The overall goal of the following experiment is to detect influenza A virus using a microfluidic chip. First, run the sample with lysis buffer through the SPE channel to lyce the virus and extract RNA load the R-T-P-C-R reagent with extracted RNA and run the reverse transcription channel to convert viral RNA into CDNA. Next, run the mixture into the PCR channel in order to amplify the DNA results are obtained that show the size and concentration of the amplicon based on the fluorescence signal from capillary electrophoresis.
The main advantage of this technology is it accepts the raw patient sample and it integrates nucleic acid extraction, purification, reverse transcription and amplification into a single chip To create two plaques, distribute approximately nine grams of XN X pellets evenly in the center of a metal plate, place on a 198 degrees Celsius heated press for five minutes. Then apply pressure slowly to 2, 500 PSI for another five minutes. Now, position one plaque onto the epoxy mold preheat at 157 degrees Celsius for 10 minutes, and apply pressure slowly to 1000 PSI for another 10 minutes.
Next, wearing thermal gloves, remove the plaque and mold from the hot press, and then remove the plaque from the mold before it cools down. Drill three holes in the embossed chip, one each at the inlet, waste port and the outlet of the microfluidic channel. Then wash the chips with IPA followed by RNAs away and dianna's water.
Dry the chips with air to bond the chips first preheat at 131 degrees Celsius for 10 minutes, and then press at 350 PSI for another 10 minutes. Using JB Weld Epoxy, attach nano ports at the inlet waste and the outlet ports separately cure for 15 to 24 hours. As per the manufacturer's instructions, rinse the SPE channel with 50 microliters of RNAs away followed with 100 microliters of nuclease free water.
Now load the SPE channel with four microliters of freshly prepared grafting solution to cross-link the methacrylate incubate for 10 minutes in a UV oven, remove the residual grafting solution with a vacuum. Break up the dried microsphere pellet by tapping on the tube with the lid closed. Then add 100 microliters of fresh prepared SPE column solution and vortex to mix.
Load four microliters of the SPE solution into the same channel cross-link by UV irradiation for 2.5 minutes on each side, resulting in an opaque white polymerized SPE column. Wash the channel with 500 microliters of 100%methanol to rinse away excess reactants and intra polymer pyrogenic solvents. Attach two thin film heaters to the bottom of the chip with thermally conductive tape.
Note that the sides of the heaters need to be aligned with the edge of the wide channels for Dene duration and a kneeling separately, place five thermocouples into the chip through the dead-ended open side channels of each chip tape. To fix the location of thermocouples, add four microliters of 25 XRNA secure to 96 microliters of 70%ethanol in one tube, and to 100%ethanol in a second tube. Also, prepare 300 microliters of channel buffer and 316 microliters of lysis buffer.
Heat the solutions at 60 degrees Celsius for 15 minutes. To activate the RNA secure, then to equilibrate the microfluidic channel. Run the channel buffer through the SPE channel at a flow rate of 0.8 milliliters per hour.
Now quick fava test sample in VTM at 37 degrees Celsius. Remove the sample as soon as it is thawed. Centrifuge the sample at 13, 000 RPM for 10 minutes.
Transfer 100 microliters of the supernatant into 300 microliters of the lysis buffer, and add six microliters of one microgram per microliter carrier, RNA vortex to mix. Then spin for five seconds and load the entire lysate into a lure lock one CC syringe. Run the lysate through the SPE channel at a flow rate of 0.8 milliliters per hour.
Then wash the SPE channel with 100 microliters of 70%ethanol, followed by 100 microliters of 100%ethanol at one milliliter per hour. Position an empty syringe at the 0.5 milliliter mark and push air through the channel to dry it at one milliliter per hour. Now run 67.5 microliters of nuclease free water through the channel to elute the bound nucleic acids at 0.5 milliliters per hour and collect 13.5 microliters from the nano port at the waste port.
Load 36.5 microliters of the R-T-P-C-R master. Mix in the nano port at the waste port and mix with template RNA for a 50 microliter R-T-P-C-R reaction. Then load the R-T-P-C-R mixture into the RT channel by gentle vacuum.
Seal the nano port with a closed fitting. Apply 35 volts to heater one. Keep the reagents in the RT channel for 30 minutes after heater one equilibrates at 50 degrees Celsius.
Keep the reagents in the RT channel for 15 minutes after heater one equilibrates at 95 degrees Celsius. Next, apply 27 volts to heater one and 50 volts to heater, two to equilibrate heater, one at 60 degrees Celsius and heater two at 95 degrees Celsius. Push the reagents into the PCR channel at 0.5 microliters per minute.
About 20 minutes later when the reagents approached the end of the serpentine channel, collect the PCR products at the outlet using an appropriately sized pipetter and tip for the Agilent high sensitivity DNA test. Remove the Agilent high sensitivity DNA kit from the refrigerator 30 minutes before testing. Turn on the bioanalyzer and open the 2, 100 expert software.
Load one microliter of the PCR products for test and follow the Agilent protocol. Analyze and record the data In this workflow. The patient's nasal pharyngeal sample is mixed with lysis buffer and applied to the chip.
The chip is run and the PCR products of influenza virus are red using a commercial capillary electrophoresis chip. Due to the different amounts of influenza virus in each patient's specimen, the final concentration of PCR product will vary. A good result should have low noise to clear ladder peaks at 35 and 10, 380 base pair and a single product peak at the designed product size of 107 base pair for the positive sample.
After watching this video, you should have a good understanding of how to fabricate and run the micro fluid chip to detect influenza a virus.
