The overall goal of this methodology is to prepare a solid state nabor for biomolecular sensing experiments by precisely controlling its size and reducing its ionic current noise characteristics. This is achieved by first mounting a solid state nanopore chip in a fluid cell to establish a nanometer size channel between two electrolyte reservoirs As a second step, a moderate electric field of approximately 0.01 volts per nanometer is applied across the nanopore to characterize its initial size and electrical noise properties. Next high electric fields of approximately 0.3 volts per nanometer are applied across the membrane in short pulses.
In order to completely wet the Nanopore reduce electrical noise and enlarge it to a desired size and discrete sub nanometer steps. Results are obtained that show that a fully wet, functional, low noise nanopore is produced based on analysis of the ionic current signal and successful translocation of biomolecules. The main advantage of this technique over existing methods for controlling the size of the nanopore, like using a transmission or scanning electron microscope, is that as performed in a salt solution using standard laboratory equipment in the same setup used for biomolecular detection.
This ensures precise control of the actual nanopore size during biomolecular sensing experiments. The procedure also reduces low frequency electrical noise and allows for clogged nanopores to be rejuvenated for further experiments. This video assumes a nanopore chip has already been mounted in a nanopore cell.
The next step is to prepare the cell for experimentation. To do this place silver, silver chloride electrodes in each reservoir. Then connect a low noise current amplifier between the reservoirs of the cell.
Put the entire nanopore cell assembly in an electrically shielded enclosure. Switch the low noise current amplifier to external command mode. To allow for automated computer control, the computer should sweep the applied potential between minus 200 millivolts and 200 millivolts and record The IV characteristics fit the IV curve to obtain the nanopore conductance in diameter for use In checking consistency lower than expected conductance seen in this example or asymmetry in the IV curve shown here.
Indicate an incompletely wet or clogged nanopore in need of conditioning. To continue with characterization, apply a 200 millivolt potential across the nanopore and record the ionic current for 30 seconds. This portion of a trace from an unconditioned Nanopore exhibits a high degree of electrical noise that makes the sample unsuitable for biomolecular sensing experiments for all measurements of the ionic current.
Quantify the low frequency noise characteristics with a power spectral density analysis. The high noise at low frequency seen here is characteristic of incompletely wet or clogged nanopores. Data from characterization may indicate that a nanopore requires conditioning.
To do this disconnect the electrodes from the low noise current amplifier. Connect one of the leads to a computer controlled power supply capable of an output voltage greater than six volts. Connect the ground electrode to an external current amplifier to test the setup.
Apply a potential difference of 400 millivolts across the nanopore for at least five seconds and measure the current, use the final one second of data to check consistency with IV curve measurements. Next, apply a 200 millisecond pulse of six volts across the nanopore. Follow this with a five second measurement period at 400 millivolts and use the last second of data to find the diameter.
Repeat this sequence as necessary until the nanopore diameter is consistent with TEM images acquired. Before mounting the nanopore or conductance measurements prior to Nanopore clogging nanopores can be enlarged to a desired size by applying longer duration pulses and or higher potentials in the conditioning setup for a 30 nanometer thick membrane. Begin by applying a 400 millivolt bias voltage across the nanopore for at least five seconds.
To provide data to estimate the nanopore size. Then apply a two second pulse of eight volts across it. Follow this with a measurement period of at least five seconds at 400 millivolts.
To determine the increased diameter cyclically, repeat the two second eight volt enlargement pulse and the five second 400 millivolt measurement steps until the desired pore size is attained. Once the target diameter is achieved, disconnect the power supply and the current amplifier. Reconnect the low noise current amplifier to the electrodes.
Acquire new IV and current trace data to confirm the diameter of the nanopore and verify low noise ionic current characteristics as seen here in comparison to the unmet and asymmetric nanopores. The IV characteristics of the enlarged nanopore exhibit a significant increase in conductance and ideal omic behavior. Here our typical conductance traces of a 10 nanometer nanopore and a 30 nanometer thick membrane before and after treatment with high electric fields.
Note the break in the conductance scale. The PO conductance shown in blue is considerably less than expected for a 10 nanometer pour, this is most likely due to incomplete wetting. After 92 second pulses of eight volts, the nanopore is fully wet and has been enlarged to 21 nanometers in diameter.
Also, the conductance is stable with little low frequency noise. A power spectral density plot shows that the low frequency noise amplitude of the unconditioned pour in blue is very high, rendering it unusable for single molecule experiments. Upon conditioning with high electric fields.
Noise power at frequencies below 10 to the four hertz is diminished by up to three ods of magnitude, making it ready for sensitive biomolecular. Sensing experiments for DNA translocation studies. Make sure the low noise current amplifier is connected to the electrodes.
Prior to adding a biomolecular sample to the reservoir. Apply a potential of positive 150 to positive 300 millivolts for at least two minutes. View the current trace and check for ionic current blockades, which are evidence of contamination.
No blockades should be present as in this trace. Access the chip and add 48.5 KILOBASE paired double stranded lambda DNA to one side of the nanopore so that its final concentration is 0.5 to two nanograms per microliter reflux gently by pipetting for at least 10 seconds to ensure homogeneous distribution of the sample throughout the reservoir. Once the DNA has been added, return the chip to the shielded enclosure.
For a 30 nanometer thick nanopore, apply a potential bias of 150 to 300 millivolts to electrophoretic Drive negatively charged DNA through the Nanopore. Use software to monitor the ionic current through the nanopore and detect transient current blockades. Due to DNA translocation, the ionic current traces can be analyzed to infer more information about the DNA sample.
Nanopores treated with high electric fields are fully functional as shown in these conductance traces showing the detection of individual lambda DNA molecules in the top trace. The double stranded DNA is driven through a nanopore that was enlarged to 32 nanometers. The bottom trace is for DNA driven through a nanopore enlarge to 11 nanometers.
Note the break in the conductance scale. In each case, the baseline conductance is extremely stable and pristine. Conductance blockades are observed as single molecules translocate through the Nanopore.
The insects show multiple discrete blockage levels as individual folded molecules. Translocate the low frequency noise properties of the nanopores provide easily resolved peaks in these histograms of the nanopore conductance for both the 32 nanometer pour above, and the 11 pour below three peaks are resolved. The first corresponds to the baseline with no DNA, the second to a single linear strand and the third to folded DNA.
After watching this video, you should have a good understanding of how to prepare solid state nanopores for single molecule sensing experiments. This technique can greatly enhance experimental yields, ultimately making solid state nanopore research more accessible and reliable for a wide range of single molecule studies.
