The overall goal of this procedure is to create nanopores in artificial membranes for use in protein absorption studies. This is accomplished by drilling nanopores in a nitride membrane using a stem. Then the nanopore is loaded into a chamber and it is wet with an ionic solution.
So that fluid forms a bridge from one side of the nanopore to the other to make the nanopore ready for experiments. A voltage is applied across it and a current of ions can be observed to pass through it. The final step is adding protein to the Nanopore chamber.
Ultimately observing long-lived blockades in the ionic current across the nanopore reveals the absorption of proteins to the sides of the Nanopore. Hi, I am Dave Wicki. I'm a graduate student in the Department of Physics at Syracuse University.
Though this method can provide insight into the absorption of proteins on inorganic surfaces. It can also be applied to other systems such as protein, nucleic acid interactions, single molecule for spectroscopy, and the study of molecules in confined spaces. Begin by bringing up the stem to an acceleration voltage.
Next load, an SPI silicon nitride window grid into the TEM sample holder. Clean the grid with oxygen plasma for 30 seconds to clear debris. Before loading the sample, load the sample holder in the TEM and allow for the vacuum to pump down.
Using the brightfield TEM mode, find the nitride window, a bright square on the raw rogram. Make sure the TEM is aligned properly. Then align and focus the sample.
Now put the system in STEM mode and use a hot off detector to image the sample and check its alignment. Set the monochromator if present to a low value, allowing for a higher current of electrons available for drilling. Select the appropriate spot size to drill the Nanopore based on the diameter of the electron probe.
With the stem aligned. Use the BROGRAM to perfectly focus on the nitride membrane at the required magnification. If there is any movement of the sample, blank, the electron beam and wait for the sample to stabilize.
This can take up to an hour. Once stabilized, place the electron probe on the sample and begin drilling the nanopore. Drilling a nanopore with a TM requires patient infidelity.
It's a process in which nitride is gradually dissipated under an electron beam probe. It's important to keep the electron probe directly above the drilling site and to confirm that the nanopore is completely drilled. Before removing the sample from the TEM, Periodically check the sample by scanning with the hot off detector.
If the sample drifts slightly, adjust the position of the electron probe to be over the nanopore. With the ROI gram in focus, the shimmering nitride film will disappear. When the nanopore forms with the roky gram out of focus, the fray nail fringe is visible.
Image the nanopore with the hot off and make an intensity profile of the image. The diameter of the nanopore can be estimated by the darkest region of the profile. The electron probe can be used to enlarge the nanopore if needed.
When it has the required diameter. Switch to TEM mode with the monochromator in the standard setting. Use brightfield TEM to confirm the pore size.
Begin by preparing degas di saline. Put the solution under vacuum and in a bath sonicate for 20 minutes Delicately, place the fragile Nanopore containing silicon nitride Chip into a 10 milliliter Pyrex beak. Then in a fume hood, place the beaker on a hot plate.
Very carefully clean the Nanopore chip with piranha solution. First, add three milliliters sulfuric acid to the container using a glass pipette. Second, carefully add one milliliter high hydrogen peroxide to the sulfuric acid.
Please take all precautions. Allow the nanopore chip to soak for 10 minutes. Then using a glass pipette, remove the piranha solution from the beaker and dispose of it properly.
Next, fill the beaker with the Degas deionized water. Using a clean glass pipette, empty the beaker of water and repeat this washing at least five times. With clean tweezers, remove the nanopore chip and dry it by light suction.
Once dry, immediately seal the nanopore chip into the chamber. The chip may be secured in the chamber by O-rings or silicone sealant. Fill the chamber with potassium chloride solution.
Connect the silver, silver chloride electrodes to the chamber. Electrodes may be made by soaking silver wire in bleach overnight. Apply a transmembrane potential across the electrodes and monitor the current response using a patch clamp amplifier.
In the voltage clamp mode, construct a current voltage curve from the measurements. The curve should be highly linear. When using one molar potassium chloride, the conductance of the nanopore should correspond to its diameter.
If the nanopore does not display a linear IV curve, the conductance is too low or the open current of the nanopore is not stable. This indicates the nanopore is not properly wedded and piha washing should be repeated. Making sure that fluid is inside the nanopore is essential.
If this step is not done properly, ion current through the nanopore will exhibit very large noise and the partitioning of proteins into the nanopore will be drastically reduced. Begin by adding a purified sample of the protein of interest into the grounded bath of the chamber. Wait for the sample to thoroughly diffuse.
Apply a transmembrane potential voltage bias with polarity opposite to that of the total charge of the protein being studied. An applied potential of 200 millivolts should be large enough to observe. Most protein analytes events will appear as transient current deflections from the baseline.
If no signal is observed, increase the concentration of the protein in the chamber. Higher voltages improve the signal to noise ratio and nitride. Nanopores can withstand several volts.
Protein absorptions will appear as long-lived changes in the baseline current of the Nanopore. Typical results for solid state nanopores will be as follows. The open pore current should be highly stable.
The IV characteristics of the nanopore should be highly linear. In one molar potassium chloride, 10 molar potassium phosphate pH 7.4, the slope of a linear fit to the IV curve will provide the unitary conductance of the nanopore. The conductance has a direct relationship to the nanopore diameter and should match the equation.
This value should match to within 30%If the value is too small, the pore is likely not wedded with addition of protein. Rapid events should ensue protein absorption is a long-lived current drop. Some proteins are highly labile and undergoes structural transformation within the pore interior.
In this case, the long-lived voltage drop will be accompanied by rapid fluctuations. After watching this video, you should have a good understanding of how to manufacture solid-state nanopores using a scanning transmission electron microscope, and using those poised SA protein absorption. Don't forget that working with piranha solution can be extremely hazardous and precautions such as working in a fume hood with gloves, A lab coat and a chemical apron should always be taken when performing this procedure.
Thanks for watching and good luck with your experiments.
