Clinical proteomics is an important new discipline promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. Here we report a new digital microfluidics or DMF method integrating several processing steps used in clinical proteomics including protein extraction, re solubilization reduction, alkylation and enzymatic digestion. DMF uses discrete micro droplets of sample proteins on an array of electrodes and can be run at room temperature, allowing parallel automated analysis of samples.
This methodology represents a significant advance over conventional methods and has the potential to be a useful new tool in clinical proteomics. Hi, I'm Steve Sche from Laboratory Professor Erin Wheeler in the Department of Chemistry and Institute of Biomaterials and Biomedical Engineering at the University of Toronto. Hi, I am is Gabriel from the Wheeler Lab at the University of Toronto.
And I'm Vivian Luke, also from the Wearer lab at the University of Toronto. I'm Erin Wheeler. I'm fortunate to work with Steve Mace and Vivian.
Today we'll show you a procedure for proteomic processing using digital microfluidics. We use this procedure In our laboratory to study proteins and biological samples. So let's get started.
Start the procedure in the fume hood by cleaning the glass substrates in Piranha solution. While wearing adequate protection, piranha solution is composed of three to one concentrated sulfuric acid to 30%hydrogen peroxide. So care should be taken while working with it.
Incubate the glass substrates in piranha for 10 minutes and after cleaning, rinse them and deionized or de water and dry the substrates with nitrogen gas. Place the substrates inside the electron beam evaporation chamber for chromium deposition to a thickness of 250 nanometers. Remove substrates from the chamber once this thickness is achieved and rinse with isopropanol, then dry on a hot plate for five minutes at 115 degrees Celsius.
Prime the dried substrates with hexa methyl xylazine or HMDS by spin coating at 30 seconds. 3000 RPM spin code again with Shipley S 1811 photo. Resist using identical parameters.
Pre-bake the substrate directly on a hot plate at 100 degrees Celsius minutes. Then pattern the photo resist by exposure to ultraviolet or UV irradiation for five seconds through a photo mask. Next, develop the UV exposed substrates in Shipley MF 3 21 developer Enough to submerge the substrate for three minutes after.
Rinse the substrate in DI water post. Bake directly on a hot plate at 100 degree Celsius for one minute. Etch the exposed chromium by immersing the substrates for 30 seconds in just enough chromium etching to completely cover them.
Rinse the substrates with DI water and immerse in a Z 300 T stripper enough to submerge the substrate for 10 minutes. To remove the remaining photoresist. Rinse in DI water and dry in nitrogen gas.
Following this deposit, two to five micrometer Paraline C.An insulating polymer onto the substrate by chemical vapor deposition deposit 50 nanometers of Teflon AF to make the surface hydrophobic by spin coating a solution. 1%weight per weight in flora nerve FC 40 at 2000 RPM for 60 seconds. Onto the substrate.
Repeat with unpassed indium tin oxide or ITO glass substrate to create the top plate post. Bake both substrates on a hot plate at 160 degrees Celsius. 10 minutes to set up the digital microfluidic device.
Remove polymer coatings from the contact pads of a bottom substrate by scraping with a scalpel. Couple the exposed pads of the bottom substrate with 40 pin connectors power on a computer running lab view. We use a home-built control box that contains relays with signals controlled by dpad.
The computer control box facilitates user control over application of 100 volts RMS per 18 kilohertz signals to the device via the 40 pin connectors. Also power on a function generator and amplifier. Assemble the device by positioning two pieces of double-sided tape of 140 micrometer total thickness on the edges of the bottom substrate.
Pipette four microliters of DI water into one of the reservoirs and enclose the device by positioning the un patterned indium tin oxide slide on top of the device with the Teflon coated side facing down. Attach a ground connector to the top plate run. The lab created initialization program in lab view to calibrate the feedback control the device is now ready for experiments.
In this protocol, we will use bovine serum albumin or BSA as a protein sample to demonstrate our digital microfluidics design and usage. BSA is diluted in working buffer or WB made of 100 millimolar tris HCL pH 7.8 with 0.08%onic F1 27 weight per volume. Prepare one milliliter of each sample and reagent solution and denote the reservoir into which it will be added.
Following reagent preparation, remove the top plate from the device and pipette four microliters of solution into its assigned reservoir. After filling the reservoirs, replace the top plate onto the device. Use the in-house lab view program to execute the following steps.
Remember, the computer interface allows the user to dispense about 600 nanoliter droplets from reservoirs and manipulate the droplets on the array of electrodes. First, dispense a droplet of protein containing sample from R one and a droplet of precipitant from R two. Merge the two droplets and allow the combined droplet to incubate for five minutes so that proteins precipitate onto the surface.
Actuate the supernatant away from the precipitated protein to the waste reservoir. R three, to wash the precipitated protein, dispense three droplets of wash buffer from R four and drive them across the precipitated protein and into the waste reservoir. Allow the precipitate to dry and dispense a droplet of re solubilizing buffer from R five onto the protein.
Allow the buffer to incubate for 20 minutes until the precipitate has dissolved. Next, dispense a droplet of reducing agent from R six and merge it with the sample droplet. Mix the combined droplet by actuating it across six electrodes in a circular pattern.
Allow the droplet to incubate for one hour at room temperature. Dispense a droplet of alkylating agent from R seven and merge it with the sample droplet, followed by mixing. Allow the droplet to incubate for 15 minutes at room temperature protected from light.
Finally, dispense a droplet of trypsin from R eight and merge it with the sample droplet, followed by mixing. Allow the droplet to incubate for three hours at 37 degrees Celsius in a Petri dish on a hot plate. To end the reaction, remove the top plate and quench digestion by pipetting.
Point six milliliters of 2.5%trichloroacetic acid in water onto the reaction droplet. Purify the quenched reaction product by siphoning up the sample droplet directly from the bottom plate. Using a C 18 zip tip, according to manufacturer's instructions, samples can now be diluted as needed and evaluated by mass spectrometry to identify the proteins in the sample Using this system.
Followed by mass spec proteins were identified with probability scores of less than one times E to the negative three, equivalent to a 99.9%confidence interval and sequence coverages of at least 30%Hence, DMF is a very useful methodology for automated proteomic sample processing. So we've been just showing today how we utilize digital microfluidic to extract and to process proteins in an automated fashion. This methodology Provides a potential solution for sample loss and contamination during protein isolation and identification.
We hope to apply this method in the future to other biological applications, including immunoassays and cell-based assays. When doing this type of experiment, the most important thing is to remember to enjoy it. So that's it.
Thanks for watching and good luck with your experiments.
