Компактный квантовых точек для одиночных молекул изображений
Summary
Мы описываем получения коллоидных квантовых точек с минимальным гидродинамическим размер одной молекулы флуоресцентной микроскопии. По сравнению с обычными квантовыми точками, эти наночастицы близки по размерам к глобулярных белков и оптимизированы для одной молекулы яркость, устойчивость против фотостарения, а также устойчивость к неспецифического связывания с белками и клетками.
Abstract
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).
Protocol
Representative Results
Discussion
По сравнению с обычными CdSe квантовых точек, тройной сплав ртути х Cd 1-X SE нанокристаллы могут быть настроены по размеру и длине волны флуоресценции самостоятельно. Размер первой выбранной в процессе синтеза нанокристаллов CdSe ядра, а длина волны флуоресценции выбран вторично…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить д-ра Hong Yi в Университете Эмори Основные микроскопии для визуализации комплексной электронной микроскопии. Эта работа была организована NIH гранты (PN2EY018244, R01 CA108468, U54CA119338, и 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:
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