This procedure begins by fabricating a micro electrode on a silicone substrate. A polyethylene glycol self-assembled monolayer is formed over the micro electrode and substrate to prevent fouling of microtubules. Then rumine labeled microtubules are prepared.
Finally, microtubules are patterned onto the ated electrodes using electrophoresis. Hi, I am John Noel from the Nana Lab in the Department of Physics and from the Molecular Biomechanics Laboratory in the Department of Biomedical Engineering at Texas a and m University. I'm Wil free Teza from the physics department and the director of the Center Skill Science and Technology at Texas a m University.
And I'm Juan Quang, the director of the Molecular Biomechanics Laboratory at Texas a and m University Department of Biomedical Engineering. Today we'll show you a procedure for fabricating micro electrodes coated with a non fouling self-assembled monolayer. We use this procedure in our laboratory to study micro tube paling using electrophoresis.
So let's get started. To begin, use a scribe or wafer cutter to cut silicon wafers into one centimeter by 1.5 centimeter pieces to use as substrates. Now clean the substrates by placing them in a beaker with enough acetone to cover them and sonicate for five minutes without allowing the substrates to dry.
Rinse them in fresh acetone and then immediately rinse them again in isopropanol and use nitrogen to dry them. Using standard positive tone photolithography or electron beam lithography, fabricate the gold micro electrodes on the clean substrates. When designing the pattern geometry, keep the diameter of each electrode pattern below 100 microns To ensure coverage by the polyethylene glycol trimeth oxy seline self-assembled monolayer or peg Sam, include contact pads positioned 300 to 500 micrometers from the pattern with a one millimeter line connecting the pad with the electrode.
After development of the pattern in the resist layer, use tweezers to scratch a line in the resist from the contact pad to the edge of the substrate. Upon metal deposition, the scratch will allow electrical contact between the electrode and the edge of the substrate. A deposition chamber, preferably with two sources, is used to deposit the metal layer.
The electrodes are formed by deposition of two nanometers of chromium, followed by six nanometers of gold Without breaking vacuum, a quartz crystal micro balance equipped inside the chamber can be used to measure the thickness of the films during deposition. We can now begin to prepare the self-assembled monolayer in a well-ventilated area, preheat and oven to 75 degrees Celsius. Prepare the ization solution by adding one milliliter of peg seline to 20 milliliters of toluene in a 250 milliliter glass beaker.
Mix well by pipetting up and down five to 10 times and then stirring with the pipette for 30 seconds. Place the pattern samples in the beaker pattern side up, making sure that they're completely submerged in the solution and are evenly spaced across the bottom of the beaker. Place the beaker in the oven and bake it for 18 to 21 hours while the Sam is forming.
Prepare the counter electrode by depositing one nanometer of chromium followed by four nanometers of gold onto a 22 by 50 millimeter glass cover slip. The counter electrode should be nearly transparent with a light gray or pink tint. Store the counter electrode coated side up in a Petri dish to keep it clean.
After 18 to 21 hours in the oven, remove the beaker from the oven. The toluene should have evaporated leaving the pattern samples in a viscous peg cline residue. Add 20 milliliters of toluene to the beaker and soak the samples for one to two minutes.
Remove the samples from the beaker and rinse them with toluene, then isopropanol and finally dry them with nitrogen. At this point, the samples should look clean and the Sam layer should be invisible to the naked eye. Sam's prepared this way are usable for up to two days when stored in cool dry conditions.
Unsuccessful Sam formation results in a cloudy or uneven hued residue coating on the surface. With the Sams prepared, we can now begin polymerization of tubulin into microtubules. Start by thawing unlabeled tubulin and r domine tubulin, and immediately combine them at a ratio of two to one unlabeled to labeled to obtain bright microtubules.
Mix them well by pipetting up and down several times. Next, polymerize the tubulin for 20 to 40 minutes in a heated water bath at 37 degrees Celsius while the tubulin is polymerizing. Prepare a pipes Taxol buffer by mixing 20 micromolar Taxol with pipee buffer.
Mix well by pipetting up and down several times and vortexing, and then place the tube into the heated bath at 37 degrees Celsius. After polymerization, dilute the microtubules in warm pipee Taxol buffer and shield them from light. Verify that the micro tubule polymerization was successful and that the microtubules are stable under several minutes of fluorescence excitation.
By imaging the prepared microtubules with an oxygen scavenging mixture with polymerization of tubulin into microtubules complete. Let's begin patterning the MTS with electrophoresis. To begin, cut a piece of double-sided tape into thin strips by placing it on a glass slide and using a razor blade to cut place two strips of double-sided tape across the patterned Sam coated sample, such that the electrode is between the strips with the connecting path running perpendicular to the tape.
Place the counter electrode on the double-sided tape with the metal side touching the tape such that the edge of the sample overhangs the counter electrode by about three millimeters. Cut several 15 centimeter lengths of insulated copper magnet wire and strip one centimeter of the insulation off of both ends. Use a small drop of silver paint to attach a wire to the exposed gold area of the substrate while taking care to avoid making electrical contact with the counter electrode.
Now use silver paint to attach a second wire to the counter. Electrode wires attached with silver paint can be further secured to the samples with tape. Next, using a pipette, introduce microtubules at the desired dilution image at 20 x magnification and locate the pattern on the silicon wafer before applying a voltage.
Because of the depth of the flow chamber, long working distance objectives may be needed to image the upper surface of the flow chamber. The distance from the sample surface to the upper surface of the flow chamber is determined by the thickness of the tape and should be around 60 to 100 micrometers. Apply a voltage of up to one volt and observe the migration of microtubules.
If reversible trapping of microtubules is desired. Use nail polish to seal the flow chamber to prevent lateral drift and use lower voltages down to about 0.5 volts. Migration can be tracked by adjusting the vertical plane of focus of the microscope with the applied voltages around 0.9 to 1.2 volts microtubules localize at the electrode surfaces.
Higher voltage will make the migration faster and adhesion stronger, but excessive voltage will induce electrolysis and depolymerize. Microtubules, We've just shown you how to prepare Sam coated micro electrodes for MT patterning because the SAM coating prevents micro tubial absorption on the micro electrodes. This procedure can also be used for reversible patterning of microtubules.
Furthermore, this process is, is not just limited to microtubules, but can also be used to pattern biomolecules in general. So that's it. Thanks for watching and good luck with your experiments.
