The overall goal of this procedure is to create a modular protein array that does not require a purification step, making it compatible with any protein library. This is accomplished by first generating synthetic genes through assembly PCR. Next, the synthetic genes are arrayed on epoxy slides.
To fabricate the microfluidic device use PDMS with silicone control and flow molds. Then the microfluidic device is aligned to the spotted DNA array. Finally, rabbit reticulocyte lysate is flowed into the microfluidic device and proteins are expressed.
Ultimately, results can be obtained that show expression of thousands of proteins through fluorescent antibody labeling. There are several advantages for using the microfluidic based technique over existing methods such as protein microarrays. We microarray DNA and then use cellis to make proteins on the device.
Thus, we do not require protein purification and the proteins never dry. They remain fresh throughout the experiment. The microfluidic technique also provides higher sensitivity To fabricate the microfluidic device, expose the silicone control and flow molds to chloro trimethyl silene vapor for 10 minutes to promote elastomer release.
After the baking steps, prepare a mixture of silicone based elastomer and curing agent in two different ratios of five to one and 20 to one for the control and flow molds respectively. Pour the five to one PDMS onto the control layer. Degas the control layer and bake it for 30 minutes at 80 degrees Celsius.
Next spin coat the 20 to one PDMS mixture on the flow layer at 2, 600 RPM for 60 seconds, and then bake it at 80 degrees Celsius for 30 minutes. Slowly separate the control layer from the mold, being careful not to peel off the SU eight pattern. Then using a scalpel, cut the device around its perimeter and use a blunt needle to punch holes to access the control channels under a microscope.
Manually align the flow and control layers beginning with the upper left corner and positioning the button valve on the control layer in the middle of the reaction chamber. Next, align the first row and gently release the control layer row by row. Ensure that all the button valves are in the middle of the reaction chambers and that the address and input valves cross the flow channels at the correct position.
Repeat the process locally until all the rows are aligned. When fully aligned, release any tension in the PDMS by carefully lifting it from the sides and corners. Then bake the device for two hours at 80 degrees Celsius after baking.
Cut around the perimeter and peel the two layer device from the flow mold punch holes to access the flow channels to generate DNA constructs for the device. Carry out PCR to produce synthetic genes composed of a T seven promoter, a ribosome binding site, an open reading frame with different epitope tags at either end and a T seven terminator. To prepare the synthetic genes for arraying, make a mixture of polyethylene glycol and D triose dihydrate Dispense two microliters per reaction into the wells of a 384.
Well plate. Add the synthetic genes into a 384 well plate. Then add distilled water to a final volume of 20 microliters.
Next, using a microarray spot, a series of synthetic genes onto epoxy coated glass substrates. Under a stereoscope, manually align the microfluidic device to the gene array with the DNA spot positioned in the middle of the DNA chambers. Then bring into line the rest of the rows.
Finishing with fine tuning of the whole device to alleviate any stress on the PDMS and ensure it bonds well to the microarray. Lift it locally. Finally, bond the device to the glass slide by incubating it overnight on a hot plate at 80 degrees Celsius.
Food dyes are used in this section for visualization purposes. The valves in the control layer are actuated from the computer using lab view. The lab view script controls a series of solenoid micro valves via an electronic control box.
The solenoid valve manifold is connected to compressed air, which controls the airflow and pressure into the control valves on the device. Using a flexible plastic tubing with internal diameter of 0.02 inches and a stainless steel pin, connect the device to the solenoid valves manifold. Fill the tubes with double distilled water and insert the pin into the access holes of the control layer.
Make sure that each tube is connected to its corresponding control channel. To activate the control channels, run the lab view application, set the air pressure to five PS.I then apply air pressure by activating the valve from the lab view script. This will push water into the PDMS control channels to identify potential crosstalk between control channels of defective devices.
Fill the sandwich and the address valves first. After all, the control channels and valves are full of water, prime the valves to block the flow channels underneath them. By first increasing the air pressure to 15 PSI then in the lab view program through a set of on off switches connected to individual solenoid valves.
Activate all the valves by turning on all the switch buttons to ensure that all the valves are open. Connect a tube to one of the flow inputs, and at four to five PSI flow air into the device. This will release any sticky valves from the glass slide.
The device is now primed and ready for visualization purposes. Yellow food dye is used to visualize the reagents in this section and the colorless solution represents the hippies to facilitate the self-assembly of a protein array on the surface and prevent non-specific absorption within the microfluidic device, chemically modify the surface by first connecting a new tube with the required solution to one of the flow channels in the device. Then connect the free side of the tube to the manual manifold and open the air pressure flow.
Load 40 microliters of biotin elated BSA in a new tube and flow approximately half of it through the device for 20 minutes. Use 50 millimolar hippies to wash away any unreactive substrate between each of the steps. Next, flow 25 microliters of streptavidin for 20 minutes.
On top of the biotin elated BSA wash with hees for five minutes to ate the surface surrounding the button, close the button valve and flow the rest of the biotinylated BSA, and then wash with he pea again for five minutes. Finally, to create an anti hiss tag array under the button, release the button valve and flow 30 microliters of Penta hiss biotin for 20 minutes to create an array of proteins on the device. Open the neck valves and flow rabbit reticulocyte.
Quick coupled transcription and translation reaction solution through the device into the DNA chamber. Next, close the sandwich valves to separate each gene from its environment and incubate the device on a hot plate for 2.5 hours at 30 degrees Celsius. Then label the end terminus of the proteins with C mix SI three antibody.
Finally, using a microarray scanner with a 532 nanometer laser and a 575 nanometer emission filter. Determine the protein levels. This figure shows the varying levels of protein expression on a protein array.
Usually 20%of a gene library fails to express to detectable levels. Background levels were determined using chambers that were not spotted with DNA. Thus, signals from the corresponding protein chambers are due to either noise or non-specific absorption of the labeling antibodies.
If it's performed properly, that device verification takes four hours and the chip experiment take five hours.
