Name: synthesis of core-shell lanthanide-doped upconversion nanocrystals for cellular applications
Uploaded: 2017-11-10T13:00:00
Description: Watch this Scientific Journal Video about Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications at JoVE.com

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
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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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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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Research

JoVE Journal

Chemistry

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, 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, ...

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.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...