Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

This procedure begins with labeling the DNA and polymer to generate a quantum dots or QD fret pear plasma DNA is conjugated with quantum dots. The energy donor and the onic polymer is labeled with an organic flora four sci five. The energy acceptor.

Upon mixing QD fret, nano complexes are formed via electrostatic self-assembly and QD mediated fret signal can then be followed as the interaction takes place. This QD fret system is introduced into a microfluidic channel where the components will only interact through diffusion on the mixing interface. The DNA nano complex self-assembly can then be monitored by the combination of QD fret and microfluidics.

Hi, I'm Megan Ho from the laboratory of Bios and single molecule dynamics in the Department of Mechanical Engineering at the Johns Hopkins University. Today we'll show you a procedure for combining quantum threat and microfluidics to monitor DNA nail self-assembly in real time. In our lab, we use this procedure to study DNNL complexes for location of gene therapy.

So let's get started. A biotin labeling kit from MIRIs bio is used to covalently biotin plasma DNA with guanine specific biotin labels, but scaled to have about one to two biotin labels per DNA. To begin, dissolve the desired amount of plasma DNA into TE buffer to make a one microgram per microliter DNA solution.

Set up the labeling reaction by mixing the plasma D-N-A-D-N-A free and RNA free water 10 x labeling buffer A and the label it reagent. The label it reagent is added last. Incubate the reaction at 37 degrees Celsius for one hour.

After the one hour incubation, purify the labeled sample by ethanol precipitation. Following standard protocols. Store the labeled DNA at minus 20 degrees Celsius until use.

The next step is to label the onic polymer. In this demonstration, we'll use Chitosan as a model onic polymer and label the free primary amines on the chitosan polymer backbone with sci-fi NHS first calculate the required amount of scifi NHS such that the molar ratio of scifi to primary means is one to 200. This will facilitate complete conjugation of the scifi dye.

Add sodium hydroxide to the chitosan solution in 25 millimolar acetate buffer to adjust its pH to about 6.5 while stirring slowly, add the calculated amount of sci-fi NHS to the Chitosan solution in a drop by drop manner. Cover the vial with aluminum foil to keep the mixture in the dark and agitate at room temperature overnight. The next day, purify the mixture by dialysis with a 10 K-M-W-C-O slide from pierce against 1%acetate buffer dialyzed for two hours at room temperature in the dark.

After two hours, replace the 1%acetate buffer and to dialyze for another two hours. At room temperature in the dark, replace the buffer again and dialyze overnight at four degrees Celsius in the dark. The next day, remove the purified labeled polymer from the dialysis cassette.

The SCI five labeled chitosan is then collected in a micro centrifuge tube and store at minus 20 degrees Celsius. Add streptavidin functionalized 6 0 5 dots into the biotinylated plasma DNA solution. Make sure you use the ITK series Q dot stripped have in conjugate as opposed to the regular Q dot series as quantum dots in the ITK series are designed for the purpose of fret.

Incubate the solution at room temperature for 15 minutes. Keep the QD labeling reaction in the dark to prevent possible photo bleaching. After 15 minutes, add the QD labeled DNA into 50 millimolar sodium sulfate solution to make the final volume 200 microliters.

To prepare the sci-fi polymer, calculate the required amount of chitosan for 10 micrograms of plasma DNA according to the desired NP ratio, which is the theoretical ratio of protonated immunes in the chitosan solution to the negative phosphates in the DNA solution dilute the sci-fi chitosan according to the desired NP ratio with Milli Q water to make the final volume 200 microliters the QD labeled DNA and sci-fi chitosan solutions are now ready for loading into the microfluidic device as will be demonstrated in section six. The silicone wafer is prepared beforehand by piranha cleaning and baking to remove moisture on the wafer for the designated master thickness of 25 microns. Spin coat the negative photoresist on the silicone wafer at 2000 RPM for 30 seconds.

Soft bake the wafer on a hot plate with a ramp of 65 degrees Celsius per hour to 95 degrees Celsius. This will evaporate the solvent in the SU eight photo. Resist and solidify it before exposure to UV light.

After the soft baking apply a mask film containing the design of micro channels on the silicon wafer exposed to UV light at 365 nanometers for 250 millijoules per centimeter squared post exposure. Bake the wafer on a hot plate with a ramp of 65 degrees Celsius per hour to 95 degrees Celsius. Develop the wafer using SU eight Photoresist developer to check for complete development.

Immerse the wafer into isopropyl alcohol. The isopropyl alcohol should not turn milky. Finally, to reinforce the SU eight structure hard bake the patterned wafer on a hot plate with a ramp of 65 degrees Celsius per hour to 200 degrees Celsius.

Maintain the wafer at 200 degrees Celsius for at least five hours. Then gradually cool the wafer down to room temperature. Place the SU eight master in a weighing boat.

Mix the silicone elastomer and curing agent poly dimethyl Sloane or PDMS in a 10 to one ratio. Pour the PDMS mixture onto the SU eight master and leave the weighing boat in a vacuum desiccate to remove bubbles. Cure the PDMS at 65 degrees Celsius for one to two hours.

After the PDMS is cured, peel the PDMS strip from the silicone master mold using a whole puncher punch, two channel inlets and one channel outlet for the fluidic device. Clean the PDMS strip and cover glass with ethanol and then air dry. Treat the cleaned PDMS strip and cover glass with oxygen plasma at 20 watts.

For one, immediately bond the PDMS strip with the cover glass. Leave the bonded microfluidic chip in the oven at 95 degrees Celsius Overnight. Plasma treatment and overnight baking are essential to enhance bonding strength.

Before loading the reagents, fill the microfluidic channel with water to ensure there are no bubbles inside. This will ensure smooth flow. During the experiment, load the previously prepared QD, labeled DNA and sci-fi labeled Chitosan solutions into two individual glass syringes through the tubing.

Connect the tubing with the two inlets of the microfluidic device. Be cautious not to introduce any air during the process. Set the flow rate at 20 nanoliters per minute Under laminar flow conditions.

Check the micro channels under the microscope when the flow is stable about 15 to 20 minutes. QD mediated fret should be observed in the center of the channel. Acquire fluorescent images at different locations along the channel with a cooled CCD camera.

Analyze these images with image J and origin lab. Fret mediated signal is monitored at different axial positions along the channel.Residence. Time is calculated based on the applied flow rate using this formula where TISA bar is the residence.

Time X is the distance from where the two streams meet to the position of the reaction under investigation. And V is the mean flow speed. The cross-sectional intensity profiles of fret mediated sci-fi signals can then be analyzed in our system.

The experimentally measured profiles exhibit a Gaussian distribution. We've just shown you how to monitor DNA nail complexes through quantum mediated thread in a simple microfluid channel. When doing these procedures, it's important to remember to design a marker on your microfluid channel so that the residents time can be straight based on the applied flow rate and the channel geometry.

So that's it. Thanks for watching and good luck with your experiment.