The authors gratefully acknowledge the Department of Chemistry and the Graduate College at Missouri State University for their support of this project. We also acknowledge the Electron Microscopy Laboratory at the Frederick National Laboratory for Cancer Research for use of their transmission electron microscope and carbon-coated grids.

1. Synthesis of Indium Phosphide/Zinc Sulfide (InP/ZnS) Quantum Dots
<ol>
	<li>Synthesis of Indium Phosphide (InP) Quantum Dot Cores
	<ol>
		<li>Fit a 100 ml round bottom, 3-neck, flask with a 12-inch condenser. Add 30 ml oleylamine (OLA), 0.398 g indium (III) chloride (InCl3), 0.245 g zinc (II) chloride (ZnCl2) and stir while evacuating at RT using a vacuum for 1 hr. The solution should appear colorless with a white precipitate.</li>
		<li>Using a heating mantle with a thermocoup...

<ol>
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The authors have nothing to disclose.

This protocol details the synthesis of highly fluorescent InP/ZnS QDs that can be used in many biological systems. The QD products synthesized here exhibited a single fluorescence emission peak centered at 600 nm with a FWHM of 73 nm (Figure 1), which is comparable to other previously described syntheses12. Reaction time and reaction temperature are extremely crucial steps due to their profound effect on QD synthesis quality and repeatability. After solubilization in water, the QDs were determ...

A correction was made to: <a href="http://www.jove.com/video/53684/synthesis-cd-free-inpzns-quantum-dots-suitable-for-biomedical" target="_blank">Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications</a>. There was an error with an author's given name. The author's name was corrected to:
Katye M. Fichter
from:
Kathryn M. Fichter

A correction was made to: <a href="http://www.jove.com/video/53684/synthesis-cd-free-inpzns-quantum-dots-suitable-for-biomedical" target="_blank">Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications</a>.

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

Quantum dots (QDs) are semiconducting nanocrystals that exhibit fluorescent properties when irradiated with light1. Due to their small size (2-5 nm), which is similar to many larger biomolecules, and ease of biofunctionalization, QDs are an extremely attractive tool for biomedical applications. They have found use in biological labeling, single-molecule live-cell imaging, drug delivery, in vivo imaging, pathogen detection, and cell tracking, among many other uses2-8.
Cd-based QDs have been most commonly used in biomedical applications because of their intense fluorescence and narrow emission peak widths9. However, concerns have been raised due to potential toxicity of Cd2+ ions10 that may be released through degradation of the nanoparticle. Recently, InP-based QDs have been explored as an alternative to Cd-based QDs because they maintain many fluorescence characteristics of Cd-based QDs and may be more biocompatible11. Cd-based QDs have been found to be significantly more toxic than InP-based QDs in in vitro assays at concentrations as low as 10 pM, after only 48 hr11.
The fluorescence emission color of QDs is size-tunable1. That is, as the size of the QD increases, the fluorescence emission is red-shifted. The size and size dispersity of the QD products can be modified by changing the temperature, reaction duration, or precursor concentration conditions during the reaction12. While the emission peak of InP QDs is typically broader and less intense than Cd-based QDs, InP QDs can be made in a large variety of colors designed to avoid spectral overlap, and are sufficiently intense for most biomedical applications12. The synthesis detailed in this protocol yields QDs with a red emission peak centered at 600 nm.
Several steps are taken after synthesis of the QD cores to maintain the optical integrity of the QDs and to make them compatible for biological applications. The surface of the QD core must be protected from oxidation or surface defects that may cause quenching; therefore, a ZnS shell is coated over the core to produce InP/ZnS (core/shell) QDs13. This coating has been shown to protect the photoluminescence of the QD product. The presence of zinc ions during InP QD synthesis has been shown to limit surface defects, as well as decrease size distribution12. Even with the presence of Zn2+ in the reaction medium, synthesis of InZnP are highly unlikely12. After coating, resulting InP/ZnS QDs are coated in hydrophobic ligands such as trioctylphosphine oxide (TOPO) or oleylamine12,14. An amphiphilic polymer can interact with hydrophobic ligands on the QD surface as well as bulk water molecules to impart water solubility15. Amphiphilic polymers with carboxylate chemical groups can be used as &#34;chemical handles&#34; to further functionalize the QDs.
This protocol details the synthesis and functionalization of water-soluble InP/ZnS QDs with very intense fluorescence emission and relatively small size-dispersity. These QDs are potentially less toxic than commonly used CdSe/ZnS QDs. Herein, the synthesis of InP/ZnS QDs provides a practical alternative to Cd-based QDs for biomedical applications.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Oleylamine</td>
			<td style="width:92px;">Acros</td>
			<td style="width:119px;">129540010</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Zinc (II) chloride</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:119px;">030-003-00-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Indium (III) chloride</td>
			<td style="width:92px;">Chem-Impex&#160;</td>
			<td style="width:119px;">24560</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:232px;">Tris(dimethylamino)phosphine</td>
			<td style="width:92px;">Encompass</td>
			<td style="width:119px;">50-901-10500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">1-dodecanethiol</td>
			<td style="width:92px;">Acros</td>
			<td style="width:119px;">117625000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Hexanes</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">H292-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Acetone</td>
			<td style="width:92px;">TransChemical</td>
			<td style="width:119px;">UN 1090</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Zinc Stearate</td>
			<td style="width:92px;">Aldrich Chem</td>
			<td style="width:119px;">307564-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tetrahydrofuran</td>
			<td style="width:92px;">Acros</td>
			<td style="width:119px;">34845-0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Molecular Water</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">BP2470-1</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Poly(maleic anhyrdride-alt-1-tetradecene), 3-(dimethylamino)-1-propylamine derivative</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:119px;">90771-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Boric acid</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">BP168-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:92px;">Fisher Sci</td>
			<td style="width:119px;">BP175-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Rhodamine B</td>
			<td style="width:92px;">Aldrich Chem</td>
			<td style="width:119px;">R95-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Nitrogen gas</td>
			<td style="width:92px;">Airgas</td>
			<td style="width:119px;">UN1066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Trypan blue</td>
			<td style="width:92px;">Thermo Sci</td>
			<td style="width:119px;">SV30084.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">3 mL plastic Luer-lock syringe</td>
			<td style="width:92px;">BD</td>
			<td style="width:119px;">309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Luer-lock Needle</td>
			<td style="width:92px;">Air-Tite</td>
			<td style="width:119px;">8300014471</td>
			<td>4 inch, 22 gauge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">50 mL polypropyene centrifuge tube</td>
			<td style="width:92px;">Falcon</td>
			<td style="width:119px;">352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">250 mL centrifuge bottle</td>
			<td style="width:92px;">Thermo Sci</td>
			<td>05-562-23</td>
			<td>Nalgene PPCO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">5 mL centrifuge tubes</td>
			<td style="width:92px;">Argos-Tech</td>
			<td style="width:119px;">T2076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">1.5 mL microcentrifuge tubes</td>
			<td style="width:92px;">Bio Plas</td>
			<td style="width:119px;">4150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">0.1 &#956;m Syringe filter</td>
			<td style="width:92px;">Whatman</td>
			<td style="width:119px;">6786-1301</td>
			<td>Puradisc 13 mm nylon filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Slide-A-Lyzer MINI Dialysis Unit</td>
			<td style="width:92px;">Thermo Sci</td>
			<td style="width:119px;">69590</td>
			<td>20,000 MWCO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Rotary Evaporator</td>
			<td style="width:92px;">Heidolph</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Centrifuge 5072</td>
			<td style="width:92px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Swinging Bucket with 50 mL tube adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Lambda 650 UV/VIS Spectrometer</td>
			<td style="width:92px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td style="width:283px;">UV-Vis Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">LS 55 Fluorescence Spectrometer</td>
			<td style="width:92px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Fluorometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Axio Observer.A1</td>
			<td style="width:92px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>epifluorescence microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">AxioCam MRm</td>
			<td style="width:92px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>CCD Camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tecnai TF20 Microscope</td>
			<td style="width:92px;">FEI</td>
			<td style="width:119px;"></td>
			<td>Transmisison Electron Miscroscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">TEM Eagle CCD</td>
			<td style="width:92px;">FEI</td>
			<td style="width:119px;"></td>
			<td>TEM CCD Camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">NanoBrook Omni DLS</td>
			<td style="width:92px;">Brookhaven</td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Dynamic Light Scattering Instrument</td>
		</tr>
	</tbody>
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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.

The uncoated InP cores do not demonstrate substantial visible fluorescence by eye. However, InP/ZnS (core/shell) quantum dots appear to fluoresce brightly by eye under UV irradiation. The fluorescence of InP/ZnS QDs was characterized using fluorescence spectroscopy. The fluorescence spectrum of QDs in hexanes (Figure 1) excited at 533 nm demonstrates one major peak centered at 600 nm with a full width at half maximum (FWHM) of 73 nm. While absorbance (0.2) offset in <stro...

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Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for 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, ...

The overall goal of this procedure is to Synthesize Highly Fluorescent Indium Phosphide Zinc Sulfide Quantum Dots that are suitable for Biomedical Applications. This method can help clarify key questions about the Synthesis of Quantum Dot Nanoparticles by demonstrating specific steps that are crucial to achieve high quality Quantum Dots. The main advantage of this technique is the production of high quality, water soluble Quantum Dots.Viable for biological systems that can be synthesized in less than one day. To begin synthesis, fit a 100 milliliter round-bottom, three-neck flask with a 12-inch condenser. 0.398 grams of Indium Chloride, 30 milliliters Oleylamine, or OLA, and 0.245 grams of Zinc Chloride.Stir while evacuating at room temperature using a vacuum for one hour. The solution should appear colorless with a white precipitate. Use a heating mantle with a Thermocouple and PID Temperature Controller to increase the temperature of the solution to 120 degrees celsius.Use of a heating mantle and PID increases the uniformity and reproducibility of the reaction product. Evacuate the solution under vacuum for 20 minutes to remove low boiling point impurities that may affect core growth. Under inert gas, reflux the solution and increase the temperature to 223 degrees celsius for 15 minutes.The Indium Chloride and Zinc Chloride completely dissolve resulting in a pale yellow solution. Allow the temperature to stabilize for 10 minutes. Next, purge a disposable three milliliter plastic syringe and 4-inch 22 gauge needle with Nitrogen gas.The injection of TDMAP is a critical step in this procedure as the injection can greatly affect the dispersity of the resulting Indium Phosphide Quantum Dots. Using the syringe, quickly deliver 0.5 milliliters of TDMAP to the Indium Chloride solution. The solution temperature decreases slightly and returns to 220 degrees celsius while the solution changes from transparent pale yellow to opaque black.After nine and a half minutes, remove the reaction flask from the heating mantle until the temperature decreases below 200 degrees celsius. To protect the integrity of the Indium Phosphide cores proceed directly to the Zinc Sulfide coating. Place the reaction flask containing the Indium Phosphide Quantum Dots on a heating mantle and stabilize the temperature at 200 degrees celsius.Slowly add 3.58 grams of DDT to the solution over the course of 15 seconds. After allowing the solution to react for one hour, remove the reaction flask from the heating mantle and allow the solution to cool for approximately 60 degrees celsius. Once the Indium Phosphide Zinc Sulfide solution reaches approximately 60 degrees celsius, add 10 milliliters of Hexanes.Then, transfer the entire solution of roughly 45 milliliters to a 50 milliliter polypropylene centrifuge tube. Centrifuge the sample to remove unreacted solid precursors. Carefully remove the Supernatant and add 200 milliliters of Acetone.Equally divide the resulting solution into four 15 milliliters centrifuge tubes. Then, centrifuge the solution to precipitate the Indium Phosphide Zinc Sulfide Quantum Dots. Decant the supernatant and dry the Quantum Dot pellet thoroughly with Nitrogen gas to remove Acetone.Then, we suspend the Quantum Dots in 20 milliliters of OLA using Sonication. Then, transfer the resuspended Quantum Dots to a 50 milliliter round-bottom three-neck flask containing 0.474 grams of Zinc Stearate and stir. Evacuate the solution under vacuum for 20 minutes at room temperature.Under Nitrogen gas, increase the temperature to 180 degrees celsius and allow the reaction to proceed for three hours. Upon completion of the reaction, remove the flask from heating mantle and allow the solution to cool to approximately 60 degrees celsius. Once the Indium Phosphide Zinc Sulfide solution reaches approximately 60 degrees celsius, add 20 milliliters of Hexanes.Then, transfer the sample to a 50 milliliter polypropylene centrifuge tube before centrifuging to remove unreacted Zinc Stearate. Following centrifugation, add 200 milliliters of Acetone to the Supernatant and centrifuge using the same procedure as before. After thoroughly drying the pellet with Nitrogen gas, to remove the Acetone, dissolve the Indium Phosphide Zinc Sulfide Quantum Dot pellet in 30 milliliters of Hexanes.Vortex and sonicate the solution briefly to ensure complete dispersion. Removing the excess ligands is a critical step in this procedure. Failure to do so will negatively impact the efficiency of transferring the Quantum Dots into aqueous solutions using PMAL-d Repeat these purification steps two more times to ensure thorough removal of excess organic ligands.Interactions between the Amphiphilic Polymer and the Quantum Dot can be compromised in the presence of excess ligands. Following purification, determine the size and concentration of the synthesized Indium Phosphide Zinc Sulfide Quantum Dots using UV-Visible Spectroscopy. Dilute a portion of the synthesized Indium Phosphide Zinc Sulfide Quantum Dots with Hexanes to obtain one milliliter of one micromolar Quantum Dots.In a centrifuge tube, transfer 0.25 milliliters of Indium Phosphide Zinc Sulfide Quantum Dots into each tube. Add one milliliter of Acetone or menthol to the centrifuge tube, and centrifuge. Carefully remove the supernatant and dissolve each precipitate in one milliliter of Tetrahydrofuran or THF.Next, transfer the Indium Phosphide Zinc Sulfide Quantum Dots dissolved in THF into a 100 milliliter round-bottom flask and dilute with 16 milliliters of THF. To reduce the number of aggregates in solution, sonicate the Quantum Dots for five to ten minutes. Then, dissolve 30 milligrams of PMAL-d and 10 milliliters of molecular grade water.Water about sonication or gentle stirring until the solution is translucent is efficent to completely dissolve the polymer. The use of Vortex or a vigorous stirring can produce many bubbles which hinders interaction of the polymer with the Quantum Dot. Next, add the 10 milliliter polymer solution to the 100 milliliter round-bottom flask containing Indium Phosphide Zinc Sulfide Quantum Dots and THF.Evaporate the THF from the Quantum Dot polymer solution using a Rotary Evaporator with the flask in an ice bath to facilitate the interaction between the polymer and Quantum Dot. Once the solution is evaporated to 10 milliliters, remove the flask from the Rotary Evaporator and add 30 milliliters of molecular grade water. Return the flask to the Rotary Evaporator and continue to evaporate to two milliliters.This final evaporation step may take many hours. Ensure the ice bath is maintained. Remove the water soluble Indium Phosphide Zinc Sulfide Quantum Dots from the round-bottom flask with a pipette.Filter the Quantum Dot solution into a five milliliter centrifuge tube using a three milliliter plastic syringe attached to a 0.1 Micron Nylon Syringe Filter. Place the Quantum Dots into a 20 thousandth molecular weight cutoff membrane dialysis unit and dialyze against 05 Molar Borate Buffer, PH 8.5, to remove excess polymer. Using a vacuum concentrator, concentrate the Quantum Dots and Borate Buffer to one milliliter.For storage, perch the solution with Nitrogen gas before sealing with Parafilm. The water soluble Indium Phosphide Zinc Sulfide Quantum Dots are stable for at least four months at four degrees celsius in the dark. Shown here is the absorbance and corrected Florescence Admission Spectra of Indium Phosphide Zinc Sulfide Quantum Dots and Hexanes excited at 533 nanometers showing a maximum absorbance at 600 nanometers and a full width at half maximum of 73 nanometers.Here a TEM, or Transmission Electron Microscopy Image, of the Indium Phosphide Zinc Sulfide Quantum Dots dissolved in water is shown. A particle size distribution historgram of the TEM results in an average diameter of 2.74 plus or minus 0.72 nanometers. Single Florescent Puncta Analysis details the presence of distinct on and off states through a blinking profile of Indium Phosphide Zinc Sulfide Quantum Dots in water.This histogram exhibits the bimodal distribution of Pixel Intensity from one Quantum Dot blinking profile. Here a graph depicts the viability of N2a cells after incubation with Indium Phosphide Zinc Sulfide Quantum Dots for 24 or 48 hours. With one nanomolar to 25 nanomolar Quantum Dots.Negligible toxicity is observed below five nanomolar. Once mastered, this techniques can be completed in 12 hours if it is performed properly. After watching this video, you should have a good understanding of how to synthesize Highly Fluorescent Quantum Dots for use in Biomedical applications.Note that Allylamine is a hazardous, corrosive organic compound. Precautions such as wearing googles, gloves and a lab coat should always be taken when performing this procedure.

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13.5K Görüntüleme.  Missouri State University. The overall goal of this procedure is to Synthesize Highly Fluorescent Indium Phosphide Zinc Sulfide Quantum Dots that are suitable for Biomedical Applications. This method can help clarify key questions about the Synthesis of Quantum Dot Nanoparticles by demonstrating specific steps that are crucial to achieve high quality Quantum Dots. The main advantage of this technique is the production of high quality, water soluble Quantum Dots.Viable for biological systems that can be synthesiz...