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 thermocouple and proportional-integral-derivative (PID) temperature controller, increase the temperature of the solution to 120 &#176;C. Evacuate the solution under vacuum for 20 min to remove low boiling point impurities that may affect core growth. 
		Note: While it is possible to use a sand bath and thermometer, using a heating mantle and PID increases the uniformity and reproducibility of the reaction products.</li>
		<li>Under inert gas (e.g., N2), reflux the solution and increase the temperature to 220 &#176;C for 15 min. The InCl3 and ZnCl2 completely dissolve, resulting in a pale yellow solution. Allow the temperature to stabilize for 10 min.</li>
		<li>Purge a disposable, 3 ml plastic syringe and 4 inch, 22 G needle with nitrogen gas. Using the syringe, quickly deliver 0.5 ml tris(dimethylamino)phosphine (TDMAP) to the InCl3 solution. The solution temperature decreases slightly and returns to 220 &#176;C. The solution changes from transparent, pale yellow to opaque, black.</li>
		<li>After 9.5 min, remove the reaction flask from the heating mantle until the temperature decreases below 200 &#176;C. To protect the integrity of the InP cores, proceed directly to the ZnS coating in step 1.2.1.</li>
	</ol>
	</li>
	<li>Synthesis of Zinc Sulfide (ZnS) Quantum Dot Shells
	<ol>
		<li>Place the reaction flask from step 1.1.5 on a heating mantle and stabilize the temperature at 200 &#176;C. Slowly add 3.58 g dodecanethiol (DDT) over the course of 15 sec to the solution containing InP QDs. Allow the solution to react for 1 hr. 
		Note: ZnS shell thickness can be varied by increasing or decreasing the amount of zinc stearate added in step 1.2.4. Altering the amount of ZnCl2 or dodecanethiol in steps 1.1.1 and 1.2.1 can significantly impact the quality of QDs by changing the reaction kinetics.
		<ol>
			<li>Afterward, remove the reaction flask from the heating mantle and allow the solution to cool to approximately 60 &#176;C.</li>
		</ol>
		</li>
		<li>Once the InP/ZnS solution reaches ~60 &#176;C, add 10 ml hexanes and transfer the entire solution of roughly 45 ml to a 50 ml polypropylene centrifuge tube. Centrifuge the sample (3,000 x g for 10 min) to remove unreacted solid precursors.</li>
		<li>Carefully transfer the supernatant to a 250 ml polypropylene centrifuge bottle, add 200 ml acetone, and centrifuge the solution (3,000 x g for 10 min) to precipitate InP/ZnS QDs. This volume can also be split evenly into four 50 ml tubes for centrifugation if a centrifuge with the necessary rotor/accessories is not available. Decant the supernatant and dry the QD pellet thoroughly with nitrogen gas to remove acetone.</li>
		<li>Resuspend the QDs in 20 ml OLA using sonication, transfer to a 50 ml round bottom, 3-neck, flask containing 0.474 g zinc stearate, and stir. Evacuate the solution under vacuum for 20 min at RT.</li>
		<li>Under nitrogen gas, increase the temperature to 180 &#176;C and allow the reaction to proceed for 3 hr. While there are no noticeable visual changes to the reaction solution that occur during this reaction, adding zinc stearate increases ZnS shell thickness, thus increasing QY by improving surface passivation of QDs12.. Once the reaction is complete, remove the flask from the heating mantle and allow the solution to cool to approximately 60 &#176;C.</li>
		<li>Once the InP/ZnS solution reaches ~60 &#176;C, add 20 ml hexanes and transfer to a 50 ml polypropylene centrifuge tube. Centrifuge the sample (3,000 x g for 10 min) to remove unreacted zinc stearate.</li>
		<li>Carefully transfer the supernatant to a 250 ml polypropylene centrifuge bottle, add 200 ml acetone, and centrifuge the solution (3,000 x g for 10 min) to precipitate InP/ZnS QDs. Carefully decant the supernatant and thoroughly dry with nitrogen gas to remove acetone.</li>
		<li>Dissolve the InP/ZnS QD pellet in 30 ml hexane. Vortex and sonicate the solution briefly to ensure complete dispersion.</li>
		<li>Repeat purification steps 1.2.6-1.2.8 two more times to ensure thorough removal of excess organic ligands. Interactions between the amphiphilic polymer and the QD in step 1.2 can be compromised in the presence of excess ligands.</li>
		<li>With calculations detailed by Xie, et al.16, determine the size and concentration of the synthesized InP/ZnS QDs using UV-Vis Spectroscopy. 
		Note: The important spectral characteristic for this analysis is the shoulder of the absorption peak. The wavelength and absorbance value of the maximum of this shoulder is used to calculate the size and the concentration of the QDs, respectively. This reaction typically yields approximately 5 &#956;mol of QDs with an emission peak at 600 nm. To synthesize QDs of different colors, the duration and/or temperature of the reaction can be changed. A longer reaction time and/or higher temperature of the solution results in a red-shifted emission peak. For example, increasing the reaction temperature to 240 &#176;C and maintaining reaction time of 10 min will result in QDs with a 680 nm maximum emissions peak. Likewise, reducing reaction time to 2 min and doubling zinc chloride used will result in QDs with a 470 nm maximum emission peak. These InP/ZnS QDs are stable for at least one month at 4 &#176;C in the dark under inert gas.</li>
	</ol>
	</li>
</ol>
2. Water Solubilization of InP/ZnS Quantum Dots Using an Amphiphilic Polymer
<ol>
	<li>Water Solubilization
	<ol>
		<li>Using the InP/ZnS QDs from step 1.2.10, dilute a portion of the QDs with hexanes to obtain 1 ml of 1 &#956;M QDs.
		<ol>
			<li>In a centrifuge tube, transfer 0.25 ml InP/ZnS QDs into each tube. Add 1 ml acetone or methanol to the centrifuge tube and centrifuge (3,000 x g for 10 min). Carefully remove the supernatant and dissolve each precipitate in 1 ml tetrahydrofuran (THF).</li>
			<li>Transfer the InP/ZnS QDs dissolved in THF into a 100 ml round bottom flask and dilute with 16 ml THF. To reduce the number of aggregates in solution, sonicate the QDs for 5-10 min.</li>
		</ol>
		</li>
		<li>Dissolve 30 mg poly(maleic andhydride-alt-1-octadecene), 3-(dimethylamino)-1-propylamine (PMAL-d) in 10 ml molecular grade water. Water bath sonication or gentle stirring until the solution is translucent is sufficient to completely dissolve the polymer. The use of vortex or vigorous stirring can produce many bubbles, which hinders interaction of the polymer with the QD. Add the 10 ml polymer solution to the 100 ml round bottom flask containing InP/ZnS QDs in THF.</li>
		<li>Evaporate THF from the QD/polymer solution using a rotary evaporator. Place the flask in an ice bath while evaporating to facilitate the interaction between the polymer and QD. Depending on the strength of the vacuum, most THF is evaporated after 10 min and the solution appears turbid.
		<ol>
			<li>Once the solution is evaporated to 10 ml, remove the flask from the rotary evaporator and add 30 ml molecular grade water. Return the flask to the rotary evaporator and continue to evaporate to 2 ml. This final evaporation step may take many hours; ensure the ice bath is maintained.</li>
		</ol>
		</li>
		<li>Remove the water-soluble InP/ZnS QDs from the round bottom flask with a pipette. Filter the QD solution using a 3 ml plastic syringe attached to a 0.1 &#956;m nylon syringe filter into a 5 ml centrifuge tube.</li>
		<li>Place the QDs into a 20,000 MWCO membrane dialysis unit and dialyze against 0.05 M borate buffer pH 8.5 to remove excess polymer. (Slowly add 0.05 M sodium tetraborate decahydrate to 0.05 M boric acid, with vigorous stirring, until the pH is 8.5 to make this borate buffer solution.) Using a vacuum concentrator, concentrate the QDs in borate buffer to 1 ml.</li>
		<li>For storage, purge the solution with nitrogen gas before sealing with Parafilm. The water-soluble InP/ZnS QDs are stable for at least 4 months at 4 &#176;C in the dark.</li>
	</ol>
	</li>
</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><tbody><tr><td>Oleylamine</td><td>Acros</td><td>129540010</td><td></td></tr><tr><td>Zinc(II) chloride</td><td>Sigma</td><td>030-003-00-2</td><td></td></tr><tr><td>Indium(III) chloride</td><td>Chem-Impex</td><td>24560</td><td></td></tr><tr><td>Tris(dimethylamino)phosphine</td><td>Encompass</td><td>50-901-10500</td><td></td></tr><tr><td>1-dodecanethiol</td><td>Acros</td><td>117625000</td><td></td></tr><tr><td>Hexanes</td><td>Fisher Sci</td><td>H292-4</td><td></td></tr><tr><td>Acetone</td><td>TransChemical</td><td>UN 1090</td><td></td></tr><tr><td>Zinc Stearate</td><td>Aldrich Chem</td><td>307564-1KG</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Acros</td><td>34845-0010</td><td></td></tr><tr><td>Molecular Water</td><td>Fisher Sci</td><td>BP2470-1</td><td></td></tr><tr><td>Poly(maleic anhyrdride-&lt;em&gt;alt&lt;/em&gt;-1-tetradecene), 3-(dimethylamino)-1-propylamine derivative</td><td>Sigma</td><td>90771-1G</td><td></td></tr><tr><td>Boric acid</td><td>Fisher Sci</td><td>BP168-500</td><td></td></tr><tr><td>Sodium Tetraborate Decahydrate</td><td>Fisher Sci</td><td>BP175-500</td><td></td></tr><tr><td>Rhodamine B</td><td>Aldrich Chem</td><td>R95-3</td><td></td></tr><tr><td>Nitrogen gas</td><td>Airgas</td><td>UN1066</td><td></td></tr><tr><td>Trypan blue</td><td>Thermo Sci</td><td>SV30084.01</td><td></td></tr><tr><td>3 ml plastic Luer-lock syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>Luer-lock Needle</td><td>Air-Tite</td><td>8300014471</td><td>4 inch, 22 gauge</td></tr><tr><td>50 ml polypropyene centrifuge tube</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>250 ml centrifuge bottle</td><td>Thermo Sci</td><td>05-562-23</td><td>Nalgene PPCO</td></tr><tr><td>5 ml centrifuge tubes</td><td>Argos-Tech</td><td>T2076</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Bio Plas</td><td>4150</td><td></td></tr><tr><td>0.1 &amp;mu;m Syringe filter</td><td>Whatman</td><td>6786-1301</td><td>Puradisc 13 mm nylon filter</td></tr><tr><td>Slide-A-Lyzer MINI Dialysis Unit</td><td>Thermo Sci</td><td>69590</td><td>20,000 MWCO</td></tr><tr><td>Rotary Evaporator</td><td>Heidolph</td><td></td><td></td></tr><tr><td>Centrifuge 5072</td><td>Eppendorf</td><td></td><td>Swinging Bucket with 50&amp;nbsp;ml tube adapters</td></tr><tr><td>Lambda 650 UV/VIS Spectrometer</td><td>Perkin Elmer</td><td></td><td>UV-Vis Spectrophotometer</td></tr><tr><td>LS 55 Fluorescence Spectrometer</td><td>Perkin Elmer</td><td></td><td>Fluorometer</td></tr><tr><td>Axio Observer.A1</td><td>Zeiss</td><td></td><td>epifluorescence microscope</td></tr><tr><td>AxioCam MRm</td><td>Zeiss</td><td></td><td>CCD Camera</td></tr><tr><td>Tecnai TF20 Microscope</td><td>FEI</td><td></td><td>Transmisison Electron Miscroscope</td></tr><tr><td>TEM Eagle CCD</td><td>FEI</td><td></td><td>TEM CCD Camera</td></tr><tr><td>NanoBrook Omni DLS</td><td>Brookhaven</td><td></td><td>Dynamic Light Scattering Instrument</td></tr></tbody></table>

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...

Watch this Scientific Journal Video about Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications at JoVE.com

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, ...

synthesis-cd-free-inpzns-quantum-dots-suitable-for-biomedical

Research

JoVE Journal

Chemistry

13.5K Views.  Missouri State University. 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.

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

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

Engineering

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 ...

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 ...

Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence1,2 3,4,5. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells6.
QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens 7. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria 8. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO2 9,10-11, but also ZnO12 and others including CuO 13. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative 10; in others it is the opposite 14. These studies have been well reviewed 15.
In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered16 17. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents.
CdTe QDs have anti-microbial effects alone18 or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates19. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water.
In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent.
The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.

Nanoparticles such as semiconductor quantum dots (QDs) can be used to create photoactivatable agents for anti-microbial or anti-cancer applications. This technique shows how to water-solubilize cadmium telluride (CdTe) QDs, conjugate them to an antibiotic, and perform a bacterial inhibition assay based upon growth curves and plate count.

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. ...

Bioengineering

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature solvents. These coordinating ligands help solubilize and stabilize the nanoparticles while in solution, and can influence the resulting size and reactivity of the nanoparticles during their formation. Despite the ubiquity of using ligands during synthesis, the presence of aliphatic ligands on the nanoparticle surface can result in a number of problems during the end use of the nanoparticles, necessitating further ligand stripping or ligand exchange procedures. We have developed a way to synthesize cadmium sulfide (CdS) nanoparticles using a unique sulfur copolymer. This sulfur copolymer is primarily composed of elemental sulfur, which is a cheap and abundant material. The sulfur copolymer has the advantages of operating both as a high temperature solvent and as a sulfur source, which can react with a cadmium precursor during nanoparticle synthesis, resulting in the generation of ligand free CdS. During the reaction, only some of the copolymer is consumed to produce CdS, while the rest remains in the polymeric state, thereby producing a nanocomposite material. Once the reaction is finished, the copolymer stabilizes the nanoparticles within a solid polymeric matrix. The copolymer can then be removed before the nanoparticles are used, which produces nanoparticles that do not have organic coordinating ligands. This nascent synthesis technique presents a method to produce metal-sulfide nanoparticles for a wide variety of applications where the presence of organic ligands is not desired.

Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction.

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature ...

Enhanced Electron Injection and Exciton Confinement for Pure Blue Quantum-Dot Light-Emitting Diodes by Introducing Partially Oxidized Aluminum Cathode

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation displays and solid-state lighting technologies. The brightness and efficiency of blue QDs-based light-emitting diodes (LEDs) remain inferior to their red and green counterparts, due to the inherently unfavorable energy levels of different colors of light. To solve these problems, a device structure should be designed to balance the injection holes and electrons into the emissive QD layer. Herein, through a simple autoxidation strategy, pure blue QD-LEDs which are highly bright and efficient are demonstrated, with a structure of ITO/PEDOT:PSS/Poly-TPD/QDs/Al:Al2O3. The autoxidized Al:Al2O3 cathode can effectively balance the injected charges and enhance radiative recombination without introducing an additional electron transport layer (ETL). As a result, high color-saturated blue QD-LEDs are achieved with a maximum luminance over 13,000 cd m-2, and a maximum current efficiency of 1.15 cd A-1. The easily controlled autoxidation procedure paves the way for achieving high-performance blue QD-LEDs.

A protocol is presented for fabricating high-performance, pure blue ZnCdS/ZnS-based quantum dots light-emitting diodes by employing an autoxidized aluminum cathode.

Stable and efficient red (R), green (G), and blue (B) light sources based on solution-processed quantum dots (QDs) play important roles in next-generation ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Inkjet Printing All Inorganic Halide Perovskite Inks for Photovoltaic Applications

A method for synthesizing photoactive inorganic&#160;perovskite quantum dot inks and an inkjet printer deposition method, using the synthesized inks, are demonstrated. The ink synthesis is based on a simple wet chemical reaction and the inkjet printing protocol is a facile step by step method. The inkjet printed thin films have been characterized by X-ray diffraction, optical absorption spectroscopy, photoluminescent spectroscopy, and electronic transport measurements. X-ray diffraction of the printed quantum dot films indicates a crystal structure consistent with an orthorhombic room temperature phase with (001) orientation. In conjunction with other characterization methods, the X-ray diffraction&#160;measurements show high quality films can be obtained through the inkjet printing method.

A protocol for synthesizing inorganic-lead-halide hybrid perovskite quantum dot inks for inkjet printing and the protocol for preparing and printing the quantum dot inks in an inkjet printer with post characterization techniques are presented.

A method for synthesizing photoactive inorganic&#160;perovskite quantum dot inks and an inkjet printer deposition method, using the synthesized inks, are ...

Synthesis of In37P20(O2CR)51 Clusters and Their Conversion to InP Quantum Dots

This text presents a method for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots. The In37P20(O2CR)51 clusters have been observed as intermediates in the synthesis of InP quantum dots from molecular precursors (In(O2CR)3, HO2CR, and P(SiMe3)3) and may be isolated as a pure reagent for subsequent study and use as a single-source precursor. These clusters readily convert to crystalline and relatively monodisperse samples of quasi-spherical InP quantum dots when subjected to thermolysis conditions in the absence of additional precursors above 200 &#176;C. The optical properties, morphology, and structure of both the clusters and quantum dots are confirmed using UV-Vis spectroscopy, photoluminescence spectroscopy, transmission electron microscopy, and powder X-ray diffraction. The molecular symmetry of the clusters is additionally confirmed by solution-phase 31P NMR spectroscopy. This protocol demonstrates the preparation and isolation of atomically-precise InP clusters, and their reliable and scalable conversion to InP QDs.

Synthesis of In37P20O2CR51 Clusters and Their Conversion to InP Quantum Dots

A protocol for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots is presented.

This text presents a method for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots. The In37P20(O2CR)51 ...

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.

Author Spotlight: High-Quality Quantum Dot Nanobeads for Sensitive Fluorescent Lateral Flow Immunoassays

Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB) and the detection of disease biomarkers using QDNB-based lateral flow immunoassay strips. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent strip reader within 15 min.

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...