镉免费的InP /硫化锌量子点的合成适合生物医学应用
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
量子点（QDs）是 半导体时与光1照射表现出荧光性质的纳米晶体。由于它们的小尺寸（2-5纳米），其类似于许多较大的生物分子，和易于biofunctionalization的，量子点是用于生物医学应用的非常有吸引力的工具。他们已经发现在生物标记的使用，单分子的活细胞成像，药物递送， 体内成像 ，病原体检测，和细胞的跟踪，许多其他用途2-8之间。
基于CD的量子点已经在生物医学领域最常用的，因为他们强烈的荧光，窄的发射峰宽9。然而，关注已经提出，由于镉的潜在毒性2+离子 10可通过纳米颗粒的降解被释放。近年来，基于InP基量子点已探索作为一种替代基于镉量子点，因为他们认为许多荧光特性的镉量子点的基础和可能更生物相容性11。基于CD的量子点已被发现比在浓度低至10 分在体外试验基于InP基量子点更毒显著，只有48小时11后。
量子点的荧光发射颜色是尺寸可调的1。也就是说，随着量子点尺寸的增加，荧光发射是红移。量子点的产品的大小和尺寸分散度可以通过在反应12期间改变温度，反应持续时间，或前体浓度的条件进行修改。虽然磷化铟量子点的发射峰通常是更广泛，比基于镉量子点不太激烈，磷化铟量子点可以在种类繁多的设计，以避免光谱重叠的颜色进行，并且是大多数生物医学应用12足够强。在本协议中详述的合成产生与600纳米为中心的红色发射峰量子点。
几个步骤采取自动对焦在QD核之三合成，保持量子点的光学完整性，使其成为生物应用程序兼容。 QD核的表面必须防止可能导致淬火氧化或表面缺陷;因此，一的ZnS壳涂覆在核心产生的InP /硫化锌（核/壳）量子点13。这种涂层已显示保护QD产物的光致发光。锌离子中的InP量子点的合成的存在已经显示出，以限制表面缺陷，以及减少尺寸分布12。甚至用Zn 2+在反应介质存在下，InZnP的合成是非常不可能12。涂布后，所得的InP / ZnS量子点被涂覆在疏水性配体诸如三辛基氧化膦（TOPO）或油胺12,14。两亲聚合物可以与量子点表面上的疏水性配位体以及本体水分子相互作用以赋予水溶性15。与鸬鹚两亲聚合物xylate化学基团可以被用作“化学柄”，进一步官能化的量子点。
该协议细节的合成和水溶性的InP / ZnS量子点的官能具有非常强烈的荧光发射和相对小的尺寸的分散性。这些量子点都可能比常用的CdSe / ZnS量子点毒性较低。在这里，磷化铟/ ZnS量子点的合成提供了基于光盘的量子点生物医学领域的实用替代。
Protocol
Representative Results
Discussion
该协议细节，可以在许多生物体系中使用高度荧光的InP / ZnS量子点的合成。此处所合成的量子点的产品表现出在600nm与FWHM为73nm（图1），这与其他先前描述合成12为中心的单个荧光发射峰。反应时间和反应温度是由于对QD合成质量和可重复性其深远的影响非常重要的步骤。在水中溶解后，量子点都确定为具有约6％的量子产率。反应时间，温度，或前体浓度的变化允许QD的尺寸和…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者非常感谢化学系和研究生院在密苏里州立大学的鼎力支持这个项目。我们也承认在弗雷德里克国家癌症研究实验室的电子显微镜实验室使用其透射电子显微镜和碳包覆电网。
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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