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>...

<ol>
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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. 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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. 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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 ...

The overall goal of this procedure is to synthesize core-shell lanthanide-doped upconversion nanocrystals based on a high-temperature co-precipitation approach and to perform biocompatible surface modifications that enable further cellular applications. In the past few years, our lab have developed a very unique lanthanide-doped upconversion of nanocrystals, which indicated very unique optical properties, so which can absorb long-wavelength infrared light and basically convert this light into multicolored emissions at short wavelength range, including UV visible to even the infrared light windows. Such unique nanocrystal structures indicated very promising the properties for biomedical applications.This method provides a feasible approach for synthesize of core-shell upconversion nanostructures and their biocompatible surface modification technique for light-gate membrane channel protein regulation upon near-infrared light illumination. To begin the procedure, prepare stock solutions of the rare-earth acetate complexes in methanol. Prepare 20 milligrams per milliliter solutions of sodium hydroxide and ammonium fluoride in methanol.Store the stock solutions at four degrees Celsius. Then, pipette three milliliters of oleic acid and seven milliliters of 1-octadecene into a 50-milliliter, three-neck, round-bottom flask. Add to this, 1.089 milliliters of the yttrium acetate solution, 0.608 milliliters of the ytterbium acetate solution, 83.6 microliters of the thulium acetate solution, and 128.5 microliters of the neodymium acetate solution.Equip the flask with a stir bar and a thermometer. Heat the flask to 100 degrees Celsius in an oil bath while stirring until the methanol has evaporated. Then, connect the flask to a Schlenk line, and pump down the flask for two to three minutes to remove residual methanol.Next, put the flask under a nitrogen atmosphere. Heat the reaction mixture at 150 degrees Celsius for one hour while stirring at 700 rpm. Then, allow the mixture to cool to room temperature.Next, place in a 15-milliliter centrifuge tube two milliliters of the sodium hydroxide stock solution and 2.965 milliliters of the ammonium fluoride stock solution. Tightly cap the tube, and vortex the sodium hydroxide and ammonium fluoride mixture for five seconds. Over the course of five minutes, use a glass pipette to slowly add the sodium hydroxide-ammonium fluoride mixture to the reaction flask while stirring.Then, heat the reaction mixture to no more than 50 degrees Celsius. Hold the mixture at that temperature for 30 minutes. Then, heat the mixture to 100 degrees Celsius to evaporate the methanol.Connect the flask to the Schlenk line, and remove residual methanol under vacuum for two to three minutes. Fill the flask with nitrogen gas, and heat the reaction mixture to 290 degrees Celsius at five degrees Celsius per minute. Hold the mixture at or slightly above 290 degrees Celsius for 1.5 hours.Then, allow the mixture to cool to room temperature while stirring. Transfer the product mixture to a 50-milliliter centrifuge tube. Rinse the residue into the tube with 30 milliliters of ethanol.Centrifuge the mixture at 4, 000 times g for eight minutes at room temperature. Discard the supernatant, and disperse the solids in 10 milliliters of hexanes by sonication for two minutes. Add 30 milliliters of ethanol to the dispersion, centrifuge the mixture again under the same conditions, and discard the supernatant.Disperse the solids in five milliliters of hexanes, and store the dispersion at four degrees Celsius. Check the upconverted emission of core-shell UCNs solution upon 808-nanometer laser irradiation in the dark. To begin preparing DBCO-modified UCNs, wash the prepared core-shell nanocrystals in 30 milliliters of ethanol by centrifugation.Discard the supernatant, and add 10 milliliters of a pH four aqueous solution of hydrochloric acid. Sonicate the mixture for 30 minutes to dissolve the solids. Transfer the mixture to a glass vial, and stir vigorously for two hours.Then, wash the mixture with four 30-milliliter portions of diethyl ether. Combine the ether fractions, and set aside the washed aqueous layer. Recover trace ligand-free UCNs from the ether layers with 10 milliliters of deionized water, and combine the aqueous layers.Add 20 milliliters of acetone to the combined aqueous layers, and vortex the mixture for five seconds. Centrifuge the mixture at 35, 000 times g for 10 minutes. Discard the supernatant, and dissolve the precipitate in two milliliters of deionized water to obtain a solution of ligand-free UCNs.Next, dissolve 200 milligrams of polyacrylic acid in 20 milliliters of deionized water by sonication for 20 minutes. Add to this the solution of ligand-free UCNs while stirring vigorously. Adjust the mixture pH to 7.4 with a one-molar solution of sodium hydroxide.Sonicate the mixture for 30 minutes, and stir the mixture for 24 hours at room temperature. Then, centrifuge the mixture at 35, 000 times g for 10 minutes. Wash the solids by centrifugation in 10 milliliters of deionized water four times.Disperse the washed solids in eight milliliters of deionized water to obtain a 10 milligram per milliliter solution of polymer-modified UCNs. Centrifuge one milligram of the polymer-modified UCNs at 35, 000 times g for 10 minutes. Store the remaining solution at four degrees Celsius.Wash the solids by centrifugation in one-milliliter portions of dry DMF three times. Then, dissolve the precipitate in 200 microliters of dry DMF. Add to this solution 12.2 milligrams of HOBT, 14 milligrams of EDC, five milligrams of DBCO-amine, and 16 microliters of DIPEA.Stir the mixture at room temperature for 24 hours. Then, centrifuge the mixture for 10 minutes at 35, 000 times g. Wash the solids four times by centrifugation in one-milliliter portions of DMSO.Disperse the DBCO-modified UCNs in 0.2 milliliters of DMSO, and store the dispersion at four degrees Celsius. First, seed one times 10 the fifth HEK293 cells in a 12-well plate, and incubate the cells at 37 degrees Celsius for 24 hours. Then, in a microcentrifuge tube, combine one microgram of the chosen plasmid with two microliters of P3000 transfection agent in 100 microliters of MEM.In another microcentrifuge tube, combine 1.5 microliters of the transfection reagent with 100 microliters of MEM. Incubate both mixtures at room temperature for 10 minutes. Then, combine the mixtures, and add 400 microliters of low-serum MEM.Next, wash the incubated cells twice with one-milliliter portions of serum-free DMEM. Distribute the 600-microliter transfection mixture into the wells of the 12-well plate. Incubate the cells at 37 degrees Celsius for four hours.Then, remove the medium, and wash the cells twice with one-milliliter portions of DMEM. Distribute among the wells one milliliter of a tetra-acetylated N-azidoacetyl-mannosamine solution in DMEM. Incubate the cells at 37 degrees Celsius for two days.Then, wash, trypsinize, and re-culture the cells in a confocal dish overnight. Next, replace the medium with one milliliter of fresh DMEM containing two microliters of a 50-milligram per milliliter solution of DBCO-UCNs. Incubate the cells at 37 degrees Celsius for two hours, and wash the cells twice with DMEM.Turn off the light of the fume hood and add the appropriate diene buffer solutions to the cells for calcium imaging, and incubate the cells for 30 minutes in the dark. Wash the cells with serum-free DMEM. Irradiate the cells with 808-nanometer, near-infrared light delivering 0.8 watts per square centimeter.Alternate five minutes of irradiation with five-minute breaks until the cells have been irradiated for 20 minutes. Image the cells with a confocal microscope. Transmission electron microscopy of the core and core-shell UCNs showed spherical structures with a shell thickness of about five nanometers.High-resolution TEM showed d-spacing consistent with highly crystallized nanostructures. Following surface modification, the UCNs were easily dispersed into buffer solution. Dynamic light scattering indicated that the hydrodynamic diameter of DBCO-UCNs in aqueous solution is about 96 nanometers.The zeta potentials of the PAA and DBCO-modified UCNs indicate negatively charged surfaces, which is consistent with solubility and stability in aqueous buffer solutions. Upconverted emission tests showed that PAA and DBCO-modified UCNs had the same emission patterns as the base core-shell UCNs when irradiated with 808-nanometer light. Azide-tagged HEK293 cells expressing light-gated channel proteins were used to test the bioapplications of DBCO-UCNs.Localization of the UCNs at the azide tags was attributed to the DBCO groups being stained with an azide-containing dye. When these cells were exposed to near-IR light, the DBCO-UCNs facilitated the flow of calcium ions across the cell membrane, as shown by confocal imaging and flow cytometry with a fluorescent calcium indicator. After watching this video, you should have a good understanding of how to prepare the core-shell upconversion nanocrystals with biocompatible surface modification and their further bio applications to activate the light-gated ion channel in living cells by following this procedure.

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JoVE Journal

Chemistry

15.1K Views.  Nanyang Technological University. The overall goal of this procedure is to synthesize core-shell lanthanide-doped upconversion nanocrystals based on a high-temperature co-precipitation approach and to perform biocompatible surface modifications that enable further cellular applications. In the past few years, our lab have developed a very unique lanthanide-doped upconversion of nanocrystals, which indicated very unique optical properties, so which can absorb long-wavelength infrared light and basically convert this light into...