 &#176;C. Remove the heating mantle immediately after injecting the precursor solution and allow the reaction mixture to cool to room temperature under ambient conditions.</li>
		<li>Add 15 mL of toluene to the crude reaction mixture and process the synthesized QDs by centrifuging for 3 min at 2,240 x g. Discard the precipitate and add an excess of methanol to the recovered supernatant. Centrifuge the mixture at 2,325 x g for 8 min and discard the supernatant.</li>
		<li>Add 5 mL of toluene to the precipitate to disperse the QDs. Carry out further processing cycles via the precipitation of the QDs using 30 mL of methanol, centrifuge the suspension at 2,240 x g for 3 min, discard the supernatant, and disperse the QDs in 5 mL of toluene.
		<ol>
			<li>Following two cycles of processing, disperse the QDs in a minimal amount (~2-3 mL) of toluene (henceforth referred to as the QD stock solution) for further use.</li>
		</ol>
		</li>
		<li>Determine the concentration of QDs in solution by taking 20 &#181;L of the stock solution and adding it to 3 mL of toluene. Measure the absorbance at 350 nm and calculate the concentration using the known molar absorptivity at 350 nm6. Scale the concentration accordingly to take into account the dilution of the stock solution. 
		NOTE: It is important to maintain vigorous stirring of the solution and to immediately remove the heating mantle after the swift injection of the precursor solution in step 2.1.5 to obtain highly size-monodisperse QDs.</li>
	</ol>
	</li>
	<li>Zinc-blende CdSe QDs. 
	Note: See7.
	<ol>
		<li>Mix 0.0384 g of cadmium oxide (CdO), 0.137 g of myristic acid (MA), and 5 mL of ODE in a three-necked RBF. Add a magnetic stir bar to the mixture and insert a temperature probe into the RBF through a punctured rubber septum. Mount a reflux condenser over the RBF and connect it to a Schlenk line via an adapter. Seal the remaining port with a rubber septum. Apply high vacuum grease at all glass-to-glass joints.</li>
		<li>Degas the solution at 90 &#176;C under constant stirring at 800 RPM.</li>
		<li>Separately, degas a mixture of 0.05 mL of oleic acid (OA), 0.5 mL of oleylamine (OM), and 2 mL of ODE in a single-necked RBF under constant stirring at 75 &#176;C and 800 RPM.</li>
		<li>Increase the temperature of the cadmium precursor solution to 250 &#176;C and maintain it for 15 min. Observe a color change in the solution, from dark brown to colorless, indicating the formation of cadmium myristate.</li>
		<li>Cool the reaction mixture to 90 &#176;C and add 12 mL of ODE. Degas the mixture again for 1 h.</li>
		<li>Let the reaction mixture cool to room temperature. Add 0.012 g of selenium powder and degas the mixture for 20 min.</li>
		<li>Increase the temperature of the reaction mixture to 240 &#176;C under N2. Observe a color change, from colorless to yellow at 150 &#176;C and then to orange-red upon reaching 240 &#176;C, signifying the formation of zb-CdSe QDs.</li>
		<li>Grow the QDs for 5 min at 240 &#176;C and then cool the mixture to room temperature. Add the degassed mixture of OM, OA, and ODE dropwise to the reaction solution. Leave the solution to cool to room temperature under ambient conditions.</li>
		<li>Process the synthesized QDs by adding acetone to the growth solution until the total volume is about 50 mL. Centrifuge the resulting suspension at 2,240 x g for 3 min. Subsequently, discard the supernatant.</li>
		<li>Add 5 mL of toluene to the precipitate to disperse the QDs. Carry out further processing cycles: precipitate the QDs using 30 mL of methanol, centrifuge the suspension at 2,240 x g for 3 min, discard the supernatant, and disperse the QDs in 5 mL of toluene.
		<ol>
			<li>Following two cycles of processing, disperse the QDs in a minimal amount (~2-3 mL) of toluene (henceforth referred to as the QD stock solution) for further use.</li>
		</ol>
		</li>
		<li>Determine the concentration of CdSe QDs in the stock solution by taking 20 &#181;L of the processed nanoparticles and adding it to 3 mL of toluene. Measure the absorbance at 350 nm and calculate the concentration using the known molar absorptivity at 350 nm6. Scale the concentration accordingly to take into account the dilution of the stock solution. 
		NOTE: For the successful synthesis of monodisperse QDs, it is critical to ensure that all the selenium powder is added to the reaction mixture and that none of it is left stuck to the neck and sidewalls of the RBF.</li>
	</ol>
	</li>
</ol>
3. Synthesis of CdSe-seeded CdS NRs
Note: See8.
<ol>
	<li>Mix 3 g of TOPO, 0.90 g of CdO, 0.80 g of hexylphosphonic acid (HPA), and 0.29 g of octadecylphosphonic acid (ODPA) in a 50-mL three-necked RBF. Add a magnetic stir bar to the mixture and insert a temperature probe through a punctured rubber septum.</li>
	<li>Mount a reflux condenser over the RBF and connect it to a Schlenk line via an adapter. Seal the remaining port with a rubber septum. Apply high vacuum grease at all glass-to-glass joints. Heat the RBF to 150 &#176;C and place it under a vacuum for 1.5 h to degas, with stirring at 800 RPM.</li>
	<li>Place 1.8 mL of the TOP-S stock solution into a 10-mL single-necked RBF and seal it with a rubber septum. Add 80 nmol of wurtzite CdSe QDs in toluene to the TOP-S solution and subsequently remove the toluene under vacuum at 70 &#176;C. Allow the solution to degas under stirring at 800 RPM for a further 30 min.</li>
	<li>Place the RBF containing the Cd precursor under nitrogen and raise the temperature to 350 &#176;C. At 320 &#176;C, add 1.8 mL of TOP to the RBF through the rubber septum.</li>
	<li>At 350 &#176;C, draw the TOP-S solution containing the w-CdSe QDs into a syringe and rapidly inject it into the RBF containing the Cd precursor. Allow the solution to stir at 800 RPM for an additional 6 min to allow for the growth of the NRs. Subsequently, remove the heating mantle and cool the solution to room temperature under ambient conditions.</li>
	<li>To process the solution of NRs, add 2 mL of toluene to the growth solution and place the entire mixture into a 50-mL centrifuge tube. Add 30 mL of methanol. Subsequently, centrifuge the resulting suspension at 2,240 x g for 3 min and then discard the supernatant.</li>
	<li>Add 5 mL of toluene to the precipitate to disperse the NRs. Carry out further processing cycles: precipitate the NRs using 30 mL of methanol, centrifuge the suspension at 2,240 x g for 3 min, discard the supernatant, and disperse the NRs in 5 mL of toluene.
	<ol>
		<li>Following 2-3 cycles of processing, disperse the NRs in 5 mL of toluene (henceforth referred to as the NR stock solution) for further use.</li>
	</ol>
	</li>
	<li>Prepare the sample for TEM analysis.5
	<ol>
		<li>Place a drop of the NR solution onto a 300-mesh copper grid covered with a continuous carbon film for electron microscopy analysis.</li>
		<li>Remove the excess solution with an adsorbent paper and dry the sample at room temperature.</li>
	</ol>
	</li>
	<li>Perform TEM imaging (and analysis) to obtain the dimensions of the NR so that the average volume of the NR and the number of moles of CdS per NR can be determined.
	<ol>
		<li>Determine the concentration of CdSe-seeded CdS NRs in the stock solution by taking 20 &#181;L of the processed NRs and adding it to 3 mL of toluene. Measure the absorbance at 350 nm and calculate the concentration of NRs using the known molar absorptivity at that wavelength (assumed to be dominated by CdS)9. Scale the concentration accordingly to account for the dilution of the stock solution. 
		NOTE: For a typical synthesis, the yield of NRs is nearly 100%, with very few byproducts such as branched structures.</li>
	</ol>
	</li>
</ol>
4. Synthesis of CdSe-seeded CdSe NRs
Note: See8.
<ol>
	<li>Mix 1.035 g of CdO, 0.1657 g of HPA, 0.1543 g of n-tetradecylphosphonic acid (TDPA), and 3 g of TOPO in a three-necked RBF. Add a magnetic stir bar to the mixture and insert a temperature probe into the RBF through a punctured rubber septum. Mount a reflux condenser over the RBF and connect it to a Schlenk line via an adapter. Seal the remaining port with a rubber septum. Apply high vacuum grease at all glass-to-glass joints.</li>
	<li>Degas the solution at 150 &#176;C for 1.5 h under constant stirring at 800 RPM.</li>
	<li>Separately, mix 2 mL of the TOPSe stock solution and 10 nmol of w-CdSe QDs (dispersed in toluene) in a single-necked RBF and degas under constant stirring over an oil bath at 800 RPM and 90 &#176;C until all the toluene is removed.</li>
	<li>After degassing, increase the temperature of the cadmium precursor mixture to 340 &#176;C. As the temperature increases, observe a color change in the solution, from dark brown to colorless, indicating the formation of the cadmium-alkyl phosphonic acid complex.</li>
	<li>As the temperature of cadmium precursor reaches 340 &#176;C, swiftly inject 1.8 mL of TOP into the mixture and allow the temperature to recover to 340 &#176;C.</li>
	<li>Upon reaching 340 &#176;C, swiftly inject 1.8 mL of the TOP-Se/CdSe QD mixture into the three-necked RBF, whereupon the temperature will drop to 320 &#176;C. Upon recovery to 340 &#176;C, maintain the reaction solution at this temperature for ~3 min before removing the heating mantle and allowing the solution to cool to room temperature under ambient conditions.</li>
	<li>After the reaction solution has cooled to room temperature, add 2 mL of toluene and transfer the entire solution to a 50-mL centrifuge tube. Top up the centrifuge tube with methanol (99%) to 50 mL and centrifuge the resulting suspension at 2,240 x g for 3 min. Subsequently, discard the supernatant.</li>
	<li>Add 5 mL of toluene to the precipitate to disperse the NRs. Carry out further processing cycles: precipitate the NRs using 30 mL of methanol (99%), centrifuge the suspension at 2,240 x g for 3 min, discard the supernatant, and disperse the NRs in 5 mL of toluene.
	<ol>
		<li>Following 2-3 cycles of processing, disperse the NRs in 5 mL of toluene (henceforth referred to as the NR stock solution) for further use.</li>
	</ol>
	</li>
	<li>Place a drop of NR solution onto a 300-mesh copper grid covered with a continuous carbon film for electron microscopy analysis. Remove the excess solution with an adsorbent paper and dry the sample at room temperature.</li>
	<li>Perform TEM imaging and analysis to obtain the dimensions of the NRs so that the average volume of the NR and the number of moles of CdSe per NR can be determined.
	<ol>
		<li>Determine the concentration of NRs in solution by taking 20 &#181;L of the stock solution and adding it to 3 mL of toluene. Measure the absorbance at 350 nm and calculate the concentration using the known molar absorptivity at that wavelength6. Scale the concentration accordingly to take into account the dilution of the stock solution. 
		NOTE: For a typical synthesis, the yield of NRs is close to 100%, with very few byproducts such as branched structures.</li>
	</ol>
	</li>
</ol>
5. Synthesis of CdSe-seeded CdSe TPs
Note: See10.
<ol>
	<li>Mix 0.43 g of CdO, 3.8 mL of OA, 2.2 mL ODE, and 0.3 mL of TOP in a three-necked RBF. Add a magnetic stir bar to the mixture and insert a temperature probe into the RBF through a punctured rubber septum. Mount a reflux condenser over the RBF and connect it to a Schlenk line via an adapter. Seal the remaining port with a rubber septum. Apply high vacuum grease at all glass-to-glass joints.</li>
	<li>Degas the solution at 90 &#176;C for 1 h under constant stirring at 800 RPM and then increase the temperature to 265 &#176;C. As the temperature reaches 265 &#176;C, observe the color of the solution changing, from dark brown to colorless, indicating the formation of the cadmium oleate complex.</li>
	<li>Reduce the temperature to 50 &#176;C and add 1.7 mL of 2 M TOPSe (separately prepared by sonicating, at 37 kHz and 320 W, a mixture of 0.316 g of Se powder in 2 mL of TOP in an 8-mL glass vial) and 0.017 g of hexadecyltrimethylammonium bromide (CTAB).</li>
	<li>Separately, mix 7 mL of ODE, 0.025 g of CTAB, 0.5 mL of TOP, 0.75 mL of OA, and 100 nmol of zinc-blende CdSe QDs (dispersed in toluene) in a three-necked RBF. Add a magnetic stir bar to the mixture and insert a temperature probe into the RBF through a punctured rubber septum.
	<ol>
		<li>Mount a condenser over the RBF and connect it to the Schlenk line via an adapter. Seal the remaining port with a rubber septum. Apply high vacuum grease at all glass-to-glass joints.</li>
	</ol>
	</li>
	<li>Degas the mixture prepared in step 5.4 at 90 &#176;C and 800 RPM under constant stirring for ~45 min until all the toluene is removed.</li>
	<li>Increase the temperature of the reaction mixture containing the zinc-blende CdSe QDs to 260 &#176;C. When the temperature reaches 260 &#176;C, add 8 mL of the cadmium precursor solution to the mixture containing zinc-blende CdSe QDs at an injection rate of 0.25 mL/min using a syringe pump. After the addition is completed, allow the solution to cool to room temperature under ambient conditions.</li>
	<li>After the solution has cooled to room temperature, transfer it to a 50-mL centrifuge tube. Add 40 mL of acetone (99%) to precipitate the TPs and centrifuge the suspension at 1,340 x g for 10 min. Subsequently, discard the supernatant.</li>
	<li>Add 5 mL of toluene to the precipitate of TPs to disperse them. Carry out further processing cycles: precipitate the TPs using 30 mL of methanol (99%), centrifuge the suspension at 2,240 x g for 3 min, discard the supernatant, and disperse the TPs in 5 mL of toluene.
	<ol>
		<li>Following 2-3 cycles of processing, disperse the TPs in 5 mL of toluene (henceforth referred to as the TP stock solution) for further use.</li>
	</ol>
	</li>
	<li>Place a drop of the TP solution onto a 300-mesh copper grid covered with a continuous carbon film for electron microscopy analysis. Remove the excess solution with adsorbent paper and dry the sample at room temperature.</li>
	<li>Perform TEM imaging and analysis to obtain the dimensions of the TPs so that the average volume of the TP and the number of moles of CdSe per TP can be determined.
	<ol>
		<li>Determine the concentration of QDs in solution by taking 20 &#181;L of the stock solution and adding it to 3 mL of toluene.</li>
		<li>Measure the absorbance at 350 nm and calculate the concentration using the known molar absorptivity at that wavelength6. Scale the concentration accordingly to account for the dilution of the stock solution. 
		Note: For a typical synthesis, the yield for the TPs is ~80%, with ~20% being bipods and tripods.</li>
	</ol>
	</li>
</ol>
6. Facet Activation and the Linking of Nanostructures
<ol>
	<li>Preparation of a dodecylamine (DDA) stock solution.
	<ol>
		<li>Prepare a DDA stock solution by adding 0.140 g of DDA to 5 mL of ethanol. Sonicate the solution at 37 kHz and 320 W for ~5 min to ensure that the DDA is fully dissolved.</li>
	</ol>
	</li>
	<li>Cation exchange and linking.
	<ol>
		<li>Prepare a 1-mL solution of NC (NR or TP) at the appropriate concentration (see Table 1). Add 6 mg of ODPA to the 1 mL of NC solution and sonicate for 10 min at 37 kHz and 320 W.</li>
		<li>Separately, mix 1 mL of DDA stock solution and 1 mL of Ag+ solution at the appropriate concentration (see Table 1) in a vial. Add a magnetic stir bar and stir the solution vigorously at 800 RPM.</li>
		<li>While stirring, add 1 mL of the NC solution to the vial and allow the reaction to proceed for the corresponding amount of time, as listed in Table 1.</li>
		<li>At the end of the reaction, stop the stirring and allow the solution to phase-separate. Extract and remove the bottom aqueous layer. Add 5 mL of methanol to the organic layer, to precipitate out the NCs. Centrifuge the vial for 3 min at 2,240 x g.</li>
		<li>After centrifugation, discard the supernatant and add 1 mL of toluene to re-disperse the product for further characterization. 
		NOTE: The cation exchange reaction occurs at different rates for different materials. Table 1 summarizes the set of conditions under which the synthesis of linked nanostructures of other materials and morphologies are synthesized. The volume of NC solution (1 mL), aqueous Ag+ solution (1 mL), and ethanol-DDA solution (1 mL) and the amount of ODPA (6 mg) added are kept the same, as mentioned above, for each set of reactions.</li>
	</ol>
	</li>
</ol>

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We do not have anything to disclose.

The linking technique described in this work allows for shape-anisotropic cadmium chalcogenide nanoparticles that can undergo cation exchange with Ag+ to be joined, facet-to-facet, into assemblies such as linear chains or branched networks. Failure to form well-dispersed, extensive assemblies of facet-to-facet linked nanoparticles is often because of two reasons: (i) the ODPA is not dispersed well in the NR-containing solution, which can be addressed by sonicating the mixture for the prescribed amount of time detailed in the protocol; or (ii) the concentration of Ag+ used is non-optimal. When the Ag+ concentration used is too low, most of the cadmium chalcogenide nanoparticles do not undergo cation exchange, resulting in little or no linking (as described in the Representative Results). When the Ag+ concentration used is too high, multiple domains of Ag2S form on each particle, resulting in severe aggregation upon linking.
Our approach to the facet-to-facet linking of shape-anisotropic inorganic nanoparticles is currently limited to metal chalcogenide nanoparticles that can undergo cation-exchange with Ag+ or Cu+ (not shown in this work) under mild reaction conditions. Efforts are underway to expand the repertoire of inorganic materials that can be linked directly via our synthetic strategy. We previously demonstrated that the facet-to-facet linked nanoparticle assemblies can undergo further cation exchange processes and can be transformed into other semiconductor materials while retaining their linked architecture5. This modified linking procedure, although more tedious, can significantly widen the diversity of semiconductor nanomaterials that can be brought together into facet-linked assemblies.
Because the assemblies of linked semiconductor nanoparticles are joined facet-to-facet, they are electronically coupled to each other. This may be advantageous for applications such as solution-processed optoelectronics, where a major problem is the poor charge transport through insulating ligands surrounding the semiconductor nanoparticles. Further work will be needed to evaluate the efficacy of assemblies of linked semiconductor nanoparticles as the active material in optoelectronic devices.

The directed assembly of colloidal semiconductor NCs offers a synthetic pathway to the fabrication of nanostructures whose physicochemical properties are either the collective sum of or radically different from their individual NC building blocks1,2,3,4. Among the various approaches to nanoparticle assembly, the method of oriented attachment - in which NCs are essentially fused with each other - stands out as one that allows for interparticle electronic coupling. However, conventional oriented attachment typically requires the delicate balancing of particle dipole-, ligand- and solvent-based interactions that are generally difficult to execute and make applicable to different NC systems.
We have recently developed a wet-chemical method of covalently joining shape-anisotropic cadmium chalcogenide NCs by introducing a reactive inorganic intermediate through a site-selective nucleation process. The particles are subsequently linked by the spontaneous fusion of the reactive inorganic intermediate domains5. Although the technique is still based on an oriented attachment mechanism, there is much less need to consider weak interparticle interactions, thus allowing for more flexibility and control. The linking of shape-anisotropic cadmium chalcogenide NCs is carried out by first converting their tip facets to silver chalcogenide via a partial cation exchange process (in solution); this is followed by the selective removal of ligands passivating the surface. The NCs then come together via the fusion of the exposed silver chalcogenide facets, resulting in assemblies of cadmium chalcogenide NCs that are linked end-to-end.
In this protocol, we demonstrate that the linking technique can be applied to a variety of shape-anisotropic cadmium chalcogenide NCs (i.e., CdSe-seeded CdS NRs and CdSe-seeded CdSe NRs or TPs), yielding long linear NR chains or highly branched TP networks. These results suggest that the technique can be extended to a wide variety of NC shapes and metal chalcogenides amenable to silver cation exchange.

<table><tbody><tr><td>Cadmium oxide (CdO), 99.5%</td><td>Sigma Aldrich</td><td></td><td>Highly toxic</td></tr><tr><td>Tri-n-octylphosphine oxide (TOPO), 90 % and 99%</td><td>Sigma Aldrich</td><td></td><td>Technical and analytical grade</td></tr><tr><td>Cadmium acetylacetonate (Cd(acac)&lt;sub&gt;2&lt;/sub&gt;), 99.9%</td><td>Sigma Aldrich</td><td></td><td>Highly toxic</td></tr><tr><td>Hexadecanediol (HDDO), 90%</td><td>Sigma Aldrich</td><td></td><td>Technical grade</td></tr><tr><td>1-octadecene (ODE), 90%</td><td>Sigma Aldrich</td><td></td><td>Technical grade</td></tr><tr><td>Dodecylamine (DDA), 98%</td><td>Sigma Aldrich</td><td></td><td>Toxic</td></tr><tr><td>Cadmium nitrate tetrahydrate ((CdNO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;.4H&lt;sub&gt;2&lt;/sub&gt;O), 98%</td><td>Sigma Aldrich</td><td></td><td>Highly toxic</td></tr><tr><td>Myristic acid (MA), 99%</td><td>Sigma Aldrich</td><td></td><td>Analytical grade</td></tr><tr><td>Octyl phosphonic acid (OPA), 97%</td><td>Sigma Aldrich</td><td></td><td>Analytical grade</td></tr><tr><td>Oleylamine (Oly), 70%</td><td>Sigma Aldrich</td><td></td><td>Technical grade</td></tr><tr><td>Hexadecyltrimethylammonium bromide (CTAB), 95%</td><td>Sigma Aldrich</td><td></td><td>Toxic</td></tr><tr><td>Selenium pellets (Se, 5 mm), 99.99%</td><td>Sigma Aldrich</td><td></td><td>Analytical grade</td></tr><tr><td>Hexadecylamine (HDA), 90%</td><td>Alfa Aesar</td><td></td><td>Technical grade, toxic</td></tr><tr><td>n-tetradecylphosphonic acid (TDPA), 98%&amp;nbsp;</td><td>Alfa Aesar</td><td></td><td>Analytical grade</td></tr><tr><td>Silver nitrate (AgNO&lt;sub&gt;3&lt;/sub&gt;), 99.9%</td><td>Alfa Aesar</td><td></td><td>Analytical grade</td></tr><tr><td>Oleic acid (OA), 90%</td><td>Alfa Aesar</td><td></td><td>Technical grade</td></tr><tr><td>Tri-n-octylphosphine (TOP), 97%</td><td>Strem</td><td></td><td>Analytical grade, toxic, air sensitive</td></tr><tr><td>n-hexylphosphonic acid (HPA), 97%</td><td>Strem</td><td></td><td>Analytical grade</td></tr><tr><td>n-octadecylphosphonic acid (ODPA), 97%</td><td>Strem</td><td></td><td>Analytical grade</td></tr><tr><td>Tellurium powder (Te), 99.9%</td><td>Strem</td><td></td><td>Air sensitive</td></tr><tr><td>Tri-n-butylphosphine (TBP), 99%</td><td>Strem</td><td></td><td>Analytical grade, highly toxic, air sensitive</td></tr><tr><td>Diisooctylphosphonic acid (DIPA), 90%</td><td>Fluka</td><td></td><td>Technical grade, toxic</td></tr></tbody></table>

facet-to-facet linking of shape-anisotropic colloidal cadmium chalcogenide nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

Using CdSe-seeded CdS NRs as a model system, as illustrated in Figure 1(a), we demonstrated that we can use a partial Ag+ exchange process to specifically transform the facets at the NR tips to Ag2S. The Ag2S facets are capped by DDA, which reacts with ODPA via an acid-base reaction to form an insoluble salt5. This causes the DDA ligands to be removed from the Ag2S facets, causing them to fuse with each other upon contact and to form linked NR chains, as shown in Figure 1(b). To ascertain whether the individual NRs are fused within the chain or are simply held together by Van der Waals forces, HRTEM analysis on the joint regions was carried out. As illustrated in Figure 1(c), there is a distinct domain in the epitaxial contact with two NRs. An FFT analysis of the HRTEM image (inset of Figure 1(c)) reveals the existence of two different lattice constants that can be ascribed to the (001) facets of Ag2S and CdS. Additionally, point energy-dispersive X-ray spectroscopy (EDX) analysis on the linkage region prominently shows the presence of Ag and the absence of Cd (Figure 1(d)), which corroborates our notion of Ag2S-tipped CdSe-seeded CdS NRs bridged via the fusion of the Ag2S domains. The yield and statistical nature of the linking process can be visualized via a histogram (Figure 1(e)) that shows the number of rods linked within a NR chain.
The mechanism for linking was previously reported by Sabyasachi et al.5 and will not be described in detail in this work. It is observed that, without the addition of ODPA, no linking takes place as seen in Figure 2(a), in accordance with the view that the addition of ODPA causes the native surface ligands of the Ag2S tip to come off. This can clearly be seen in Figure 2(c), as the histogram for the reaction shows a large proportion of single, unlinked NRs. The presence of dimers might be due to residual ODPA left over from the NR synthesis reaction, allowing a small amount of linking to occur. Aside from ODPA, the concentration of AgNO3 was also found to be extremely important, as illustrated in Figure 2(b), where only short chains were obtained under non-optimal concentrations of AgNO3. Where the Ag+ concentration used was too low, the histogram shows that the distribution was heavily weighted towards shorter chain lengths. Indeed, the linking statistics for the chains shown in Figure 2(b) feature a substantial proportion of dimers, followed by monomers, as seen in Figure 2(d).
In addition to CdSe-seeded CdS NRs, we show that the Ag+-mediated linking process can be extended to CdSe-seeded CdSe NRs and TPs, which are shown in Figure 3(a) and (b), respectively. Under the reaction conditions summarized in Table 1, we show that we can achieve similar chained networks of CdSe-seeded CdSe NRs and TPs, as exemplified in Figure 3(c) and (d). As in the case of Ag2S-linked CdSe-seeded CdS NRs, the CdSe-seeded CdSe nanoparticles are linked via Ag2Se intermediates.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56009/56009fig1.jpg" /> 
Figure 1. Characterization of linked CdSe-seeded CdS NRs. (a) Low-resolution TEM image showing as-synthesized CdSe-seeded CdS NR. (b) Low-resolution TEM image showing Ag2S-linked CdSe-seeded CdS NRs. (c) High-resolution TEM (HRTEM) image of a single joint between two NRs. Inset is a Fast Fourier Transform (FFT) of the HRTEM image, showing two different lattice points that are attributed to CdS and Ag2S. (d) Point EDX analysis on a single linkage point, confirming its composition as Ag2S. (e) Histogram showing the distribution of the number of NRs per chain in a typical linked NR sample. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56009/56009fig1large.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/56009/56009fig2.jpg" /> 
Figure 2. Results of non-optimal reaction conditions. (a) TEM image of CdSe-seeded CdS NRs with Ag2S tips that underwent the linking procedure, with the exception that no ODPA was used. (b) TEM image showing short chains of Ag2S-linked CdSe-seeded CdS NRs when the Ag+ concentration was not optimized. (c) Histogram showing that most of the species present are single NRs when ODPA is absent. (d) Histogram showing that when the Ag+ concentration is not optimized, only short chains are present. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56009/56009fig2large.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/56009/56009fig3.jpg" /> 
Figure 3. Extension of the linking process to CdSe-seeded CdSe nanostructures. (a) Low-resolution TEM image showing as-synthesized CdSe-seeded CdSe NRs. (b) Low-resolution TEM image showing as-synthesized CdSe-seeded CdSe TPs. (c) Low-resolution TEM image showing the CdSe-seeded CdSe NRs after linking. (d) Low-resolution TEM image showing the CdSe-seeded CdSe TPs after linking. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56009/56009fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Nanoparticle</td>
			<td>Concentration of NC solution (mM)</td>
			<td>Concentration of Ag+ solution (mM)</td>
			<td>Stirring time (hours)</td>
		</tr>
		<tr>
			<td>CdSe seeded CdS NRs</td>
			<td>3</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>CdSe seeded CdSe NRs</td>
			<td>5</td>
			<td>0.8</td>
			<td>1</td>
		</tr>
		<tr>
			<td>CdSe seeded CdSe TPs</td>
			<td>5</td>
			<td>0.6</td>
			<td>0.5</td>
		</tr>
	</tbody>
</table>
Table 1. Summary of the approximate concentrations of the NC solution and the Ag+ solution and of the required stirring time.

Watch this Scientific Journal Video about Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures at JoVE.com

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

facet-to-facet-linking-shape-anisotropic-colloidal-cadmium

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.6K Views.  National University of Singapore. A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Video: Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Engineering

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted significant interests across the research fields of materials science, physics, chemistry and biology. The key enabling technology is a liquid cell. We fabricate liquid cells with thin viewing windows through a sequential microfabrication process, including silicon nitride membrane deposition, photolithographic patterning, wafer etching, cell bonding, etc. A liquid cell with the dimensions of a regular TEM grid can fit in any standard TEM sample holder. About 100 nanoliters reaction solution is loaded into the reservoirs and about 30 picoliters liquid is drawn into the viewing windows by capillary force. Subsequently, the cell is sealed and loaded into a microscope for in situ imaging. Inside the TEM, the electron beam goes through the thin liquid layer sandwiched between two silicon nitride membranes. Dynamic processes of nanoparticles in liquids, such as nucleation and growth of nanocrystals, diffusion and assembly of nanoparticles, etc., have been imaged in real time with sub-nanometer resolution. We have also applied this method to other research areas, e.g., imaging proteins in water. Liquid cell TEM is poised to play a major role in revealing dynamic processes of materials in their working environments. It may also bring high impact in the study of biological processes in their native environment.

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

The recent development for in situ transmission electron microscopy, which allows imaging through liquids with high spatial resolution, has attracted ...

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous networks, ribbons, worm-like aggregates, toroidal structures, and spherical particles) is described. These aggregates are morphologically influenced by extensive hydrophobic side chain-side chain interactions of the singular polycarbodiimide strands, as inferred by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. Polycarbodiimide-g-polystyrene copolymers (PS-PCDs) were prepared by a combination of synthetic methods, including coordination-insertion polymerization, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) &#34;click&#34; chemistry, and atom transfer radical polymerization (ATRP). PS-PCDs were found to form specific toroidal architectures at low concentrations in CHCl3. To determine the influence of a more polar solvent medium (i.e., THF and THF/EtOH) on polymer aggregation behavior, a number of representative PS-PCD composites have been tested to show discrete concentration-dependent spherical particles. These fundamental studies are of practical interest to the development of experimental procedures for desirable architectures by directed self-assembly in thin film. These architectures may be exploited as drug carriers, whereas other morphological findings represent certain interest in the area of novel functional materials.

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

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 ...

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.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Bioengineering

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...