A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

This method can help answer key questions in the combinatorial technique of the microfluidic system and the molecular biotechnology field, such as microfluidic devices and asymmetry PCR method. The main advantages of this technique are efficient generation of highly homogenized microspheres and the production of single-stranded DNA without any additional separation steps. The implications of this technique extend toward the diagnosis or therapy of various diseases.

Because generation of single-strand DNA is key for ap-ta-mo-sis assays, it can be used for detection and treatment. Though this method can provide insight into three-dimensional microfluidic system in MEMS, it can also be applied to other systems used for detection and treatment of disease. Generally, individuals new to this method will struggle to properly control fluids from flowing in the reverse direction.

Visual demonstration of this method is critical to observe the microsphere formation in the flow-focusing geometry and solidification of generated microspheres. First, prepare 20 milliliters of liquid PDMS pre-polymer by mixing base polymer and catalyst in a 10-to-one ratio. Pour 10 milliliters of the liquid PDMS onto a prepared SU-8 mold on a silicon wafer for the upper part of the microfluidic network.

For the bottom flat part, pour the same volume of liquid PDMS on the silicon wafer without a mold structure. Place two silicon wafers coated with liquid PDMS pre-polymer on a preheated hotplate and cure at 75 degrees Celsius for 30 minutes. Following this, manually peel off the cured PDMS layer from the SU-8 mold.

Align a 1.5 millimeter diameter round hole-punching tool to the oil port on the replicated microfluidic network for interfacing micron-scale flow channels with the macro-fluid samples. Then manually punch out the through-hole. After repeating the punching process three times, perform hydrophilic surface treatment on both the upper and bottom PDMS layers using a handheld corona treater for several seconds per sample.

Stack two plasma-treated PDMS layers and heat at 90 degrees Celsius for 30 minutes on a hotplate for the PDMS-to-PDMS bonding process. Vortex and briefly centrifuge a standard single-stranded DNA acrydite-labeled probe and a 19-to-one acrylamide bis stock solution. Prepare solution one by mixing 25 microliters of 40%acrylamide bis solution, 10 microliters of single-stranded DNA, 10 microliters of 5X TBE buffer, and five microliters of water.

Prepare solution two with 50 microliters of 20%ammonium persulfate. Prepare two syringes individually filled with solution one and solution two and mount them onto the syringe pump to introduce solution flows into the microfluidic platform. Prepare mineral oil mixed with 0.4%TEMED for surface solidification of the microsphere.

After filling a glass bottle with the mineral oil, insert two tubes to the pneumatic and fluid ports in the cap of the glass bottle as a microfluidic reservoir. Connect tubes between the glass bottle and the oil port in the microfluidic device. Connect the tubes to the two solution ports in the microfluidic device in order to supply the two solutions from the syringe pumps.

Then insert the tubes to the outlet port in order to transfer the generated microsphere into the beaker. Next, set the flow rate of the syringe pump to 0.4 to 0.7 milliliters per hour. Adjust the compressed air pressure of the compressor using a regulator.

Place a glass beaker with magnetic stir bar on a hotplate and set the rotational speed. Operate the syringe pump and supply the compressed air generated by an external compressor into the glass bottle using the on-off control of an electromagnetic valve. Using a digital microscope, observe the formation of microspheres in the flow-focusing geometry and solidification of generated microspheres in the glass beaker.

Observing the formation of microspheres is one of the most critical steps because it is a major process in this study to produce microspheres and microfluidic system. Following this, transfer 100 microliters of the microspheres to a 1.5 milliliter micro-centrifuge tube. Then remove the supernatant through gentle centrifuging and pipetting.

Resuspend the complimentary probe-modified cyanine-3 using 100 microliters of 1X TE buffer in order to achieve a final concentration of 100 micromolar. Next, add 100 micromolar of complementary probe-modified cyanine-3 to a sterile 1.5 milliliter micro-centrifuge tube containing AP copolymerized microspheres. Tap the tube a few times to mix and incubate at room temperature in the dark for one hour.

Following incubation, discard the supernatant and remove the residual buffer through pipetting. Then rinse three times with 500 microliters of TE buffer. Place the microspheres on a 75-by-50 millimeter glass slide and cover with aluminum foil prior to imaging.

Obtain approximately 25 microspheres through microscopic counting using a light microscope with 40X magnification. Following this, combine all reagents as described in the text protocol. Place the samples into a thermocycler and start the asymmetric PCR under the appropriate conditions.

The collection of a supernatant after asymmetric PCR is one of the most critical step because it is a major process for generating single-strand DNA with microsphere. Following amplification, add eight microliters of 6X loading buffer to each sample. Load 15 microliters of each sample into 2%agarose gel.

Then perform electrophoresis at 100 volts for 35 minutes in 1X TAE buffer. Place the hybridized microspheres in a holder and attach the holder to the stage of a confocal microscope. Select the laser and turn it on in the laser control.

Select the objective lens in the microscope control. Then select the desired filter for cyanine-3 and channel in the configuration control. Finally, start the experiment and observe the sample.

The fabricated polymeric droplet-based microfluidic platform consists of two PDMS layers. Three kinds of microfluidic channel networks are used for generating microspheres, which are flow-focusing geometry, a serpentine channel for mixing solutions one and two, and a polymerization channel for microsphere solidification. A lab-based pneumatic control system for continuous flow of the mineral oil and two syringe pumps for the solution flow were used for generating microspheres in the microfluidic platform.

If the microspheres are correctly functionalized with the five-prime acrydite DNA probe, they should result in coverage of fluorescent activity on the surface during the hybridization experiment as shown in these fluorescent images. If copolymerization does not occur correctly, the optical microsphere image would exhibit internal contamination inside the microspheres. Manipulating the 3D surface of microspheres with a DNA oligo-probe is a much faster process and an on-flow microsphere synthesis platform can provide a tool for single-stranded DNA amplification and purification.

The initially copolymerized five-prime AP provides a three-prime hydroxyl end for DNA polymerization after annealing to its complementary template. Double-stranded DNA contaminants were observed in the asymmetric PCR experiment. The resulting single-stranded DNA accumulated from microsphere PCR was demonstrated by comparison to the synthetic size markers through gel electrophoresis analysis.

While attempting this procedure, it's important to remember that prior knowledge of protocols and adjustments that are commonly necessary for optimizing PCR experiments are required for obtaining the amplification cycle, annealing temperature, and probe sequence in this method. After its development, this technique paved the way for researchers in the field of diagnosis and therapy to connecting two regions in molecular biotechnology and biomedicine. Don't forget that working with acrylamide solution can be hazardous and precautions such as wearing gloves and lab coats should always be taken while performing this procedure.