The authors would like to thank John Grazul (Cornell University), Andre Marziali (The University of British Columbia at Vancouver) and Vincent Tabard-Cossa (The University of Ottawa) for their advice. This work is funded in part by grants from the US National Science Foundation (DMR-0706517 and DMR-1006332) and the National Institutes of Health (R01-GM088403). The nanopore drilling was performed at the Electron Microscopy Facility of the Cornell Center for Materials Research (CCMR) with support from the National Science Foundation - Materials Research Science and Engineering Centers (MRSEC) program (DMR 0520404). The preparation of the silicon nitride membranes was performed at the Cornell NanoScale Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS-0335765).

1. Manufacture of solid-state nanopores in silicon nitride membranes
<ol>
 <li>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</li>
 <li>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.</li>
 <li>Load the sample into the S/TEM and allow for the vacuum to pump down. Once the S/T...

<ol>
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Spontaneous adsorption of proteins onto solid-state surfaces 27-29 is fundamentally important in a number of areas, such as biochip applications and design of a new class of functional hybrid biomaterials. Previous studies have shown that proteins adsorbed to solid-state surfaces do not show lateral mobility or significant desorption rates, and therefore protein adsorption is generally considered an irreversible and nonspecific process 30-32. Protein adsorption onto solid state surfaces is thought t...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tecnai F20 S/TEM</td>
 <td>FEI</td>
 <td>&nbsp;</td>
 <td>S/TEM requires acceleration voltage &ge;200kV and field-emission source. </td>
 </tr>
 <tr>
 <td>20 nm thick silicon nitride membrane window for TEM</td>
 <td>SPI</td>
 <td>4163SN-BA </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Axopatch 200B patch-clamp amplifier</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Digidata 1440A</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pCLAMP 10 software</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>Electrophysiology Data Acquisition and Analysis Software</td>
 </tr>
 <tr>
 <td>Sulfuric Acid</td>
 <td>Fisher Scientific</td>
 <td>A300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hydrogen peroxide</td>
 <td>Fisher Scientific</td>
 <td>H325</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>silicone O-rings</td>
 <td>McMaster-Carr</td>
 <td>003 S70</td>
 <td>Alternatively use PDMS </td>
 </tr>
 <tr>
 <td>silver wire</td>
 <td>Sigma-Aldrich</td>
 <td>348759</td>
 <td>For electrodes</td>
 </tr>
 <tr>
 <td>SPC Technology, D sub contact, pin</td>
 <td>Newark</td>
 <td>9K4978 </td>
 <td>For electrodes</td>
 </tr>
 <tr>
 <td>potassium chloride</td>
 <td>Sigma</td>
 <td>P9541</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>potassium phosphate dibasic</td>
 <td>Sigma</td>
 <td>P2222</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>potassium phosphate monobasic</td>
 <td>Sigma</td>
 <td>P5379</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PDMS</td>
 <td>Dow Corning</td>
 <td>&nbsp;</td>
 <td>Sylgar 184 Elastomer set. For making chamber.</td>
 </tr>
 <tr>
 <td>Kwik-Cast Sealant</td>
 <td>World Precision Instruments</td>
 <td>KWIK-CAST</td>
 <td>Fast acting silicone sealant</td>
 </tr>
 <tr>
 <td>hot plate</td>
 <td>Fisher Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday cage</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</table>

monitoring protein adsorption with solid-state nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, 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.

Watch this Scientific Journal Video about Monitoring Protein Adsorption with Solid-state Nanopores at JoVE.com

Monitoring Protein Adsorption with Solid-state Nanopores

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

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13.4K ビュー.  Syracuse University. 無機表面へのタンパク質の非特異的吸着を監視するための固体ナノ細孔を用いる方法が説明されています。方法は、リアルタイムで、単一分子レベルで検出対象にする吸着を可能に、抵抗パルスの原理を採用しています。単一タンパク質吸着のプロセスがはるかに平衡からなので、我々は同様にタンパク質吸着の定数見かけ一次反応速度の定量測定と一?...