Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications
Summary
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.
Abstract
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.
Introduction
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 "chemical handles" 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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Oleylamine | Acros | 129540010 | |
| Zinc (II) chloride | Sigma | 030-003-00-2 | |
| Indium (III) chloride | Chem-Impex | 24560 | |
| Tris(dimethylamino)phosphine | Encompass | 50-901-10500 | |
| 1-dodecanethiol | Acros | 117625000 | |
| Hexanes | Fisher Sci | H292-4 | |
| Acetone | TransChemical | UN 1090 | |
| Zinc Stearate | Aldrich Chem | 307564-1KG | |
| Tetrahydrofuran | Acros | 34845-0010 | |
| Molecular Water | Fisher Sci | BP2470-1 | |
| Poly(maleic anhyrdride-alt-1-tetradecene), 3-(dimethylamino)-1-propylamine derivative | Sigma | 90771-1G | |
| Boric acid | Fisher Sci | BP168-500 | |
| Sodium Tetraborate Decahydrate | Fisher Sci | BP175-500 | |
| Rhodamine B | Aldrich Chem | R95-3 | |
| Nitrogen gas | Airgas | UN1066 | |
| Trypan blue | Thermo Sci | SV30084.01 | |
| 3 mL plastic Luer-lock syringe | BD | 309657 | |
| Luer-lock Needle | Air-Tite | 8300014471 | 4 inch, 22 gauge |
| 50 mL polypropyene centrifuge tube | Falcon | 352098 | |
| 250 mL centrifuge bottle | Thermo Sci | 05-562-23 | Nalgene PPCO |
| 5 mL centrifuge tubes | Argos-Tech | T2076 | |
| 1.5 mL microcentrifuge tubes | Bio Plas | 4150 | |
| 0.1 μm Syringe filter | Whatman | 6786-1301 | Puradisc 13 mm nylon filter |
| Slide-A-Lyzer MINI Dialysis Unit | Thermo Sci | 69590 | 20,000 MWCO |
| Rotary Evaporator | Heidolph | ||
| Centrifuge 5072 | Eppendorf | Swinging Bucket with 50 mL tube adapters | |
| Lambda 650 UV/VIS Spectrometer | Perkin Elmer | UV-Vis Spectrophotometer | |
| LS 55 Fluorescence Spectrometer | Perkin Elmer | Fluorometer | |
| Axio Observer.A1 | Zeiss | epifluorescence microscope | |
| AxioCam MRm | Zeiss | CCD Camera | |
| Tecnai TF20 Microscope | FEI | Transmisison Electron Miscroscope | |
| TEM Eagle CCD | FEI | TEM CCD Camera | |
| NanoBrook Omni DLS | Brookhaven | Dynamic Light Scattering Instrument |
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