Name: monitoring protein adsorption with solid-state nanopores
Uploaded: 2011-12-02T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Protein Adsorption with Solid-state Nanopores at JoVE.com

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>
	<li>Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-beam+sculpting+at+nanometre+length+scales.">Ion-beam sculpting at nanometre length scales.</a> Nature. 412, 166-169 (2001).</li><li>Li, J., Gershow, M., Stein, D., Brandin, E., Golovchenko, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+molecules+and+configurations+in+a+solid-state+nanopore+microscope.">DNA molecules and configurations in a solid-state nanopore microscope.</a> Nat. Mater. 2, 611-615 (2003).</li><li>Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-state+nanopores.">Solid-state nanopores.</a> Nature. Nanotechnology. 2, 209-215 (2007).</li><li>Branton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+and+challenges+of+nanopore+sequencing.">The potential and challenges of nanopore sequencing.</a> Nat. Biotechnol. 26, 1146-1153 (2008).</li><li>Wanunu, M., Morrison, W., Rabin, Y., Grosberg, A. Y., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrostatic+focusing+of+unlabelled+DNA+into+nanoscale+pores+using+a+salt+gradient.">Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient.</a> Nat. Nanotechnol. 5, 160-165 (2010).</li><li>Peng, H., Ling, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+DNA+translocation+through+a+solid-state+nanopore+by+magnetic+tweezers.">Reverse DNA translocation through a solid-state nanopore by magnetic tweezers.</a> Nanotechnology. 20, 185101-185101 (2009).</li><li>Talaga, D. S., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+protein+unfolding+in+solid+state+nanopores.">Single-molecule protein unfolding in solid state nanopores.</a> J. Am. Chem. Soc. 131, 9287-9297 (2009).</li><li>Zhao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+SNPs+using+a+synthetic+nanopore.">Detecting SNPs using a synthetic nanopore.</a> Nano. Lett. 7, 1680-1685 (2007).</li><li>Storm, A. J., Chen, J. H., Ling, X. S., Zandbergen, H. W., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+solid-state+nanopores+with+single-nanometre+precision.">Fabrication of solid-state nanopores with single-nanometre precision.</a> Nat. Mater. 2, 537-540 (2003).</li><li>Sexton, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistive-pulse+studies+of+proteins+and+protein/antibody+complexes+using+a+conical+nanotube+sensor.">Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor.</a> J. Am. Chem. Soc. 129, 13144-13152 (2007).</li><li>Martin, C. R., Siwy, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry.+Learning+nature's+way:+biosensing+with+synthetic+nanopores.">Chemistry. Learning nature's way: biosensing with synthetic nanopores.</a> Science. 317, 331-332 (2007).</li><li>Sexton, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adsorption-based+model+for+pulse+duration+in+resistive-pulse+protein+sensing.">An adsorption-based model for pulse duration in resistive-pulse protein sensing.</a> J. Am. Chem. Soc. 132, 6755-6763 (2010).</li><li>Niedzwiecki, D. J., Grazul, J., Movileanu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+observation+of+protein+adsoption+onto+an+inorganic+surface.">Single-molecule observation of protein adsoption onto an inorganic surface.</a> J. Am. Chem. Soc. 132, 10816-10822 (2010).</li><li>Wanunu, M., Meller, A., Selvin, P. R., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-molecule techniques: a laboratory manual. , 395-420 (2008).</li><li>Han, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+detection+of+single+protein+molecules+and+protein-protein+interactions+using+synthetic+nanopores.">Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores.</a> Anal. Chem. 80, 4651-4658 (2008).</li><li>Han, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+protein+molecules+using+nanofabricated+pores.">Sensing protein molecules using nanofabricated pores.</a> Appl. Phys. Lett. 88, (2006).</li><li>Fologea, D., Ledden, B., McNabb, D. S., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+characterization+of+protein+molecules+by+a+solid-state+nanopore.">Electrical characterization of protein molecules by a solid-state nanopore.</a> Appl. Phys. Lett. 91, (2007).</li><li>Firnkes, M., Pedone, D., Knezevic, J., Doblinger, M., Rant, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrically+Facilitated+Translocations+of+Proteins+through+Silicon+Nitride+Nanopores:+Conjoint+and+Competitive+Action+of+Diffusion,+Electrophoresis,+and+Electroosmosis.">Electrically Facilitated Translocations of Proteins through Silicon Nitride Nanopores: Conjoint and Competitive Action of Diffusion, Electrophoresis, and Electroosmosis.</a> Nano. Lett. , (2010).</li><li>Pedone, D., Firnkes, M., Rant, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+analysis+of+translocation+events+in+nanopore+experiments.">Data analysis of translocation events in nanopore experiments.</a> Anal. Chem. 81, 9689-9694 (2009).</li><li>Oukhaled, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Completely+Unfolded+and+Native+Proteins+through+Solid-State+Nanopores+as+a+Function+of+Electric+Driving+Force.">Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force.</a> ACS. Nano.. , (2011).</li><li>Yusko, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+protein+translocation+through+nanopores+with+bio-inspired+fluid+walls.">Controlling protein translocation through nanopores with bio-inspired fluid walls.</a> Nat. Nanotechnol. 6, 253-260 (2011).</li><li>Arafat, A., Schroen, K., de Smet, L. C., Sudholter, E. J., Zuilhof, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailor-made+functionalization+of+silicon+nitride+surfaces.">Tailor-made functionalization of silicon nitride surfaces.</a> J. Am. Chem. Soc. 126, 8600-8601 (2004).</li><li>Wanunu, M., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+modified+solid-state+nanopores.">Chemically modified solid-state nanopores.</a> Nano. Lett. 7, 1580-1585 (2007).</li><li>Movileanu, L., Cheley, S., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+individual+flexible+polymers+into+a+nanoscopic+protein+pore.">Partitioning of individual flexible polymers into a nanoscopic protein pore.</a> Biophys. J. 85, 897-910 (2003).</li><li>Aksimentiev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+ionic+current+signatures+of+DNA+transport+through+a+nanopore.">Deciphering ionic current signatures of DNA transport through a nanopore.</a> Nanoscale. 2, 468-483 (2010).</li><li>Timp, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopore+Sequencing:+Electrical+Measurements+of+the+Code+of+Life.">Nanopore Sequencing: Electrical Measurements of the Code of Life.</a> IEEE Trans. Nanotechnol. 9, 281-294 (2010).</li><li>Roach, P., Farrar, D., Perry, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+tailoring+for+controlled+protein+adsorption:+effect+of+topography+at+the+nanometer+scale+and+chemistry.">Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry.</a> J. Am. Chem. Soc. 128, 3939-3945 (2006).</li><li>Roach, P., Farrar, D., Perry, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+protein+adsorption:+surface-induced+conformational+changes.">Interpretation of protein adsorption: surface-induced conformational changes.</a> J. Am. Chem. Soc. 127, 8168-8173 (2005).</li><li>Brewer, S. H., Glomm, W. R., Johnson, M. C., Knag, M. K., Franzen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+BSA+binding+to+citrate-coated+gold+nanoparticles+and+surfaces.">Probing BSA binding to citrate-coated gold nanoparticles and surfaces.</a> Langmuir. 21, 9303-9307 (2005).</li><li>Schon, P., Gorlich, M., Coenen, M. J., Heus, H. A., Speller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonspecific+protein+adsorption+at+the+single+molecule+level+studied+by+atomic+force+microscopy.">Nonspecific protein adsorption at the single molecule level studied by atomic force microscopy.</a> Langmuir. 23, 9921-9923 (2007).</li><li>Rabe, M., Verdes, D., Zimmermann, J., Seeger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+organization+and+cooperativity+during+nonspecific+protein+adsorption+events.">Surface organization and cooperativity during nonspecific protein adsorption events.</a> J. Phys. Chem. B. 112, 13971-13980 (2008).</li><li>Rabe, M., Verdes, D., Rankl, M., Artus, G. R., Seeger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+study+of+concepts+and+phenomena+of+the+nonspecific+adsorption+of+beta-lactoglobulin.">A comprehensive study of concepts and phenomena of the nonspecific adsorption of beta-lactoglobulin.</a> Chemphyschem. 8, 862-872 (2007).</li><li>Micic, M., Chen, A., Leblanc, R. M., Moy, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+Electron+Microscopy+Studies+of+Protein-Functionalized+Atomic+Force+Microscopy+Cantilever+Tips.">Scanning Electron Microscopy Studies of Protein-Functionalized Atomic Force Microscopy Cantilever Tips.</a> Scanning. 21, 394-397 (1999).</li><li>Grant, A. W., Hu, Q. H., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+electron+microscopy+'windows'+for+nanofabricated+structures.">Transmission electron microscopy 'windows' for nanofabricated structures.</a> Nanotechnology. 15, 1175-1181 (2004).</li><li>Giannoulis, C. S., Desai, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+proteins+and+fibroblasts+on+thin+inorganic+films.">Characterization of proteins and fibroblasts on thin inorganic films.</a> J. Mater. Sci: Mater. Med. 13, 75-80 (2002).</li><li>Gustavsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modifications+of+silicon+nitride+for+cellular+biosensors+applications.">Surface modifications of silicon nitride for cellular biosensors applications.</a> J. Mater. Sci: Mater. Med. 19, 1839-1850 (2008).</li><li>Shirshov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+response+of+planar+polarization+interferometer+to+molecular+layer+formation:+fibrinogen+adsorption+on+silicon+nitride+surface.">Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface.</a> Biosens. Bioelectron. 16, 381-390 (2001).</li><li>Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+layer+deposition+to+fine-tune+the+surface+properties+and+diamters+of+fabricated+nanopores.">Atomic layer deposition to fine-tune the surface properties and diamters of fabricated nanopores.</a> Nano. Lett. 4, 1333-1337 (2004).</li><li>Siwy, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosensors+based+on+biofunctionalized+conical+gold+nanotubes.">Protein biosensors based on biofunctionalized conical gold nanotubes.</a> J. Am. Chem. Soc. 127, 5000-5001 (2005).</li><li>Ding, S., Gao, C., Gu, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+Single+Molecules+of+Immunoglobulin+and+Ricin+with+an+Aptamer-Encoded+Glass+Nanopore.">Capturing Single Molecules of Immunoglobulin and Ricin with an Aptamer-Encoded Glass Nanopore.</a> Anal. Chem. , (2009).</li><li>Kim, M. -. J., Wanunu, M., Bell, C. D., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Fabrication+of+Uniform+Size+Nanopores+and+Nanopore+Arrays+for+Parallel+DNA+Analysis.">Rapid Fabrication of Uniform Size Nanopores and Nanopore Arrays for Parallel DNA Analysis.</a> Adv. Mater. 18, 3149-3153 (2006).</li><li>Bezrukov, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+as+molecular+Coulter+counters+to+probe+metabolite+transport.">Ion channels as molecular Coulter counters to probe metabolite transport.</a> J. Membr. Biol. 174, 1-13 (2000).</li></ol>

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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Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or ...