Compact Quantum Dots for Single-molecule Imaging

The overall goal of this procedure is to prepare fluorescent quantum dots with optimized brightness and stability, as well as minimized size and non-specific binding for use in single molecule imaging. This is accomplished by first preparing small cadmium selenide quantum dot cores. The second step is to alloy these cores with mercury to shift their fluorescence into the red spectrum and increase their brightness.

Next, a thin alloy shell is grown on the nano crystals to stabilize their fluorescence emission. The final step is to transfer these particles from organic solvents to aqueous buffers using a multi dentate polymer, which can be modified with biologically inert polyethylene glycol. Ultimately, fluorescence spectrometry, gel chromatography and gel electrophoresis are used to show that these particles have bright fluorescence, a compact hydrodynamic size, and a neutral electrostatic charge attributes well suited for single molecule fluorescence imaging in biology.

The main advantage of these quantum dots over existing commercial materials is that these nanoparticles have a significantly decreased hydrodynamic size. Although smaller quantum dots are negatively impacted by decreased fluorescence intensity, decreased fluorescence stability, and fluorescence only in the blue spectrum, we have offset these effects using a mercury cation exchange process. These nano Crystals can help answer key questions in biophysics and cellular signaling by enabling dynamic imaging of single biomolecules in crowded biological environments.

To begin, prepare a 0.4 molar solution of selenium in top by adding selenium to a 50 milliliter. Three neck flask, evacuating the flask under a high vacuum and filling with argon using a sch shank line under air free conditions. Add 10 milliliters of top and heat to 100 degrees Celsius while stirring for one hour to yield a clear colorless solution.

Cool the solution to room temperature and set the flask aside. Also, prepare a solution of cadmium oxide TDPA and ODE in a 250 milliliter three neck flask using the quantities listed in the written protocol. Accompanying this video, evacuate the solution using a shank line while stirring.

Increase the temperature to 100 degrees Celsius and evacuate for an additional 15 minutes. To remove low boiling point impurities under Argonne gas, heat the mixture to 300 degrees Celsius for one hour to fully dissolve the cadmium oxide. The solution will change from a reddish color to clear and colorless cool the solution to room temperature.

Next, add HDA to the cadmium solution, heat to 70 degrees Celsius and evacuate. Once a constant pressure is attained, increase the temperature and reflux the solution for 30 minutes. Switch the schlink line valve to inert gas and insert the thermocouple directly into the solution.

Under air free conditions, add DPP to the cadmium solution and increase the temperature to 310 degrees Celsius. Now use a disposable plastic syringe attached to a 16 gauge needle to remove 7.5 milliliters of the 0.4 molar top selenium solution. Once the temperature equilibrates to 310 degrees Celsius, set the temperature controller to zero degrees Celsius and swiftly inject the top selenium solution directly into the cadmium solution.

The solution will change from colorless to yellow, orange, and the temperature will quickly drop and increase again to approximately 280 degrees Celsius. The most difficult step in this procedure is to inject the entire volume of the syringe as quickly as possible into the cadmium solution. This ensures that the particles homogeneously nucleate.

After one minute of reaction, remove the flask from the heating mantle and quickly cool with a stream of air until the temperature is less than 200 degrees Celsius. When the temperature reaches approximately 40 degrees Celsius, dilute with 30 milliliters of hexane, most of the remaining cadmium precursor will settle out of solution. Remove this precipitate by centrifugation.

In each of six 50 milliliter polypropylene conical centrifuge tubes dilute 12 milliliters of the resulting crude nano Krystal solution. With 40 milliliters of acetone following centrifugation with the same parameters, carefully decant and discard the supernatant. Next, dissolve the nano krysttal pellets in hexane.

Extract the solution with an equal volume of methanol retaining the top phase. Repeat this extraction twice more for the third extraction. The volume of methanol may be adjusted to approximately 15 milliliters to obtain a concentrated hexane solution of pure cadmium selenide quantum dots at roughly 200 micromolar.

The typical yield of this reaction is three micromolar cadmium selenide crystals with a diameter of two three nanometers determine the nano Krystal diameter and concentration by measuring the ultraviolet visible absorption spectrum as described in the written procedure, the nano crystals may be partially exchanged with mercury to redshift, the absorption and fluorescence emission. To do this in a 20 milliliter glass vial with stir bar mix hexane and chloroform, then add the cadmium selenide quantum dot solution OLA and mercury octane violate. After purifying the nano crystals and determining their concentration as described in the written procedure, allow the nano crystals to age for at least 24 hours at room temperature for shell growth.

Prepare 0.1 molar shell precursor solutions in 50 milliliter, three neck flasks under vacuum heat solutions of cadmium precursor, zinc precursor, and sulfur precursor to reflux for one hour to yield clear solutions, then charge with argon to a three necked flask. Add the topo ODE and prepared mercury cadmium selenide quantum dots. Evacuate off the hexine at room temperature using the flank line.

Increase the temperature to 100 degrees Celsius and reflux for 15 minutes. Change thele line valve to Argonne gas and insert the thermocouple in the nano Krystal solution. After increasing the temperature to 120 degrees Celsius, add 0.5 monolayers or 140 microliters of sulfur precursor solution and allow the reaction to proceed for 15 minutes.

Increase the temperature to 140 degrees Celsius. Add 0.5 monolayers or 140 microliters of cadmium precursor solution and allow the reaction to proceed for 15 minutes. Then add 500 microliters of anhydrous OLA to the reaction solution.

Add 160 degrees Celsius, add 0.5 monolayers, or 220 microliters of sulfur precursor solution, followed by an equal quantity of zinc precursor solution at 170 degrees Celsius with 15 minutes between each addition. Then at 180 degrees Celsius, add 0.25 monolayers, or 150 microliters of sulfur precursor solution and zinc precursor solution in 15 minute intervals.Cool. The solution to room temperature and a new extinction coefficient for these particles.

Using a UV vis spectrum, assuming that the number of nano crystals has not changed, store the reaction solution as a crude mixture in a freezer. At this stage, the nano crystals may be characterized using electron microscopy, UV vis absorption spectroscopy and fluorescence spectroscopy. Add purified core shell quantum dots to a 50 milliliter three neck flask and remove the hexine under a high vacuum.

To yield a dry film, fill the flask with argonne. Add anhydrous purine to the nanoparticle film and heat the slurry to 80 degrees Celsius over the course of one to two hours. The nanoparticles will fully dissolve.

Add one milliliter of one FIO glycerol to the solution and stir at 80 degrees Celsius for two hours. After cooling the solution to room temperature, add 0.5 milliliters of triethylamine to de protonate the thio glycerol and stir for 30 minutes. The solution may become cloudy after the addition of triethylamine due to the poor solubility of polar nano crystals in this solvent mixture.

Transfer the quantum dot solution into a 50 milliliter conical centrifuge tube containing a mixture of 20 milliliters of hexane and 20 milliliters of acetone and mix well. Isolate the precipitated nano crystals via centrifugation, followed by pellet wash with acetone, dissolve the quantum dot pellet in five milliliters of DMSO with bath sonication and follow with centrifugation. To remove possible aggregates, then determine the nanoparticle concentration from a UV V absorption spectrum.

The solution of pure quantum dots should be used within three hours as the surface ths can slowly oxidize under ambient conditions in air. Dilute the quantum dot solution to 10 micromolar or less with DMSO and transfer to a 50 milliliter flask. Add a prepared five milligram per milliliter solution of dilated polyacrylic acid in DMSO, dropwise to the quantum dot solution while stirring and degas the solution at room temperature for five minutes.

Purge the quantum dot polymer solution with Argonne and heat to 80 degrees Celsius for 90 minutes. After cooling the solution to room temperature, add an equal volume of 50 millimolar sodium ate pH eight dropwise and stir for 10 minutes. Purify the quantum dots as described in the written procedure and determine the concentration from a UV vis absorption spectrum in a four milliliter glass vial with stir bar mix one mole quantum dots in borate buffer with a 40, 000 times molar excess of 750 Dalton mono amino polyethylene glycol.

Instructions can be found in the written procedure on how to add specific chemical functionality to the nano crystals. Quickly add a 25, 000 times molar excess of a freshly prepared solution of the activating agent solution to the quantum dot solution and stir it room temperature for 30 minutes. Repeat this step four more times to saturate the nano krysttal surface with PEG.

Finally, add 200 microliters of one molar tris buffer to quench the reaction before purifying the nano crystals Using dialysis centrifugal filters or ultracentrifugation, The resulting nano chrystal can be analyzed for mono dispersity hydrodynamic size and surface charge using liquid chromatography, gel electrophoresis and fluorescence microscopy. Shown here is a representative absorption and fluorescent spectra for cadmium selenide nano crystals, mercury, cadmium, selenide, nano crystals after cadion exchange, and a core mercury cadmium selenide, shell cadmium, zinc sulfide nano crystals after shell growth. The core cadmium selenide nano crystals have a quantum yield of fluorescence near 15%However, this efficiency drops to less than 1%after mercury exchange, likely due to charge carrier traps introduced through surface atom disruption.

Growth of a thin shell of cadmium zinc sulfide boosts this efficiency to more than 70%which is largely maintained after transfer to water. In contrast, core cadmium selenide, shell cadmium, zinc sulfide nano crystals without mercury incorporation lose a substantial fraction of their quantum yield in water unless a thick shell is grown. It is important to note that capping with cadmium zinc sulfide shifts the spectra to the red due to leakage of the electronic charge carriers into the shell material.

This shift is around 20 to 30 nanometers for core cadmium cores and increases with increasing mercury content in the core. Thus, by incorporating mercury into the core nano crystal, the small size of the nano Krystal can be maintained without sacrificing brightness. The small size is demonstrated in this transmission electron micrograph and particle size distribution of core mercury, cadmium, selenide, shell cadmium, zinc sulfide nano crystals showing an average diameter of 3.2 plus or minus 0.6 nanometers.

The use of a two-step phase transfer to water is critical for obtaining a homogenous population of nano crystals that do not require further size sorting to remove clusters and aggregates the size exclusion. Chromatogram depicted here confirms that the size is similar to that of con albumin. At 75 kilodaltons and after modification with 750 Dalton amino PEG, the size is increased to just 12 nanometers, similar to that of an IgG antibody.

PEG modification neutralizes the surface charge as confirmed in the aros gel electrophoresis experiment depicted here, the well is marked with an arrow and electrode polarities are indicated on the right showing that before conjugation the nano crystals migrate as onic particles and that the pegylated nano crystals are electrostatically neutral. Shown here is an epi fluorescence micrograph of these nano crystals deposited on a glass cover slip and excited with 545 nanometer visible light. These nano crystals are readily observed at the single molecule level at 30 frames per second with an electron multiplying CCD camera.

This plot shows that the number of fluorescent particles observed in each frame fluctuates over time with continuous excitation. This is due to a combination of blinking and photo degradation. Blinking dominates for the first seven minutes before oxidative photo degradation slowly becomes apparent.

After watching this video, you should have a good understanding how to chemically synthesize quantum dots, how to transfer them into aqueous buffers, and how to modify them for bioimaging applications. Don't forget that working with reagents containing cadium and mercury can be extremely hazardous, and extra precautions should be taken to prevent personal exposure.