This work was partially supported by NTU-AIT-MUV NAM/16001, RG110/16 (S), (RG 11/13) and (RG 35/15) awarded in Nanyang Technological University, Singapore and National Natural Science Foundation of China (NSFC) (No. 51628201).

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Please use all appropriate safety practices when performing the synthesis of UCNs at a high temperature (~290 &#176;C), including the use of engineering controls (fume hood) and personal protective equipment (e.g., safety goggles, gloves, lab coat, full length pants, and closed-toe shoes).
1. Synthesis of NaYF4:Yb/Tm/Nd(30/0.5/1%)@NaYF4:Nd(20%) core-shell nanocrystals
<ol>
	<li>Synthesis of NaYF4:Yb/Tm/Nd(30/0.5/1%) core nanostructure
	<ol>
		<li>Preparation of the RE(CH3CO2)3, NaOH, NH4F methanol stock solution 
		NOTE: The rare-earth (RE) lanthanide ions include Yttrium (Y), Ytterbium (Yb), Thulium (Tm), and Neodymium (Nd).
		<ol>
			<li>Dissolve 500 mg Yttrium(III) acetate hydrate in 5 mL methanol (100 mg/mL), 250 mg Ytterbium (III) acetate hydrate in 5 mL methanol (50 mg/mL), 10 mg Thulium (III) acetate hydrate in 1 mL methanol (10 mg/mL), and 10 mg Neodymium (III) acetate hydrate in 1 mL methanol (10 mg/mL) in glass vials with an ultrasonic cleaning bath for 2 min.</li>
			<li>Combine 400 mg NaOH in a 50 mL centrifuge tube with 20 mL methanol with ultrasonic cleaning bath to prepare NaOH stock solution (20 mg/mL).</li>
			<li>Combine 600 mg NH4F in a 50 mL centrifuge tube with 30 mL methanol with ultrasonic cleaning bath to prepare NH4F stock solution (20 mg/mL). 
			NOTE: Place the stock solution of lanthanide complexes, NaOH, and NH4F in glass vials, seal with parafilm and store them in a refrigerator at ~4 &#176;C until needed. The prepared methanol stock solutions are replaced once every 2 weeks.</li>
		</ol>
		</li>
		<li>Preparation of NaYF4:Yb/Tm/Nd core nanostructure
		<ol>
			<li>Pipette 3 mL of oleic acid and 7 mL of 1-octadecene into a 50-mL three-neck flask.</li>
			<li>Combine 1.089 mL of the Y(CH3CO2)3 stock solution, 0.608 mL of the Yb(CH3CO2)3 stock solution, 83.6 &#181;L of the Tm(CH3CO2)3 stock solution, and 128.5 &#181;L of the Nd(CH3CO2)3 stock solution into the flask.</li>
			<li>Fit a thermometer (0 - 360 &#176;C range) in the flask and let its tip touch the solution. Place the flask in a glass container with phenylmethyl silicone oil.</li>
			<li>Heat the solution to 100 &#176;C with a hot plate to evaporate off the methanol. Connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual methanol and keep the reaction mixture under vacuum for 2 - 3 min while stirring.</li>
			<li>Increase the temperature to 150 &#176;C and keep this temperature for 60 min. Maintain a stirring speed of 700 rpm during the synthesis. 
			NOTE: Set a moderate stirring speed to avoid splashing the solution.</li>
			<li>Stop the hot plate and keep the flask at room temperature to allow the solution cool down slowly. 
			NOTE: The protocol can be paused here.</li>
			<li>Combine 2 mL of NaOH-methanol stock solution and 2.965 mL of NH4F-methanol stock solution into a 15 mL centrifuge tube. Tighten the tube with its cap and mix the solution by powerful vortexing for 5 s.</li>
			<li>Add the NaOH-NH4F mixture slowly into the flask by a glass pipette over 5 min.</li>
			<li>Increase the temperature of the mixture to 50 &#176;C and keep this temperature for 30 min. 
			NOTE: Set the temperature at no more than 50 &#176;C because the high temperature will promote crystal nucleation and growth.</li>
			<li>Increase the temperature (~100 &#176;C) to evaporate off the methanol and connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual methanol and keep the reaction mixture under vacuum for 2 - 3 min.</li>
			<li>Switch the position of the stopcock to fill the flask with nitrogen.</li>
			<li>Increase the temperature to 290 &#176;C at a heating rate of ~5 &#176;C/min. Keep the reaction mixture at 290 &#176;C for 1.5 h.</li>
			<li>Stop the hot plate and remove the flask to allow the reaction mixture to cool down slowly in room temperature while stirring. 
			CAUTION: Be careful of the hot plate with high temperature (&#62;400 &#176;C) to avoid severe burns upon skin contact.</li>
			<li>Transfer the mixture in the flask into a 50 mL centrifuge tube. Rinse the flask with 30 mL of ethanol and transfer the solution to the centrifuge tube.</li>
			<li>Centrifuge the product at 4,000 &#215; g for 8 min at room temperature and discard the supernatant.</li>
			<li>Add 10 mL of hexane to the centrifuge tube and re-disperse the product with sonication (60 kHz, 240 W) for 2 min.</li>
			<li>Add 30 mL of ethanol to the tube. Spin down the product at 4,000 &#215; g for 8 min and then discard the supernatant.</li>
			<li>Re-disperse the solid products in the bottom of centrifuge tube with 5 mL hexane. Store the solution in a refrigerator at ~4 &#176;C for a next step. 
			NOTE: The upconversion emission of core-shell UCNs is tested by irradiating the solutions with an 808 nm laser (2 W).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of NaYF4:Yb/Tm/Nd(30/0.5/1%)@NaYF4:Nd(20%) core-shell nanocrystals
	<ol>
		<li>Combine 3 mL of oleic acid and 7 mL of 1-octadecene into a 50-mL three-neck flask. Add 1.082 mL of the Y(CH3CO2)3 stock solution and 2.87 mL of the Nd(CH3CO2)3 stock solution into the flask.</li>
		<li>Add the obtained core nanostructure (80 mg in 5 mL hexane from step 1.1.2.18) into the flask while stirring.</li>
		<li>Fit a thermometer (0 - 360 &#176;C range) in the flask and let its tip touch the mixture. Place the flask in a glass container with phenylmethyl silicone oil.</li>
		<li>Heat the mixture at 100 &#176;C on the top of hot plate to evaporate off the methanol and hexane. Connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual solvent and keep the reaction mixture under vacuum for 2 - 3 min while stirring.</li>
		<li>Increase the temperature to 150 &#176;C and keep it for 60 min. Maintain the stirring speed at 700 rpm in the synthesis. 
		NOTE: Set a moderate stirring speed to avoid splashing the solution.</li>
		<li>Stop the hot plate and remove the flask to allow the solution to cool down slowly at room temperature. 
		NOTE: The protocol can be paused here.</li>
		<li>Pipette 2 mL of NaOH-methanol stock solution and 2.965 mL of NH4F-methanol stock solution into a 15 mL centrifuge tube. Tighten the tube with its cap and mix the solution by powerful vortexing for 5 s.</li>
		<li>Add the mixture into the flask by a glass pipette slowly over 5 min.</li>
		<li>Increase the temperature of the mixture to 50 &#176;C and keep it at 50 &#176;C for 30 min. 
		NOTE: Set the temperature no higher than 50 &#176;C because the high temperature will promote crystal nucleation and growth.</li>
		<li>Increase the temperature (~100 &#176;C) to evaporate off the methanol and connect the flask to a Schlenk line with a dual vacuum/gas manifold to remove the residual methanol, keeping the reaction mixture under vacuum for 2 - 3 min.</li>
		<li>Switch the position of the stopcock to fill the flask with nitrogen.</li>
		<li>Increase the temperature to 290 &#176;C at a heating rate of ~5 &#176;C/min. Keep the reaction mixture at 290 &#176;C for 1.5 h.</li>
		<li>Stop the hot plate and remove the flask to allow the reaction mixture to cool down slowly at room temperature while stirring. 
		CAUTION: Be careful of the hot plate with high temperature (&#62;400 &#176;C) to avoid severe burns upon skin contact.</li>
		<li>Transfer the mixture in the flask into a 50 mL centrifuge tube. Rinse the flask with 30 mL of ethanol and transfer the solution to the centrifuge tube.</li>
		<li>Centrifuge the product at 4,000 &#215; g for 8 min at room temperature and discard the supernatant.</li>
		<li>Add 10 mL of hexane to the centrifuge tube and re-disperse the product with sonication (60 kHz, 240 W) for 2 min.</li>
		<li>Add 30 mL of ethanol to the tube. Spin down the product at 4,000 &#215; g for 8 min and then discard the supernatant.</li>
		<li>Re-disperse the solid products in the bottom of centrifuge tube with 5 mL hexane. Store the solution in a refrigerator at ~4 &#176;C until needed. 
		NOTE: The upconversion emission of core-shell UCNs is tested by irradiating the solutions with an 808 nm laser (2 W).</li>
	</ol>
	</li>
</ol>
2. Synthesis of Biocompatible UCNs Nanostructures
<ol>
	<li>Preparation of ligand-free UCNs nanoparticle
	<ol>
		<li>Combine 30 mL ethanol with the as-prepared oleate-capped UCNs solution (step 1.2.18) in a 50 mL centrifuge tube. Centrifuge the mixture at 4,000 &#215; g for 8 min at room temperature and discard the supernatant.</li>
		<li>Combine 10 mL acid aqueous solution (pH = 4) adjusted by HCl (0.1 M) with the precipitate in 50 mL centrifuge tube and dissolve the precipitate by sonication (60 kHz, 240 W) for 30 min.</li>
		<li>Transfer the solution in a glass vial with vigorous stirring for 2 h.</li>
		<li>Extract the aqueous solution with 30 mL diethyl ether to remove the oleic acid, repeat three times.</li>
		<li>Add 10 mL water in the combined ether layers with gentle shaking.</li>
		<li>Collect the aqueous phase together (20 mL) and add 20 mL acetone with vortexing for 5 s.</li>
		<li>Spin down the product at 35,000 &#215; g for 10 min and then discard the supernatant. Dissolve the precipitate in 2 mL water.</li>
	</ol>
	</li>
	<li>Preparation of polymer modified UCNs (PAA-UCNs)
	<ol>
		<li>Combine 200 mg of polyacrylic acid (PAA, Mw = 1,800) with 20 mL water by sonication (60 kHz, 240 W) for 20 min. Add the aforementioned ligand-free UCNs in the PAA solution with vigorous stirring.</li>
		<li>Adjust the pH value to 7.4 by NaOH solution (1 M) with sonication (60 kHz, 240 W) for 30 min. Keep the mixture for 24 h at room temperature while stirring.</li>
		<li>Collect the precipitate by centrifugation at 35,000 &#215; g for 10 min. Re-suspend the product in 10 mL water by sonication (60 kHz, 240 W) for 5 min and centrifuge again at 35,000 &#215; g for 10 min. Repeat this step three times.</li>
		<li>Re-disperse the product in 8 mL water by sonication (60 kHz, 240 W) for 5 min. Store the solution in a refrigerator at ~4 &#176;C until needed.</li>
	</ol>
	</li>
	<li>Preparation of functional DBCO-UCNs nanoparticle
	<ol>
		<li>Spin down the as-prepared PAA@UCNs (1 mg) by centrifugation at 35,000 &#215; g for 10 min. Re-suspend the precipitate in 1 mL dry DMF by sonication (60 kHz, 240 W) for 1 min and centrifuge again at 35,000 &#215; g for 10 min. Repeat this step twice.</li>
		<li>Dissolve the precipitate in 200 &#181;L dry DMF in a glass vial. Add HOBT (12.2 mg), EDC (14 mg), DBCO-NH2 (5 mg), and DIPEA (16 &#181;L) in the vial with magnetic stirring for 24 h.</li>
		<li>Collect the product by centrifugation at 35,000 &#215; g for 10 min. Remove the supernatant and re-suspend the precipitate in 1 mL DMSO and centrifuge again at 35,000 &#215; g for 10 min. Repeat this step three times.</li>
		<li>Disperse the product in 0.2 mL DMSO and store in a refrigerator at ~ 4 &#176;C before use.</li>
	</ol>
	</li>
</ol>
3. Bioapplications of DBCO-UCNs in the Regulation of Membrane Channels in Living Cells
<ol>
	<li>Culture the HEK293 cells in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 10% FBS, 100 units/mL penicillin, 100 &#181;g/mL streptomycin and maintain in a humidified incubator with 5% CO2 at 37 &#176;C. Seed 1&#215;105 cells in 1 mL DMEM/well in a 12-well plate and keep it in the incubator for 24 h.</li>
	<li>Combine the plasmid (pCAGGS-ChR2-Venus, 1 &#181;g) with P3000 transfection reagent (2 &#181;L) in Eagle&#39;s Minimum Essential Media (MEM) (100 &#181;L) in microcentrifuge tube A, and add transfection reagent (1.5 &#181;L) in MEM (100 &#181;L) in tube B.</li>
	<li>Incubate the mixture of solutions in tubes A and B for 10 min at room temperature.</li>
	<li>Combine the solution in tube A and B with 400 &#181;L MEM. Wash the cells with 1 mL serum-free DMEM twice.</li>
	<li>Add the 600 &#181;L transfection mixture into each well of a 12-well plate and incubate the cells at 37 &#176;C incubator for 4 h. Remove the medium and wash twice with 1 mL DMEM.</li>
	<li>Add 1 mL DMEM containing 1 &#181;L Ac4ManNAz (50 mM in DMSO) in the well and keep it in the incubator for 2 days.</li>
	<li>Remove the medium and wash once with 1 mL PBS. Add 1 mL Trypsin-EDTA (0.25%) solution in each well of the 12-well plate and incubate at 37 &#176;C until cells have detached (~2 min). Re-culture the cells in a confocal dish at 1&#215;105 cells/well in 1 mL DMEM for overnight.</li>
	<li>Remove the medium and add 1 mL fresh DMEM with 2 &#181;L DBCO-UCNs (50 mg/mL) in the dish for 2 h at 37 &#176;C. For confocal imaging, wash cells twice with DMEM and add 1 &#181;L Rhod-N3 (10 mM) for 30 min.</li>
	<li>For intracellular calcium analysis, incubate the cells with the mixture of 1 &#181;L Rhod-3 AM (10 mM), 10 &#181;L Probenecid (250 mM), and 10 &#181;L loading buffer (Pluronic surfactant polyols,0.1% w/v)) in 1 mL DMEM medium for 30 min in the dark.</li>
	<li>Wash the cells with serum-free DMEM and irradiate with 808 nm NIR light at the dosage of 0.8 W/cm2 for 20 min (5 min break after 5 min illumination).</li>
	<li>Record the imaging results on the confocal microscope (laser source: 561 nm laser; detector range: 610/75 nm; lens: 100x 1.4 NA oil, exposure time: 100 ms). Add 1 mL Trypsin-EDTA (0.25%) solution in each well and incubate at 37 &#176;C until cells have detached (~2 min). Re-suspend the cells in 1 mL PBS for flow cytometry (FCM) analysis (Ex: 561 nm, Em: 582/15 nm).</li>
</ol>

<ol>
	<li>Wang, F., 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=Tuning+upconversion+through+energy+migration+in+core-shell+nanoparticles.">Tuning upconversion through energy migration in core-shell nanoparticles.</a> Nat Mater. 10 (12), 968-973 (2011).</li><li>Liu, Y., 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=Amplified+stimulated+emission+in+upconversion+nanoparticles+for+super-resolution+nanoscopy.">Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy.</a> Nature. 543 (7644), 229-233 (2017).</li><li>Fan, W., Bu, W., Shi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+The+Latest+Three-Stage+Development+of+Nanomedicines+based+on+Upconversion+Nanoparticles.">On The Latest Three-Stage Development of Nanomedicines based on Upconversion Nanoparticles.</a> Adv Mater. 28 (24), 3987-4011 (2016).</li><li>Zhu, X., 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=Temperature-feedback+upconversion+nanocomposite+for+accurate+photothermal+therapy+at+facile+temperature.">Temperature-feedback upconversion nanocomposite for accurate photothermal therapy at facile temperature.</a> Nat Commun. 7, 10437-10446 (2016).</li><li>Li, W., Wang, J., Ren, J., Qu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+upconversion+controls+photocaged+cell+adhesion.">Near-infrared upconversion controls photocaged cell adhesion.</a> J Am Chem Soc. 136 (6), 2248-2251 (2014).</li><li>Min, Y., Li, J., Liu, F., Yeow, E. K., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+light-mediated+photoactivation+of+a+platinum+antitumor+prodrug+and+simultaneous+cellular+apoptosis+imaging+by+upconversion-luminescent+nanoparticles.">Near-infrared light-mediated photoactivation of a platinum antitumor prodrug and simultaneous cellular apoptosis imaging by upconversion-luminescent nanoparticles.</a> Angew Chem Int Ed. 53 (4), 1012-1016 (2014).</li><li>Yang, D., Ma, P., Hou, Z., Cheng, Z., Li, C., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+lanthanide+ion+(Ln(3+))-based+upconversion+nanomaterials+for+drug+delivery.">Current advances in lanthanide ion (Ln(3+))-based upconversion nanomaterials for drug delivery.</a> Chem Soc Rev. 44 (6), 1416-1448 (2015).</li><li>Wang, C., Cheng, L., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upconversion+nanoparticles+for+photodynamic+therapy+and+other+cancer+therapeutics.">Upconversion nanoparticles for photodynamic therapy and other cancer therapeutics.</a> Theranostics. 3 (5), 317-330 (2013).</li><li>Li, L. 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=Biomimetic+surface+engineering+of+lanthanide-doped+upconversion+nanoparticles+as+versatile+bioprobes.">Biomimetic surface engineering of lanthanide-doped upconversion nanoparticles as versatile bioprobes.</a> Angew Chem Int Ed. 51 (25), 6121-6125 (2012).</li><li>Wang, J., Ming, T., Jin, Z., Wang, J., Sun, L. D., Yan, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+energy+upconversion+through+thermal+radiation+with+the+power+efficiency+reaching+16%.">Photon energy upconversion through thermal radiation with the power efficiency reaching 16%.</a> Nat Commun. 5, 5669-5678 (2014).</li><li>Zou, W., Visser, C., Maduro, J. A., Pshenichnikov, M. S., Hummelen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadband+dye-sensitized+upconversion+of+near-infrared+light.">Broadband dye-sensitized upconversion of near-infrared light.</a> Nat Photonics. 6 (8), 560-564 (2012).</li><li>Liu, Y., Tu, D., Zhu, H., Li, R., Luo, W., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strategy+to+achieve+efficient+dual-mode+luminescence+of+Eu(3+)+in+lanthanides+doped+multifunctional+NaGdF(4)+nanocrystals.">A strategy to achieve efficient dual-mode luminescence of Eu(3+) in lanthanides doped multifunctional NaGdF(4) nanocrystals.</a> Adv Mater. 22 (30), 3266-3271 (2010).</li><li>Min, Y., Li, J., Liu, F., Padmanabhan, P., Yeow, E. K., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advance+of+Biological+Molecular+Imaging+Based+on+Lanthanide-Doped+Upconversion-Luminescent+Nanomaterials.">Recent Advance of Biological Molecular Imaging Based on Lanthanide-Doped Upconversion-Luminescent Nanomaterials.</a> Nanomaterials. 4 (1), 129-154 (2014).</li><li>Li, X., Zhang, F., Zhao, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+on+upconversion+nanoparticles:+optical+properties+and+applications+engineering+via+designed+nanostructure.">Lab on upconversion nanoparticles: optical properties and applications engineering via designed nanostructure.</a> Chem Soc Rev. 44 (6), 1346-1378 (2015).</li><li>Gu, Z., Yan, L., Tian, G., Li, S., Chai, Z., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+design+and+fabrication+of+upconversion+nanoparticles+and+their+safe+theranostic+applications.">Recent advances in design and fabrication of upconversion nanoparticles and their safe theranostic applications.</a> Adv Mater. 25 (28), 3758-3779 (2013).</li><li>Dong, H., Sun, L. D., Yan, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+transfer+in+lanthanide+upconversion+studies+for+extended+optical+applications.">Energy transfer in lanthanide upconversion studies for extended optical applications.</a> Chem Soc Rev. 44 (6), 1608-1634 (2015).</li><li>Ai, X., 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=In+vivo+covalent+cross-linking+of+photon-converted+rare-earth+nanostructures+for+tumour+localization+and+theranostics.">In vivo covalent cross-linking of photon-converted rare-earth nanostructures for tumour localization and theranostics.</a> Nat Commun. 7, 10432-10440 (2016).</li><li>Lu, S., 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=Multifunctional+Nano-Bioprobes+Based+on+Rattle-Structured+Upconverting+Luminescent+Nanoparticles.">Multifunctional Nano-Bioprobes Based on Rattle-Structured Upconverting Luminescent Nanoparticles.</a> Angew Chem Int Ed. 54 (27), 7915-7919 (2015).</li><li>Bogdan, N., Vetrone, F., Ozin, G. A., Capobianco, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+ligand-free+colloidally+stable+water+dispersible+brightly+luminescent+lanthanide-doped+upconverting+nanoparticles.">Synthesis of ligand-free colloidally stable water dispersible brightly luminescent lanthanide-doped upconverting nanoparticles.</a> Nano Lett. 11 (2), 835-840 (2011).</li><li>Zheng, W., Huang, P., Tu, D., Ma, E., Zhu, H., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lanthanide-doped+upconversion+nano-bioprobes:+electronic+structures,+optical+properties,+and+biodetection.">Lanthanide-doped upconversion nano-bioprobes: electronic structures, optical properties, and biodetection.</a> Chem Soc Rev. 44 (6), 1379-1415 (2015).</li><li>Chen, X., Peng, D., Ju, Q., Wang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+upconversion+in+core-shell+nanoparticles.">Photon upconversion in core-shell nanoparticles.</a> Chem Soc Rev. 44 (6), 1318-1330 (2015).</li><li>Wang, F., Deng, R., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+core-shell+NaGdF4+nanoparticles+doped+with+luminescent+lanthanide+ions+to+be+used+as+upconversion-based+probes.">Preparation of core-shell NaGdF4 nanoparticles doped with luminescent lanthanide ions to be used as upconversion-based probes.</a> Nat Protoc. 9 (7), 1634-1644 (2014).</li><li>Chen, G., Agren, H., Ohulchanskyy, T. Y., Prasad, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+upconverting+core-shell+nanostructures:+nanophotonic+control+for+emerging+applications.">Light upconverting core-shell nanostructures: nanophotonic control for emerging applications.</a> Chem Soc Rev. 44 (6), 1680-1713 (2015).</li><li>Yang, Y., 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=In+vitro+and+in+vivo+uncaging+and+bioluminescence+imaging+by+using+photocaged+upconversion+nanoparticles.">In vitro and in vivo uncaging and bioluminescence imaging by using photocaged upconversion nanoparticles.</a> Angew Chem Int Ed. 51 (13), 3125-3129 (2012).</li><li>Sedlmeier, A., Gorris, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+and+characterization+of+photon-upconverting+nanoparticles+for+bioanalytical+applications.">Surface modification and characterization of photon-upconverting nanoparticles for bioanalytical applications.</a> Chem Soc Rev. 44 (6), 1526-1560 (2015).</li><li>Hu, M., 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=Near+infrared+light-mediated+photoactivation+of+cytotoxic+Re(I)+complexes+by+using+lanthanide-doped+upconversion+nanoparticles.">Near infrared light-mediated photoactivation of cytotoxic Re(I) complexes by using lanthanide-doped upconversion nanoparticles.</a> Dalton Trans. 45 (36), 14101-14108 (2016).</li><li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci USA. 100 (24), 13940-13945 (2003).</li><li>Ai, X., 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=Remote+Regulation+of+Membrane+Channel+Activity+by+Site-Specific+Localization+of+Lanthanide-Doped+Upconversion+Nanocrystals.">Remote Regulation of Membrane Channel Activity by Site-Specific Localization of Lanthanide-Doped Upconversion Nanocrystals.</a> Angew Chem Int Ed. 56 (11), 3031-3035 (2017).</li><li>Xie, 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=In+vivo+metabolic+labeling+of+sialoglycans+in+the+mouse+brain+by+using+a+liposome-assisted+bioorthogonal+reporter+strategy.">In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy.</a> Proc Natl Acad Sci USA. 113 (19), 5173-5178 (2016).</li><li>Bansal, A., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocontrolled+nanoparticle+delivery+systems+for+biomedical+applications.">Photocontrolled nanoparticle delivery systems for biomedical applications.</a> Acc Chem Res. 47 (10), 3052-3060 (2014).</li><li>Yang, Y., Aw, J., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructures+for+NIR+light-controlled+therapies.">Nanostructures for NIR light-controlled therapies.</a> Nanoscale. 9 (11), 3698-3718 (2017).</li><li>Ai, X., Mu, J., Xing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+of+Light-Mediated+Theranostics.">Recent Advances of Light-Mediated Theranostics.</a> Theranostics. 6 (13), 2439-2457 (2016).</li></ol>

We have nothing to disclose.

This article has presented a method for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their surface modification with functional moieties for cellular applications. This novel nanomaterial possesses outstanding optical properties, which can emit UV and visible light upon NIR light excitation through a multi-photon upconversion process. In this protocol, the core-shell UCNs nanostructures (NaYF4:Yb/Tm/Nd (30/0.5/1%)@NaYF4:Nd (20%)) are prepared by a high-temperatur...

In recent years, lanthanide-doped upconversion nanocrystals (UCNs) have been widely used as an alternative to conventional organic dyes and quantum dots in biomedical applications, which are mainly based on their outstanding chemical and optical properties, including great biocompatibility, high resistance to photobleaching, and narrow-bandwidth emission1,2,3. More importantly, they can serve as a promising nanotransducer with excellent tissue penetration depth in vivo to convert near-infrared (NIR) excitation into a broad range of emissions from the UV, visible, and the NIR regions through a multi-photon upconversion process4,5. These unique properties make lanthanide-doped UCNs serve as a particularly promising vector for biological sensing, biomedical imaging, and diseases theranostics6,7,8.
The general components of UCNs are mainly based on the doped lanthanide ions in the insulating host matrix containing a sensitizer (e.g., Yb3+, Nd3+) and an activator (e.g., Tm3+, Er3+, Ho3+) within the crystal homogeneously9. The different optical emission from the nanocrystals is attributed to the localized electronic transition within the 4f orbitals of the lanthanide dopants due to their ladder-like arranged energy level10. Therefore, it is critical to precisely control the size and morphology of synthesized UCNs with multicomponent lanthanide dopants. By right, some promising methods have been well established for the preparation of lanthanide-doped UCNs, including thermal decomposition, high-temperature co-precipitation, hydrothermal synthesis, sol-gel processing, etc.11,12,13 Among these approaches, the high-temperature co-precipitation method is one of the most popular and convenient strategies for UCNs synthesis, which can be strictly controlled to prepare desired high-quality nanocrystals with uniform shape and size distribution in a relatively short reaction time and low-cost14. However, most nanostructures synthesized by this method are mainly capped with hydrophobic ligands such as oleic acid and oleylamine, which generally hinder their further bioapplication due to the limited of hydrophobic ligand solubility in aqueous solution15. Therefore, it is necessary to perform suitable surface modification techniques to prepare biocompatible UCNs in biological applications in vitro and in vivo.
Herein, we present the detailed experimental procedure for the synthesis of core-shell UCNs nanostructures through the high-temperature co-precipitation method and a feasible modification technique to functionalize biocompatible polymer on UCNs surface for further cellular applications. This UCNs nanoplatform incorporates three lanthanide ions (Yb3+, Nd3+, and Tm3+) into the nanocrystals to acquire strong blue emission (~480 nm) upon NIR light excitation at 808 nm, which has greater penetration depth in living tissue. It is well known that Nd3+-doped UCNs display minimized water absorption and overheating effects at this spectral window (808 nm) as compared to conventional UCNs upon 980 nm irradiation16,17,18. Moreover, to utilize the UCNs in biological systems, the hydrophobic ligands (oleic acid) on the surface of UCNs are firstly removed by sonication in acid solution19. Then the ligand-free UCNs are further modified with a biocompatible polymer (polyacrylic acid, PAA) to acquire great solubility in aqueous solutions20. Furthermore, as a proof-of-concept in cellular applications, the hydrophilic UCNs are further functionalized with molecular ligands (dibenzyl cyclooctyne, DBCO) for specific localization on the N3-tagged cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission at 480 nm can effectively activate a light-gated channel protein, channelrhodopsins-2 (ChR2), on cell the surface and thus facilitate cation (e.g., Ca2+ ion) influx across the membrane of living cells.
This video protocol provides a feasible methodology for lanthanide-doped UCNs synthesis, biocompatible surface modification, and UCNs bioapplication in living cells. Any differences in the synthesis techniques and chemical reagents used in nanocrystal growth will influence the size distribution, morphology, and upconversion luminescence (UCL) spectra of final UCNs nanostructures used in cell experiments. This detailed video protocol is prepared to help new researchers in this field to improve the reproducibility of UCNs with the high-temperature co-precipitation method and avoid the most common mistakes in UCNs biocompatible surface modification for further cellular applications.

<table>
	<tbody>
		<tr>
			<td>1-Octadecene</td>
			<td>Sigma Aldrich</td>
			<td>O806</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>oleic acid</td>
			<td>Sigma Aldrich</td>
			<td>364525</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A412</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A405</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>A18</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Sigma Aldrich</td>
			<td>H292</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>Thulium (III) acetate hydrate (Tm(CH3CO2)3)</td>
			<td>Sigma Aldrich</td>
			<td>367702</td>
			<td>99.9% trace metals basis</td>
		</tr>
		<tr>
			<td>Neodymium (III) acetate hydrate (Nd(CH3CO2)3)</td>
			<td>Sigma Aldrich</td>
			<td>325805</td>
			<td>99.9% trace metals basis</td>
		</tr>
		<tr>
			<td>Ytterbium (III) acetate hydrate (Yb(CH3CO2)3)</td>
			<td>Sigma Aldrich</td>
			<td>326011</td>
			<td>99.9% trace metals basis</td>
		</tr>
		<tr>
			<td>Yttrium(III) acetate hydrate (Y(CH3CO2)3)</td>
			<td>Sigma Aldrich</td>
			<td>326046</td>
			<td>99.9% trace metals basis</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>reagent grade</td>
		</tr>
		<tr>
			<td>Ammonium fluoride (NH4F)</td>
			<td>Sigma Aldrich</td>
			<td>338869</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>Hydrogen chloride (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144</td>
			<td>reagent grade</td>
		</tr>
		<tr>
			<td>polyacrylic acid (PAA)</td>
			<td>Sigma Aldrich</td>
			<td>323667</td>
			<td>average Mw 1800</td>
		</tr>
		<tr>
			<td>1-Hydroxybenzotriazole hydrate (HOBT)</td>
			<td>Sigma Aldrich</td>
			<td>54802</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)</td>
			<td>Sigma Aldrich</td>
			<td>E7750</td>
			<td>commercial grade</td>
		</tr>
		<tr>
			<td>Dibenzocyclooctyne-amine (DBCO-NH2)</td>
			<td>Sigma Aldrich</td>
			<td>761540</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine (DIPEA)</td>
			<td>Sigma Aldrich</td>
			<td>D125806</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>BP231</td>
			<td>Technical grade</td>
		</tr>
		<tr>
			<td>HEK293 cell line</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td>human embryonic kidney</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma Aldrich</td>
			<td>F1051</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td>10,000 U/mL</td>
		</tr>
		<tr>
			<td>plasmid (pCAGGS-ChR2-Venus)</td>
			<td>Addgene</td>
			<td>15753</td>
			<td>Plasmid sent as bacteria in agar stab</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher</td>
			<td>11965092</td>
			<td>High glucose</td>
		</tr>
		<tr>
			<td>opti-Modified Eagle Medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>51985034</td>
			<td>Reduced Serum Media</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Thermo Fisher</td>
			<td>L3000015</td>
			<td>Lipid-Based Transfection</td>
		</tr>
		<tr>
			<td>N-Azidoacetylmannosamine, Acetylated (Ac4ManNAz)</td>
			<td>Sigma Aldrich</td>
			<td>A7605</td>
			<td>ACS reagent</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td>Phenol red</td>
		</tr>
		<tr>
			<td>Rhod-3 AM Calcium Imaging Kit</td>
			<td>Thermo Fisher</td>
			<td>R10145</td>
			<td>Fluorescence dye</td>
		</tr>
		<tr>
			<td>5-carboxytetramethylrhodamine-azide (Rhod-N3)</td>
			<td>Sigma Aldrich</td>
			<td>760757</td>
			<td>Azide-fluor 545</td>
		</tr>
		<tr>
			<td>Confical dish</td>
			<td>ibidi GmbH</td>
			<td>81158</td>
			<td>Glass Bottom, 35 mm</td>
		</tr>
		<tr>
			<td>50 ml conical centrifuge tubes</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>Polypropylene</td>
		</tr>
		<tr>
			<td>15 ml conical centrifuge tubes</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>Polypropylene</td>
		</tr>
		<tr>
			<td>1.5 ml conical microcentrifuge tubes</td>
			<td>Greiner Bio-One</td>
			<td>616201</td>
			<td>Polypropylene</td>
		</tr>
		<tr>
			<td>Phenylmethyl silicone oil</td>
			<td>Clearco Products</td>
			<td>63148-52-7</td>
			<td>Less than 320 degrees Celsius</td>
		</tr>
		<tr>
			<td>Glass thermometer</td>
			<td>GH Zeal</td>
			<td>L0111/10</td>
			<td>From -10 to 360 degrees Celsius</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Sigma Aldrich</td>
			<td>Z707775</td>
			<td>Polystyrene</td>
		</tr>
	</tbody>
</table>

synthesis of core-shell lanthanide-doped upconversion nanocrystals for cellular applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

The schematic synthesis process of core-shell lanthanide-doped UCNs are shown in Figure 1 and Figure 2. The transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images of core and core-shell UCNs nanostructures were collected respectively (Figure 1). The ligand-free UCNs are prepared by removing the hydrophobic oleic acid on the surface of UCNs in acid s...

Watch this Scientific Journal Video about Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications at JoVE.com

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

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15.2K Views.  Nanyang Technological University. A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Video: Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

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 ...

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.