This nanopore-based detection method has demonstrated the potential to physically characterize and identify individual molecules. Working with metallo-nanoparticles, which is the purpose of this video, is challenging our understanding of what limits the measurement's accuracy and precision. The main advantage of this technique is that this apparatus has greater bandwidth than traditional lipid bilayer memory set-up, which lets us see faster results and more reactions in nanopore.
This technique was made possible because in the early 1990s it was shown that gating in protein nanopores, that is, switching between different states, can be controlled and molecules can bind to the pore wall. This technique is being developed for commercial DNA-sequencing applications because it uses minimal samples. It doesn't require fluorescent labels and uses much longer read lengths than conventional methods do.
This technique has also been developed to discriminate between polymers based on their size with greater accuracy and speed than gel electrophoresis and chromatography. First, prepare 500 milliliters of a solution of one-molar sodium chloride and 10-millimolar monosodium phosphate in ultrapure water as the electrolyte. It is best to use fresh solutions and samples with this particular set-up.
In addition, we found that high-quality deionized water is very important for successfully forming membranes. Next, combine one milligram of lyophilized wild-type monomeric S.aureus alpha hemolysin powder with one milliliter of ultrapure water. Don't forget that working with Staph aureus alpha hemolysin toxin protein can be hazardous.
Follow the precautions in the MSDS when performing this procedure. Next, prepare four milliliters of five milligrams per milliliter DPhyPC in endecaine in a glass scintillation vial. Cap the vial with a polytetrafluoroethylene-lined cap and store it at four degrees Celsius for up to a month.
After that, dissolve 57.6 milligrams of 12-phosphotungstic acid hydrate in 10 milliliters of the electrolyte to make a two-millimolar polyoxometalate solution. Divide the POM solution into two five-milliliter portions. Use three-molar sodium hydroxide to adjust the pH of one POM solution to 5.5.
To begin preparing for the test, assemble the test cell of a planar lipid bilayer electrophysiology apparatus. First, abrade a silver wire with 600-grit sandpaper and soak it in commercially available sodium hypochlorite-based bleach for 10 minutes to make the silver-silver chloride wire electrode. Rinse and dry the wire electrode before inserting it into the quartz capillary of the apparatus.
Keep the electrode inside the quartz-nanopore membrane, or QNM, separating the capillary and the reservoir. Then place a cylindrical silver-silver chloride pellet electrode mounted on a silver wire in the reservoir. Connect both electrodes to the amplifier circuit board.
Next, open the data acquisition program and start the power supply at zero millivolts. Confirm that no DC current is detected between the electrodes in the empty cell. During this demonstration it's important to watch how the transcapillary pressure can impact membrane formation and subsequent nanopore formation.
Without doing these two steps correctly, the experiments won't work. Load about one milliliter of electrolyte into a syringe and connect it to the fluid line connected to the reservoir. Add electrolyte to the reservoir until the face of the QNM is submerged, as indicated by the ionic current between the electrodes saturating the amplifier.
If the amplifier does not saturate, unclog the QNM by applying a pop voltage of plus or minus one volt or increasing the capillary pressure by about 300 millimeters of mercury. Use both techniques if necessary. Once the amplifier saturates, withdraw electrolyte from the reservoir until the solution levels drops below the QNM, as indicated by the current returning to zero.
To begin the lipid bilayer formation process, raise the electrolyte solution level well above the face of the QNM and note about how much solution that took. Then dip a 10-microliter pipette tip into the DPHyPC solution and draw all visible lipid into the tip. Touch the tip of the pipette to the surface of the electrolyte solution and allow the lipid to flow from the pipette across the air-water interface.
Wait two to five minutes for the lipid to spread uniformly across the solution. Next, slowly withdraw the electrolyte until the solution's surface is below the QNM and then slowly add enough electrolyte to raise the solution above the QNM to form the insulating lipid bilayer membrane. If the bilayer does not form after three attempts, apply another portion of lipid as previously described and repeat the process.
Once the bilayer seems to have formed, increase the pressure to the capillary to pop the bilayer and then reform it by slowly lowering and raising the solution level in the same way as before. Repeat this process several times to ensure that the QNM is not clogged and a bilayer has formed. Next, thaw an aliquot of alpha hemolysin protein on ice or at room temperature.
Fill the reservoir with 250 nanograms of monomeric alpha hemolysin protein and about 250 microliters of ultrapure water. Then, apply a 200 to 400 millivolt bias to induce pore formation. To facilitate pore formation, increase the capillary pressure by 40 to 200 millimeters of mercury to expand the bilayer from the QNM.
Once a nanopore forms, reduce the applied bias to the measurement voltage and reduce the pressure to about half of the insertion pressure. To begin the test, readjust the DC-offset voltage to ensure that there is no measured current when the applied potential is set to zero. Next, acquire an ionic current trace for an applied potential of 120 to negative 120 millivolts relative to the reservoir.
Confirm that there are no contaminants, which would be indicated by spontaneous current blockades. Add one to six microliters of pH 5.5 two-millimolar POM cluster solution to the reservoir. Apply ionic current time-series measurements at an applied voltage of negative 120 millivolts.
The movement of individual anionic phosphotungstic acid molecules through the alpha hemolysin channel produced transient blockades that decreased the mean open-pore ionic current by about 80%The duration and blockade current are both directly related to the particle-pore interaction, allowing ionic species to be differentiated in histograms of the relative blockade depth ratios. At pH 5.5, two peaks were observed with depth ratios of about 0.06 and 0.16. Based on phosphorous NMR, the minor peak was the six-minus phosphotungstate species and the major peak was the seven-minus species.
At pH 7.5, the relative concentrations shifted to a higher ratio of the 6-minus species to the seven-minus species. The 20-fold decrease in blockade events relative to pH 5.5 was attributed to degradation to free phosphate and tungstate ions. Fitting the residence-time distributions required multiple exponential functions, indicating multiple types of particle-pore interactions within each ionic species.
The shorter residence times at pH 7.5 suggested weaker particle-pore interactions than pH 5.5, consistent with prior studies showing pH-dependent changes in the relative number of fixed charges in or near the alpha hemolysin channel lumen. High-purity deionized water is critical for this particular set-up. A spent organics filter cartridge was the likely cause of our lab being unable to form membranes on the QNMs for months.
Individuals new to this particular version of the method might also struggle because the membranes are so small and there is a trick to getting nanopores to form. This method, which was initiated several decades ago, has proven useful for analyzing ions, nucleic acids, and synthetic polymers as well as DNA sequencing. It could also find applications in biophysics and basic cell biology.
For example, this procedure can be used to study the physical properties of synthetic polymers and biopolymers to investigate their size, interactions with other molecules, and other important questions.
