The authors acknowledge The Netherlands Organization for Scientific research (NWO ECHO-STIP Grant 717.013.005, NWO VIDI Grant 723.014.006) for the financial support.

1. Synthesis of Core-shell Silica Particles
Note: Silica particles are synthesized according to the following procedure, which is based on the St&#246;ber method24,25.
<ol>
	<li>Synthesis of fluorescent silica seeds
	<ol>
		<li>Dissolve 105 mg (0.27 mmol) of fluorescein-isothiocyanate in 5 ml of ethanol.</li>
		<li>Add 100 &#181;l of (3-aminopropyl) triethoxysilane (APTES, 0.43 mmol) to the previous solution.</li>
		<li>Sonicate the solution durin...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+roughness+directed+self-assembly+of+patchy+particles+into+colloidal+micelles.">Surface roughness directed self-assembly of patchy particles into colloidal micelles.</a> PNAS. 109 (27), 10787-10792 (2012).</li><li>Rossi, L., 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=Cubic+crystals+from+cubic+colloids.">Cubic crystals from cubic colloids.</a> Soft Matter. 7 (9), 4139-4142 (2011).</li><li>Erb, R. M., Son, H. S., Samanta, B., Rotello, V. M., Yellen, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+assembly+of+colloidal+superstructures+with+multipole+symmetry.">Magnetic assembly of colloidal superstructures with multipole symmetry.</a> Nature. 457 (7232), 999-1002 (2009).</li><li>Vutukuri, H. 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=Colloidal+analogues+of+charged+and+uncharged+polymer+chains+with+tunable+stiffness.">Colloidal analogues of charged and uncharged polymer chains with tunable stiffness.</a> Angew. Chem. Int. Edit. 51 (45), 11249-11253 (2012).</li><li>De Greef, T. F. A., Meijer, E. 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The authors have nothing to disclose.

When cyclohexane, with a refractive index of 1.426, is used as a solvent to disperse the BTA-colloids, van der Waals interactions are very weak, since the refractive indices of colloids and solvent are nearly the same. Note that the concentration of functionalized colloids used for the SLS experiments in cyclohexane is much higher compared to the bare silica colloids in water. This is necessary to obtain a sufficiently strong scattering due to the low contrast as the refractive indices are almost matched. Trace amounts o...

Mesostructured colloidal materials find widespread application in science and technology, as model systems for fundamental studies on atomic and molecular materials1,2, as photonic materials3,4, as drug delivery systems5,6, as coatings7 and in lithography for surface patterning8,9. Since lyophobic colloids are metastable materials that eventually aggregate irreversibly due to the omnipresent van der Waals interactions, their manipulation into specific target structures is notoriously difficult. Numerous strategies have been developed to control colloidal self-assembly including the use of additives to tune the electrostatic10,11 or depletion interactions12,13, or external triggers such as magnetic14 or electric15 fields. A sophisticated alternative strategy to achieve control over the structure, dynamics and mechanics of these systems is their functionalization with molecules interacting through specific and directional forces. Supramolecular chemistry offers a comprehensive toolbox of small molecules exhibiting site-specific, directional and strong yet reversible interactions, which can be modulated in strength by solvent polarity, temperature and light16. Since their properties have been studied extensively in bulk and in solution, these molecules are attractive candidates to structure soft materials into exotic phases in a predictable manner. Despite the clear potential of such an integrated approach to orchestrate colloidal assembly via supramolecular chemistry, these disciplines have rarely interfaced to tailor the properties of mesostructured colloidal materials17,18.
A solid platform of supramolecular colloids must fulfill three main requirements. Firstly, coupling of the supramolecular moiety should be done under mild-conditions to prevent degradation. Secondly, surface forces at separations larger than direct contact should be dominated by the tethered motifs, which means that uncoated colloids should nearly exclusively interact via excluded-volume interactions. Therefore, the physico-chemical properties of the colloids should be tailored to suppress other interactions inherent in colloidal systems, such as van der Waals or electrostatic forces. Thirdly, characterization should allow for an unequivocal attribution of the assembly to the presence of the supramolecular moieties. To meet these three prerequisites, a robust two-step synthesis of supramolecular colloids was developed (Figure 1a). In a first step, hydrophobic NVOC-functionalized silica particles are prepared for dispersion in cyclohexane. The NVOC group can be easily cleaved, yielding amine-functionalized particles. The high reactivity of amines enables straightforward post-functionalization with the desired supramolecular moiety using a wide range of mild reaction conditions. Herein, we prepare supramolecular colloids by functionalization of silica beads with stearyl alcohol and a benzene-1,3,5-tricarboxamide (BTA) derivative20. The stearyl alcohol plays several important roles: it makes the colloids organophilic and it introduces short-range steric repulsions which aids to reduce the nonspecific interaction between colloids21,22. van der Waals forces are further reduced because of the close match between the refractive index of the colloids and the solvent23. Light-and thermoresponsive short-range attractive surface forces are generated by incorporation of o-nitrobenzyl protected BTAs20. O-nitrobenzyl moiety is a photo-cleavable group that blocks the formation of hydrogen bonds between adjacent BTAs when incorporated on the amides in the discotics (Figure 1b). Upon photocleavage by UV-light, the BTA in solution is able to recognize and interact with identical BTA molecules through a 3-fold hydrogen bond array, with a binding strength that is strongly temperature dependent17. Since the van der Waals attractions are minimal for stearyl coated silica particles in cyclohexane as well as light- and temperature-independent, the observed stimuli-responsive colloidal assembly must be BTA-mediated.
This detailed video demonstrates how to synthesize and characterize supramolecular colloids and how to study their self-assembly upon UV-irradiation by confocal microscopy. In addition, a simple image analysis protocol to distinguish colloidal singlets from clustered colloids and to determine the amount of colloids per clusters is reported. The versatility of the synthetic strategy allows to readily vary particle size, surface coverage as well as the introduced binding moiety, which opens up new avenues for the development of a large family of colloidal building blocks for mesostructured advanced materials.

<table border="0" cellpadding="0" cellspacing="0" width="1805">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">APTES</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">FTIC</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">TEOS</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">LUDOX AS-40</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td>Silica particles of 13 nm in radius</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">MilliQ</td>
			<td style="width:128px;">---</td>
			<td style="width:119px;">---</td>
			<td style="width:1035px;">18.2 M&#937;&#183;cm at 25 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Ethanol</td>
			<td style="width:128px;">SolvaChrom</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Ammonia (25% in water)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Chloroform</td>
			<td style="width:128px;">SolvaChrom</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Cyclohexane</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Dimethylformamide (DMF)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Stearyl alcohol</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Diisopropylethylamine (DIPEA)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinimidyl 3-(2-pyridyldithio)propionate (SPDP)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:1035px;">---</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">NVOC-C11-OH</td>
			<td style="width:128px;">Synthesized</td>
			<td style="width:119px;">---</td>
			<td style="width:1035px;">I. de Feijter, 2014 Responsive materials from adaptive supramolecular constructs, Doctoral thesis, Technical University of Eindhoven, The Netherlands</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">BTA</td>
			<td style="width:128px;">Synthesized</td>
			<td style="width:119px;">---</td>
			<td style="width:1035px;">I. de Feijter, 2014 Responsive materials from adaptive supramolecular constructs, Doctoral thesis, Technical University of Eindhoven, The Netherlands</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Centrifuge</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Heraeus Megafuge 1.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Ultrasound bath</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;"></td>
			<td>Ultrasonic cleaner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic pumps</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td>PHD Ultra Syringe Pump</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:524px;">UV-oven</td>
			<td style="width:128px;">Luzchem</td>
			<td style="width:119px;"></td>
			<td>LZC-a V UV reactor equipped with 8x8 UVA light bulbs (&#955;max=354 nm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Stirrer-heating plate</td>
			<td style="width:128px;">Heidolph</td>
			<td style="width:119px;"></td>
			<td>MR-Hei Standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Light Scattering</td>
			<td style="width:128px;">ALV</td>
			<td style="width:119px;"></td>
			<td>CGS-3 MD-4 compact goniometer system, equipped with a Multiple Tau digital real time correlator (ALV-7004) and a solid-state laser (&#955;=532 nm, 40 mW)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-Vis spectrophotometer</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>NanoDrop 1000 Spectrophotometer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Confocal microscope</td>
			<td style="width:128px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Ti Eclipse with an argon laser with &#955;excitation=488 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Slide spacers</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td>Grace BioLabs Secure seal imaging spacer (1 well, diam. &#215; thickness 13 mm &#215; 0.12 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Syringes</td>
			<td style="width:128px;">BD Plastipak</td>
			<td style="width:119px;"></td>
			<td>20 mL syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Plastic tubing</td>
			<td style="width:128px;">SCI</td>
			<td style="width:119px;">BB31695-PE/5</td>
			<td>Ethylene oxide gas sterilizable micro medical tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:524px;">Pulsating vortex mixer</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;"></td>
			<td>Electrical: 120V, 50/60Hz, 150W Speed Range: 500&#8211;3000 rpm</td>
		</tr>
	</tbody>
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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.

&#160;Given that the two-step procedure used to synthesize the supramolecular colloids (Figure 1a), couples the BTA- derivatives (Figure 1b) in a second step at room temperature and in mild-reaction conditions, its stability is ensured.
<img alt="Figure 1" src="/files/ftp_upload/53934/53934fig1.jpg" /> 
Figure 1. Scheme of the synthesis of supramolecular colloids. A) Coupli...

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Synthesis and Characterization of Supramolecular Colloids

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 ...

The overall goal of this protocol is to describe how to direct colloidal self-assembly by anchoring supramolecular moieties onto the colloids whose interactions are strong, directional, and reversible. This method can help to answer key questions in the material science field, such as how to accurately control the colloidal self-assembly to create complex and mesostructured materials with interesting properties. The main advantage of this technique is that colloidal association is driven by the supramolecular moieties that remain responsive to light and to temperature upon surface immobilization.Therefore, colloids self-assemble upon photoactivation of the hydrogen bonds of the molecules by UV light. To synthesize the florescent silica seeds, first mix 2.5 milliliters of dye-functionalized aptes with 25 millileters of 25%ammonia and 250 milliliters of ethanol in a one liter round-bottom flask. To obtain monodisperse silica seeds it is really important to add the tetraethyl orthosilicate under the meniscus of the reaction mixture.Using a glass pipette, add 10 milileters of TEOS while stirring with a magnetic stirrer. Similarly, after five hours, add another 1.75 milliliters of TEOS and stir the mixture overnight under an argon atmosphere. The following day, wash the seeds as described in the text protocol.To synthesize the core-shell silica particles, first add ethanol, deionized water, 25%ammonia, and the seed dispersion to a one liter round-bottom flask. Then, fill a plastic syringe with five milliliters of TEOS and 10 milliliters of ethanol. Fill a second plastic syringe with 1.34 milliliters of 25%ammonia, 3.4 milliliters of deionized water, and 10.25 milliliters of ethanol.Connect both syringes to the round-bottom flask with plastic tubing. Equip the flask with an argon flow. The argon inlet has to be next to the outlet of the second syringe to avoid contact between ammonia gases from the TEOS droplets and prevent secondary nucleation.Add the content of both syringes at the same time at 1.7 milliliters per hour using peristaltic pumps while stirring the mixture. Ensure to obtain free-falling droplets to avoid colliding on the walls and therefore secondary nucleation. Stop the addition after seven hours to obtain core-shell particles of approximately 300 nanometers in radius before washing the particles as described in the text protocol.For the synthesis of NVOC-functionalized colloids, disperse 10 milligrams of core-shell silica particles in one milliliter of ethanol together with 12 milligrams of the NVOC-protected linker molecule and 31 milligrams of stearyl alcohol in a 50 milliliter round-bottom flask. Sonicate the mixture for 10 minutes to ensure that all molecules are disolved and the particles are well dispersed. Add a magnetic stir bar to the mixture and evaporate the ethanol with a steady stream of argon at room temperature.Before proceeding, ensure that there is no ethanol left, otherwise it could react with the silanol groups of the particles. Next, heat the flask up to 180 degrees Celsius for six hours under continuous stirring and under a steady stream of argon, before washing the silica colloids as described in the text protocol. To synthesize the BTA colloids, disperse 10 milligrams of the functionalized particles in three milliliters of chloroform.To cleave the NVOC group homogeneously, place the sample in the UV oven and stir it gently with a magnetic stirrer while deprotecting. Irradiate the dispersion for one hour to yield the amine functionalized particles. Then, dissolve nine milligrams of BTA, 8.7 microliters of DIPEA, and 5.2 milligrams of PyBOP in one milliliter of chloroform.Add the solution to the amine functionalized particle dispersion and stir overnight at room temperature and under an argon atmosphere. Following centrifugation of the dispersion, remove the supernatant and add three milliliters of fresh chloroform. Sonicate the new dispersion for three minutes before centrifuging again and removing the supernatant.Dry the particles at 70 degrees Celsius in vacuo for 48 hours. Disperse 20 milligrams of the small functionalized particles in one milliliter of chloroform and irradiate the dispersion in a UV oven for one hour to cleave the NVOC group. Stir the dispersion gently with a magnetic stir bar while deprotecting.Spin down the resulting amine functionalized particles and remove the supernatant. Then, dry the particles at 70 degrees Celsius for two hours. Next, dissolve 0.5 milligrams of SPDP in 200 microliters of DMF.Add the SPDP solution to the 20 milligrams of dried amine functionalized particles and vortex the system for 30 minutes. Wash the particles with one milliliter of DMF, six times. In the last washing step, try to remove as much supernatant as possible.Then, dissolve 0.53 milligrams of DTT in 50 microliters of DMF. Add the DTT solution to the particles and vortex the dispersion for 30 minutes, within this time, the pyridine 2 thione group is cleaved. Determine the absorbence of the free pyridine 2 thione liberated in the supernatant at a wave length of 233 nanometers with a microvolume UV-Vis spectrophotometer.To monitor the colloidal assembly by confocal microscopy, first prepare 400 microliters of a dispersion of 0.1 weight percent of BTA functionalized particles in cyclohexane and sonicate the sample for 20 minutes. Then irradiate the sample vial in the UV oven to cleave off the ortho-nitrobenzyl group of the BTA. Take 25 microliter aliquots at different times of irradiation, for example, from zero up to 30 minutes, to monitor the clustering process.Place the different aliquots on different glass slides with the help of a spacer, and close the chambers with a cover slip. After closing the chamber, turn the cover slip upside-down to let the particles sediment and absorb onto the glass, which facilitates the imaging. Take several images of each sample with the confocal microscope as soon as possible after sample preparation for each irradiation time.To produce supramolecular colloids, first silica colloids are hydrophobized by functionalization with stearyl alcohol, and the NVOC-protected linker. The NVOC-protective group is then cleaved, and colloids can be post-functionalized with the supramolecular moiety. Static light scattering measurements allow for calculation of the refractive index of the colloids.By fitting the date, values of 1.391 for the bare colloid and 1.436 for the stearyl alcohol coated colloids were determined. This clearly shows how the functionalization of the colloids has an impact on their refractive index. To assess the amount of active sites per particle, the amount of cleaved pyridine 2 thione groups are directly related to the number of amines per particle.Here, one amine per 46.4 square nanometers was found. confocal image analysis allows for quantification of the number of singlets as a function of the UV irradiation time. Before UV irradiation to photocleave the ortho-nitrobenzyl moiety, 80%of the colloids are present as singlets.Irradiation at a maximum wavelength of 354 nanometers initiates colloidal aggregation as the hydrogen bonds of the BTA are activated. While attempting this procedure, it's important to keep the system water-free, as colloids might cluster due to capillary forces. Therefore, water traces should be illiminated from all synthesis steps.This work demonstrates that bridging the gap between colloidal and supramolecular science paves the way for researchers in the field of material science to create complex materials sensitive to external stimuli. After watching this video, you should have a good understanding on how to direct colloidal self-assembly using hydrogen bonds by anchoring supramolecular motifs on colloidal particles.

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9.6K visualizações.  Eindhoven University of Technology. The overall goal of this protocol is to describe how to direct colloidal self-assembly by anchoring supramolecular moieties onto the colloids whose interactions are strong, directional, and reversible. This method can help to answer key questions in the material science field, such as how to accurately control the colloidal self-assembly to create complex and mesostructured materials with interesting properties. The main advantage of this technique is that colloidal association is driven by t...