Production and Targeting of Monovalent Quantum Dots

The overall goal of this procedure is to provide an easy and generalizable protocol for the preparation of monovalent quantum dots for single molecule imaging of target membrane proteins in live cells. This is accomplished by first introducing a stoichiometric amount of a long poly phospho oligonucleotide ligand to a quantum dot. The long DNA completely wraps the quantum dot and prevents the binding of a second DNA molecule, thereby generating monovalent quantum dots exclusively and quantitatively.Separately.

Benzo guine functionalized, DNA bearing complementary sequences is prepared to target snap tagged membrane proteins expressed on live cells. Next, the sequential treatment of live cells expressing snap tagged membrane proteins with benzo guine, DNA, followed by monovalent quantum dots leads to selective labeling of the targeted receptor. The final step is to image the target protein distribution and diffusion trajectories via single molecule fluorescence microscopy.

Ultimately real-time dynamic spatiotemporal information of target proteins in live cells is extracted to reveal how the target molecules work in vivo. The main advantage of this approach to synthesizing monovalent quantum dots is its simplicity and general accessibility. Unlike many other approaches that require special skills or equipment, this approach is relatively straightforward.

As a consequence, it should be available to any lab and for any discipline. We first had the idea for this method when Zeva and I began discussing how our expertise on DNA nanoparticle synthesis could be combined to better control the reactivity of nanoparticles. We carried out the initial approval principle experiments together over a weekend.

Increasingly, our first trial worked successfully. In this video, we use monovalent quantum dots to study the single particle dynamics of the notch receptor in live mammalian cells. However, these monovalent quantum dots should find broad utility for any studies that require single particle tracking.

Generally, individuals new to this method will struggle because not all commercially available quantum dots are the same, either geometrically or chemically. Therefore, some optimization will be required. Visual demonstration of this method is critical at your phase transfer and phosphorous I related DNA addition steps could be intimidating to a laboratory without multi synthetic chemistry experience.

To begin, dilute 200 microliters of a one micromolar solution of organic phase quantum dots with 400 microliters of chloroform in a five milliliter glass vial. Combine these quantum dots with a mixture of 400 microliters of 0.3 molar tetra butyl ammonium bromide chloroform solution, and 36 microliters of neat pec thiol and shake overnight. Then add 800 microliters of a 0.2 molar sodium hydroxide aqueous solution and shake for 30 seconds.

A phase transfer occurs within a few minutes indicated by the transfer of the colored particles to the aqueous phase above the denser organic phase. If the particles aggregate in a third phase between the aqueous and organic phases, increase the incubation time with the pec dial. If the aqueous phase remains clear, the quantum dots did not phase transfer.

Increase the concentration of pec dial to alleviate poor phase transfer. Next, recover the colored aqueous phase. Then concentrate the collected quantum dots with the racon spin column to one milliliter.

Add the concentrated quantum dot solution into a cidex NEP 10 column pree equilibrated with 10 millimolar tris buffer containing 30 millimolar sodium chloride. pH eight elute the quantum dots with 1.5 milliliters of elution buffer by gravity flow. Proceed to measure the concentration of quantum dots with absorption spectroscopy at 350 nanometers.

To prepare a monovalent targeted quantum dots purchase or synthesize phospho eight DNA, prepare one milliliter of 100 ano molar quantum dot solution in 10 millimolar tris buffer containing 30 millimolar sodium chloride pH eight. Next, add 500 microliters of the 100 nanomolar phospho IO eight DNA solution. Dropwise to the aqueous quantum dots for one minute while vigorously stirring.

Stir or place the mixture on a shaker for an additional nine hours. Remove approximately 10 microliters of the quantum dot mixture to run on an analytical agros gel. Also, remove a similar concentration of unconjugated quantum dots in aqueous phase.

Then at approximately two microliters of six x fi call loading buffer to increase the solution density. Run these two samples together on a 0.8%weight volume AROS gel in sodium bide buffer for 15 minutes at 150 volts. The result should be a single band in the unconjugated control lane migrating close to the well and two bands in the lane with conjugated quantum dots.

Calculate the unconjugated quantum dots fraction using the relative intensity of the two bands. Then use that fraction to calculate the additional volume of 100 nanomolar phospho eight DNA solution required to match the number of quantum quantum dots. Repeat the procedure once more with this calculated volume or until the conjugated quantum dots collapse into a single band on the gel indicating complete conjugation of all quantum dots.

Next, add 100 microliters of 10 millimolar carboxy. Peg six alkane thiol to the conjugated monovalent targeted quantum dots and shake for 10 minutes. To remove excess alkane pegol, add 0.5 milliliters of the quantum dot solution to ACE fitex knap five column pre equilibrated with elucian buffer.

Then collect the monovalent targeted quantum dots with one milliliter of elu buffer by gravity flow. Concentrate the collected quantum dots with a centric con spin column. These monovalent targeted quantum dots can be stored at four degrees Celsius per months, ate the monovalent quantum dots just prior to an imaging experiment using reagents such as casein or bovine serum albumin plate cells expressing the snap tagg protein on total internal reflection, fluorescence or turf quality glass.

In this particular experiment, we use a U2 OS cell line expressing a snap tagged notch one receptor and high quality glass surfaces after the cells have attached to the glass, remove the growth media and wash with phosphate buffered saline or PBS. Then incubate the cells for 10 to 30 minutes at room temperature in PBS or cell media containing benzoyl guine DNA, prepared as detailed in the text protocol after incubation with benzo Guin, DNA, carefully wash the cells with PBS or cell media, then incubate the cells for approximately five to 10 minutes with the monovalent quantum dots. Wash the unbound monovalent quantum dots and return the cells to a buffer or media suitable for both imaging and culture image.

The cells using a turf microscope because of the brightness of monovalent, quantum dots collect images at high frame rates in turf while imaging the basal side of a live cell. Once the quantum dots are coated with the negatively charged DNA, they should migrate on a gel separately from the non rapped quantum dots using an aliquot of unwrapped quantum dots as a control. A second faster migrating band should appear upon addition of the Phosphol IO eight DN.A complete formation of the monovalent quantum dots is demonstrated with the loss of the immobile band and its collapse into the mobile band.

If an aliquot of the monovalent quantum dots product migrates as a single band separable from the unwrapped quantum dots, then the quantum dots are indeed monovalent and ready to be used in further steps. Shown here is representative labeling of cells expressing a snap notch M cherry construct at various monovalent quantum dot concentrations. Monovalent quantum dots passivated with PEG 12 co localized with M cherry indicating specific labeling.

Lower labeling densities are generally preferable for single particle tracking. After watching this video, you should have a good understanding how to synthesize a fluorescent nanoparticle that is bright modular monovalent and targeted Once mastered, this technique can be done in two data preparation of the monovalent quantum dots and one hour photo cell labeling If it is performed properly. It's important to remember that using commercially available starting reagents, we can produce monovalent quantum dots with excellent photo physical properties that can be used for long single particle tracking experiments.

Following this procedure, other proteins can be labeled with snap tag fusions to follow their molecular dynamics on live cells. Working with quantum dot can be extremely hard to dust, so don't forget to wear safety goggle gloves and lab coats while performing this procedure.