Monitoring Protein Adsorption with Solid-state Nanopores

Abstract

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids [1][2][3][4][5][6] , the unfolding of proteins 7 , and binding affinity of different ligands 8 . By coupling these nanopores to the resistive-pulse technique [9][10][11][12] , such measurements can be done under a wide variety of conditions and without the need for labeling 3 . In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13 . Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy. Video LinkThe video component of this article can be found at http://www.jove.com/video/3560/ Protocol 1. Manufacture of solid-state nanopores in silicon nitride membranes 1. Bring the FEI Tecnai F20 S/TEM to an acceleration voltage of 200 kV. If using a different S/TEM, the acceleration voltage should be greater than or equal to 200 kV 9 2. Load a 20 nm thick SPI silicon nitride window grid into the TEM sample holder and clean with Oxygen plasma for 30 seconds to remove any contaminants from the holder. 3. Load the sample into the S/TEM and allow for the vacuum to pump down. Once the S/TEM has pumped down to vacuum, find the nitride window in bright-field TEM mode by looking for a bright square on the Ronchigram. Make sure the TEM is aligned properly, then align and focus the sample. 4. Switch the S/TEM into STEM mode. Use the HAADF (or equivalent) detector to image the sample and make sure it is properly aligned. 5. Set the Monochromator to a low value. A lower value of the Monochromator allows for a higher current of electrons available for drilling. If the S/TEM does not have a Monochromator, ignore this step. 6. Select an appropriate spot size for drilling the nanopore. This corresponds to the diameter of the electron probe. A 3 nm probe works well for drilling 5 nm diameter pores. A larger probe (5 nm) will drill more quickly and create a larger pore. A smaller probe (1 nm) will take longer to drill and create a smaller pore. 7. Adjust the STEM alignments, if necessary. 8. Focus the nitride membrane a...

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

“…While the protocol described can be applied to solid-state nanopores of various material fabricated using any method, they are commonly drilled by TEM using previously established protocols 11,14 . Nanopores drilled by TEM are typically between 4-8 nm in diameter (Figure 2).…”

“…Even with this treatment, however, nanopores often do not wet or continue to exhibit high noise, and the suggested solution for failed attempts is to perform additional cleaning which can be extremely time consuming 14 . With the application of high electric fields, these lengthy protocols may not be necessary depending on the application.…”

“…Deposition of carbonaceous residues during drilling and imaging can have detrimental effects on the electrical signal quality, often making complete wetting a challenge and causing the formation of nanobubbles that can be difficult to remove 12 . Furthermore, clogging of the nanopore by analyte molecules degrades signal quality rendering pores unusable for further experiment 13,14 . Altogether, these effects greatly reduce yield of functional nanopore devices and increase the cost associated with solid-state nanopore research.…”

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

“…The nanopores used in this study were drilled in 30-nm or 10-nm thick silicon nitride membrane windows. While the protocol described can be applied to solid-state nanopores of various material fabricated using any method, they are commonly drilled by TEM using previously established protocols 11,14 . Nanopores drilled by TEM are typically between 4-8 nm in diameter (Figure 2).…”

“…Deposition of carbonaceous residues during drilling and imaging can have detrimental effects on the electrical signal quality, often making complete wetting a challenge and causing the formation of nanobubbles that can be difficult to remove 12 . Furthermore, clogging of the nanopore by analyte molecules degrades signal quality rendering pores unusable for further experiment 13,14 . Altogether, these effects greatly reduce yield of functional nanopore devices and increase the cost associated with solid-state nanopore research.…”