Overvågning Protein Adsorption med Solid-state Nanopores
Summary
En metode til ved hjælp af solid-state nanopores til at overvåge ikke-specifikke adsorption af proteiner ud på en uorganisk overflade er beskrevet. Metoden anvender resistive-puls princip, der giver mulighed for adsorption til probed i real-time og på enkelt-molekyle niveau. Fordi processen med enkelt protein adsorption er langt fra ligevægt, foreslår vi ansættelse af parallelle arrays af syntetiske nanopores, der gør det muligt for kvantitativ bestemmelse af den tilsyneladende første-ordens reaktion hastighedskonstanten af protein adsorption samt og Langmuir adsorption konstant.
Abstract
Solid-state nanopores er blevet brugt til at udføre målinger på enkelt-molekyle niveau for at undersøge den lokale struktur og fleksibilitet af nukleinsyrer 1-6, udfoldelsen af proteiner 7, og bindende affinitet for forskellige ligander 8. Ved at koble disse nanopores til resistive-puls teknik 9-12, kan sådanne målinger ske under en lang række betingelser og uden behov for mærkning 3. I den resistive-puls teknik, er en ionisk salt-opløsning, der indføres på begge sider af nanopore. Derfor er ioner drevet fra den ene side af salen til den anden ved en påført transmembrane potentiale, hvilket resulterer i en lind strøm. Opdelingen af en analyt i nanopore forårsager en veldefineret afbøjning i denne strøm, som kan analyseres for at udtrække en enkelt-molekyle oplysninger. Ved hjælp af denne teknik, kan adsorption af enkelte proteiner til nanopore væggene blive overvåget i henhold til en bred vifte afbetingelser 13. Protein adsorption vokser i betydning, for som mikrofluidenheder skrumpe i størrelse, samspillet mellem disse systemer med enkelte proteiner bliver et problem. Denne protokol beskriver en hurtig analyse for protein binding til nitrid film, som let kan udvides til andre tynde film gøres til genstand for nanopore boring, eller at funktionaliserede nitrid overflader. En række af proteiner kan undersøges under en bred vifte af løsninger og denaturering betingelser. Derudover kan denne protokol bruges til at udforske mere grundlæggende problemer med at bruge nanopore spektroskopi.
Protocol
Discussion
Spontan adsorption af proteiner på solid-state overflader 27-29 er fundamentalt vigtige i en række områder, såsom biochip applikationer og design af en ny klasse af funktionelle hybrid biomaterialer. Tidligere undersøgelser har vist, at proteiner adsorberes til solid state-overflader ikke viser lateral mobilitet eller væsentlige desorption satser, og derfor er protein adsorption er generelt betragtes som en irreversibel og uspecifik proces 30-32. Protein adsorption på solid state overflader …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke John Grazul (Cornell University), André Marziali (University of British Columbia i Vancouver) og Vincent Tabard-Cossa (The University of Ottawa) for deres rådgivning. Dette arbejde er finansieret delvist af tilskud fra den amerikanske National Science Foundation (DMR-0706517 og DMR-1006332) og National Institutes of Health (R01-GM088403). Den nanopore boring blev udført ved Electron Microscopy facilitet i Cornell Center for Materials Research (CCMR) med støtte fra National Science Foundation – Materialeforskning Science and Engineering Centers (MRSEC) program (DMR 0.520.404). Forberedelsen af silicium nitrid membranerne blev udført på Cornell nanoskala Facility, et medlem af National Nanotechnology Infrastructure Network, som er støttet af National Science Foundation (Grant ECS-0335765).
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Tecnai F20 S/TEM | FEI | S/TEM requires acceleration voltage ≥200kV and field-emission source. | |
| 20 nm thick silicon nitride membrane window for TEM | SPI | 4163SN-BA | |
| Axon Axopatch 200B patch-clamp amplifier | Molecular Devices | ||
| Axon Digidata 1440A | Molecular Devices | ||
| pCLAMP 10 software | Molecular Devices | Electrophysiology Data Acquisition and Analysis Software | |
| Sulfuric Acid | Fisher Scientific | A300 | |
| hydrogen peroxide | Fisher Scientific | H325 | |
| silicone O-rings | McMaster-Carr | 003 S70 | Alternatively use PDMS |
| silver wire | Sigma-Aldrich | 348759 | For electrodes |
| SPC Technology, D sub contact, pin | Newark | 9K4978 | For electrodes |
| potassium chloride | Sigma | P9541 | |
| potassium phosphate dibasic | Sigma | P2222 | |
| potassium phosphate monobasic | Sigma | P5379 | |
| PDMS | Dow Corning | Sylgar 184 Elastomer set. For making chamber. | |
| Kwik-Cast Sealant | World Precision Instruments | KWIK-CAST | Fast acting silicone sealant |
| hot plate | Fisher Scientific | ||
| Faraday cage |
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