紧凑型量子点的单分子成像
Summary
我们描述了准备的胶体量子点与最小化的流体力学尺寸为单分子荧光成像。相比传统的量子点，这些纳米颗粒的尺寸类似于球状蛋白质单分子反对光降解，亮度，稳定性和耐非特异性结合的蛋白质和细胞进行了优化。
Abstract
单分子成像是一个重要的工具，对于理解生物分子功能的机制和可视化的空间和时间异质性的分子行为的基础细胞生物学1-4。要图像的个别分子的利益，它是典型的共轭到一个荧光标记（染料，蛋白质，小珠，或量子点），并与落射荧光或总内部反射荧光显微镜（TIRF）观察。它们的荧光染料和荧光蛋白质已被几十年来的荧光成像的主体，是不稳定的必要观察单个分子的高光子通量下，得到信号完全丧失之前只需要几秒钟的观察。乳胶珠和染料标记的珠提供改进的信号的稳定性，但在急剧放大的流体动力学尺寸，它可以有害地改变所研究的分子的扩散和行为的牺牲。
ntent“量子点（量子点）之间的平衡这两个问题的制度。这些纳米粒子组成的半导体材料，可以设计一个水动力紧凑的尺寸，优异的抗光降解5。因此，近年来，量子点已经在使长期观察单分子水平上的复杂大分子的行为。然而，这些粒子仍然表现出受损的扩散分子在拥挤的环境中，如细 胞的细胞质和神经元突触间隙，它们的尺寸仍然过大，4,6 ，7。最近，我们已设计的流体动力学尺寸最小化的量子点的芯和表面涂层，而平衡的偏移量的胶体稳定性，耐光性，亮度，和在过去8,9阻碍紧凑量子点的实用程序的非特异性结合。这篇文章的目的是展示这些优化的纳米晶的合成，修饰和表征，由汞的合金X CD-X SE核心涂有绝缘CDŸ锌1-Y的外壳，再涂上一多与短聚乙二醇修饰的高分子配体（ PEG）链（ 图1）。与传统的CdSe纳米晶体相比，汞X CD 1-X SE合金提供了更大的荧光量子产率，在增强的信号噪声在细胞中，和在非细胞毒性的可见光波长的激发的红光和近红外波长的荧光。多齿聚合物涂层的纳米晶体的表面，在一个封闭的和平面的构象结合的流体动力学尺寸最小化，并，PEG中和表面电荷，以尽量减少非特异性结合的细胞和生物分子。最终的结果是一个明亮的荧光纳米晶与排放之间550-800 nm和总的水动力大小近12处。这是在sAME许多可溶性的球形细胞中的蛋白质，远小于传统的聚乙二醇化量子点（25-35纳米）的尺寸范围。
Protocol
Representative Results
Discussion
相对于传统的CdSe量子点，三元合金汞X CD-X SE纳米晶体可调谐的大小和荧光波长独立。在合成过程中的CdSe纳米晶体核的大小是第一次选择，和荧光的波长选择在二次汞阳离子交换步骤中，基本上不改变纳米晶体尺寸9。重要的是要允许纯化汞X CD 1-X SE纳米晶体在室温下孵育，在至少24小时前封盖。这允许一些弱吸附汞阳离子扩散到的纳米晶体晶格。不使这个过程中发生，在近…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者想感谢香港易在埃默里大学综合显微镜核心的电子显微成像。这项工作是由美国国立卫生研究院拨款赞助（PN2EY018244，R01 CA108468，U54CA119338的，1K99CA154006-01）。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Selenium | Sigma-Aldrich | 229865 | |
| Tri-n-octylphosphine | Strem | 15-6655 | 97% pure, unstable in air |
| Cadmium oxide | Sigma-Aldrich | 202894 | Highly toxic: use caution |
| Tetradecylphosphonic acid | PCI Synthesis | 4671-75-4 | |
| Octadecene | Alfa Aesar | L11004 | Technical grade |
| Hexadecylamine | Sigma-Aldrich | H7408 | |
| Diphenylphosphine | Sigma-Aldrich | 252964 | Pyrophoric |
| Mercury acetate | Sigma-Aldrich | 456012 | Highly toxic: use caution |
| 1-Octanethiol | Sigma-Aldrich | 471836 | Strong odor |
| Oleic acid | Sigma-Aldrich | W281506 | |
| Zinc acetate | Alfa Aesar | 35792 | |
| Cadmium acetate hydrate | Sigma-Aldrich | 229490 | Highly toxic: use caution |
| Oleylamine | Fisher Scientific | AC12954 | Unstable in air |
| Sulfur | Sigma-Aldrich | 344621 | |
| Trioctylphosphine oxide | Strem | 15-6661 | 99% |
| Pyridine | VWR | EM-PX2012-6 | Anhydrous |
| Thioglycerol | Sigma-Aldrich | M1753 | Strong odor |
| Triethylamine | Sigma-Aldrich | 471283 | Anhydrous |
| Dialysis tubing | Spectrum Labs | 131342 | 20 kDa cutoff |
| Centrifugal filter | Millipore | UFC801024 | 10 kDa cutoff |
| Monoamino-PEG | Rapp Polymere | 12 750-2 | 750 Da |
| DMTMM, 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride hydrate | Alfa Aesar | H26333 | |
| AKTAprime Plus Chromatography System | GE HealthCare | ||
| Superose 6 10/300 GL chromatography column | GE HealthCare | 17-5172-01 | |
| Agarose, OmniPur | VWR | EM-2120 | |
Appendix Synthesis of mercury octanethiolate: Slowly add a methanol solution of mercury acetate (1 eq.) to a stirring solution of 1-octanethiol (3 eq.) and potassium hydroxide (3 eq.) in methanol at room temperature. Isolate the mercury(II) octanethiolate precipitate via filtration, wash two times with methanol and once with ether, and then dry under vacuum. Synthesis of multidentate polymer: Dissolve polyacrylic acid (1 g, 1,773 Da) in 25 ml dimethylformamide (DMF) in a 150 ml three-necked flask and bubble with argon for 30 min. Add an anhydrous solution of cysteamine (374 mg, 4.87 mmol) in 10 ml DMF. At room temperature with vigorous stirring, slowly add anhydrous diisopropylcarbodiimide (DIC, 736 mg, 5.83 mmol) over 30 min, followed by triethylamine (170 μl, 1.22 mmol), and allow the reaction to proceed for 72 hr at 60 °C. Add mercaptoethanol (501 mg, 6.41 mmol) to quench the reaction, and stir for 2 hr at room temperature. Remove DMF via rotary evaporation and isolate the polymer with the addition of a 2:1 mixture of ice-cold acetone:chloroform, followed by centrifugation. Dissolve the polymer in ~5 ml anhydrous DMF, filter, precipitate again with diethyl ether, and repeat. Dry the product under vacuum and store under argon. Determination of CdSe core diameter: From the UV-Vis absorption spectrum determine the wavelength of the first exciton peak (λ, in nm), which is the longest-wavelength peak (e.g. roughly 498 nm for CdSe in Figure 2a), and use the sizing curve of Mulvaney and coworkers 12:
Determination of CdSe nanocrystal concentration: From a background-subtracted UV-Vis spectrum of an optically clear solution of CdSe nanocrystals, determine the absorption at 350 nm wavelength. Serial dilutions can be used to determine if the optical absorption is within the linear range of Beer’s Law. The nanocrystal concentration (QD, in M) can be determined by plugging in the nanocrystal diameter (D, in nm), the optical absorption value (A3sa), and the cuvette path length (l, in cm) into the following equation from the empirical correlation of Bawendi and coworkers 13:
|
Referências
- Toprak, E., Selvin, P. R. New fluorescent tools for watching nanometer-scale conformational changes of single molecules. Annu. Rev. Biophys. Biomol. Struct. 36, 349-369 (2007).
- Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C., Ha, T. J. Advances in single molecule fluorescence methods for molecular biology. Annu. Rev. Biochem. 77, 51-76 (2008).
- Pinaud, F., Clarke, S., Sittner, A., Dahan, M. Probing cellular events, one quantum dot at a time. Nat. Method. 7, 275-285 (2010).
- Smith, A. M., Wen, M. M., Nie, S. M. Imaging dynamic cellular events with quantum dots. Biochemist. 32, 12-17 (2010).
- Smith, A. M., Duan, H. W., Mohs, A. M., Nie, S. M. Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv. Drug Deliv. Rev. 60, 1226-1240 (2008).
- Smith, A. M., Nie, S. M. Next-generation quantum dots. Nature Biotech. 27, 732-733 (2009).
- Groc, L., Lafourcade, M., Heine, M., Renner, M., Racine, V., Sibarita, J. -. B., Lounis, B., Choquet, D., Cognet, L. Single trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies. J. Neurosci. 27, 12433-12437 (2007).
- Smith, A. M., Nie, S. M. Minimizing the hydrodynamic size of quantum dots with multifunctional multidentate polymer ligands. J. Am. Chem. Soc. 130, 11278-11279 (2008).
- Smith, A. M., Nie, S. M. Bright and compact alloyed quantum dots with broadly tunable near-infrared absorption and fluorescence spectra through mercury cation exchange. J. Am. Chem. Soc. 133, 24-26 (2011).
- Shriver, D. F., Drezdzon, M. A. . The Manipulation of Air-Sensitive Compounds. , (1986).
- Errington, R. J. . Advanced Practical Inorganic and Metalorganic Chemistry. , (1997).
- Jasieniak, J., Smith, L., van Embden, J., Mulvaney, P., Califano, M. Re-examination of the size-dependent absorption properties of CdSe quantum dots. J. Phys. Chem. C. 113, 19468-19474 (2009).
- Leatherdale, C. A., Woo, W. K., Mikulec, F. V., Bawendi, M. G. On the absorption cross section of CdSe nanocrystal quantum dots. J. Phys. Chem. B. 106, 7619-7622 (2002).
- Smith, A. M., Mohs, A. M., Nie, S. M. Tuning the optical and electronic properties of colloidal nanocrystals by lattice strain. Nature Nanotech. 4, 56-63 (2009).
- Demas, J. N., Crosby, G. A. The measurement of photoluminescence quantum yields. A review. J. Phys. Chem. 75, 991-1024 (1971).
- Van Embden, J., Jasieniak, J., Mulvaney, P. Mapping the optical properties of CdSe/CdS heterostructure nanocrystals: the effects of core size and shell thickness. J. Am. Chem. Soc. 131, 14299-14309 (2009).
- Smith, A. M., Duan, H. W., Rhyner, M. N., Ruan, G., Nie, S. M. A systematic examination of surface coatings on the optical and chemical properties of semiconductor quantum dots. Phys. Chem. Chem. Phys. 8, 3895-3903 (2006).
- Zhang, X., Mohandessi, S., Miller, L. W., Snee, P. T. Efficient functionalization of aqueous CdSe/ZnS nanocrystals using small-molecule chemical activators. Chem. Comm. 47, 3532-3534 (2011).
- Bucio, L., Souza, V., Albores, A., Sierra, A., Chavez, E., Carabez, A., Guiterrez-Ruiz, M. C. Cadmium and mercury toxicity in a human fetal hepatic cell line (WRL-68 cells). Toxicol. 102, 285-299 (1995).
- Han, S. G., Castranova, V., Vallyathan, V. J. Comparative cytotoxicity of cadmium and mercury in a human bronchial epithelial cell line (BEAS-2B) and its role in oxidative stress and induction of heat shock protein 70. J. Toxicol. Environ. Health Part A. 70, 852-860 (2007).
- Strubelt, O., Kremer, J., Tilse, A., Keogh, J., Pentz, R. J. Comparative studies on the toxicity of mercury, cadmium, and copper toward the isolated perfused rat liver. J. Toxicol. Environ. Health Part A. 47, 267-283 (1996).
