Indsigt i samspillet mellem aminosyrer og peptider med uorganiske materialer Brug Single-Molecule force spektroskopi
Summary
Her præsenterer vi en protokol til at måle kraft af samspillet mellem en veldefineret uorganisk overflade og enten peptider eller aminosyrer ved en enkelt-molekyle force spektroskopi målinger ved hjælp af en atomic force mikroskop (AFM). Opnået fra måleinformationen er vigtigt for bedre at forstå peptid-uorganiske materiale interfase.
Abstract
Samspillet mellem proteiner eller peptider og uorganiske materialer føre til flere interessante processer. For eksempel kombinerer proteiner med mineraler fører til dannelse af kompositmaterialer med unikke egenskaber. Desuden er den uønskede proces med biologisk begroning initieret ved adsorption af biomolekyler, især proteiner, på overflader. Denne organiske lag er et klæbelag for bakterier og giver dem mulighed for at interagere med overfladen. Forståelse af de fundamentale kræfter, der styrer samspillet på organiske-uorganiske interface er derfor vigtigt for mange områder af forskning og kan føre til design af nye materialer til optiske, mekaniske og biomedicinske anvendelser. Dette demonstrerer et enkeltstrenget molekyle force spektroskopi teknik, der anvender en AFM at måle adhæsionskraft mellem enten peptider eller aminosyrer og veldefinerede uorganiske overflader. Denne teknik indebærer en protokol til fastgørelse biomolekylet til AFMtip gennem en kovalent fleksibel linker og enkelt-molekyle force spektroskopi målinger ved atomic force mikroskop. Desuden er en analyse af disse målinger medfølger.
Introduction
Samspillet mellem proteiner og uorganiske mineraler fører til konstruktionen af kompositmaterialer med særlige egenskaber. Dette inkluderer materialer med høj mekanisk styrke eller unikke optiske egenskaber. 1, 2 Fx kombinationen af proteinet kollagen med mineralet hydroxyapatit genererer enten bløde eller hårde knogler for forskellige funktionaliteter. 3 Korte peptider kan også binde uorganiske materialer med høj specificitet. 4, har 5, 6 Specificiteten af disse peptider blevet anvendt til at designe nye magnetiske og elektroniske materialer, 7, 8, 9 fabrikere nanostrukturerede materialer, voksende krystaller, 10 og syntetisere nanopartikler. 11 Forståelse af mekanismen bag interaktioner mellem peptider eller proteiner og uorganiske materialer vil derfor tillade os at designe nye kompositmaterialer med forbedrede adsorberende egenskaber. Eftersom interfasen af implantater med et immunrespons medieres af proteiner, bedre forståelse af interaktioner af proteiner med uorganiske materialer vil forbedre vores evne til at designe implantater. Et andet vigtigt område, der involverer proteiner interagerer med uorganiske overflader er fremstillingen af antifouling materialer. 12, 13, 14, 15 Biofouling er en uønsket proces, hvor organismer tillægger en overflade. Det har mange skadelige konsekvenser for vores liv. For eksempel, begroning af bakterier på medicinsk udstyr fører til hospitalserhvervede infektioner. Begroning af marine organismer på både og større skibe øger forbrug af brændstof. 12, 16, 17, 18
Single-molekyle force spektroskopi (Standard MIDI), ved anvendelse af en AFM, kan direkte måle vekselvirkningerne mellem en aminosyre eller et peptid med et substrat. 19, 20, 21, 22, 23, 24, 25, 26 Andre fremgangsmåder såsom fagdisplay, 27, 28 kvartskrystalmikrovægt (QCM) 29 eller overfladeplasmonresonans (SPR) 29, 30, 31, 32,ref "> 33 foranstaltning samspillet mellem peptider og proteiner til uorganiske overflader i løs vægt. 34, 35, 36 Det betyder, at der opnås ved disse metoder resultater vedrører samlinger af molekyler eller aggregater. I Standard MIDI, er en eller meget få molekyler fastgjort til AFM spids og deres samspil med det ønskede substrat måles. Denne fremgangsmåde kan udvides til at studere proteinfoldning ved at trække proteinet fra overfladen. Desuden kan den anvendes til at måle interaktioner mellem celler og proteiner, og bindingen af antistoffer til deres ligander. 37, 38, 39, 40 Dette papir beskriver i detaljer, hvordan man vedhæfte enten peptider eller aminosyrer til AFM spids hjælp silanol kemi. Desuden papiret forklarer, hvordan man udfører kraftmålinger, og hvordan man analysereresultater.
Protocol
Representative Results
Discussion
Steps 1.3, 1.4 and 1.7 in the protocol should be carried out with extensive care and in a very gentle manner. In step 1.3, the tip should not be in contact with the silane mixture and the silanization process should be carried out in an inert atmosphere (moisture free).45 This is done in order to prevent multilayer formation and because silane molecules readily undergo hydrolysis in the presence of moisture.45
In step 1.4, the temperature and tim…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Marie Curie International Reintegration Grant (EP7). P. D. acknowledges the support of the Israel Council for Higher Education.
Materials
| Silicon nitride (Si3N4) AFM cantilevers with silicon tips | Bruker (Camarilo, CA, USA) | MSNL10, nominal cantilevers radius ~2 nm | |
| Methyltriethoxysilane | Acros Organics (New Jersey, USA) | For Silaylation of the AFM tip | |
| 3-(Aminopropyl) triethoxysilane | Sigma-Aldrich (Jerusalem, Israel) | Used for tip modification | |
| Triisopropylsilane | Sigma-Aldrich (Jerusalem, Israel) | Used for tip modification | |
| N-Ethyldiisopropylamine | Alfa-Aesar (Lancashire, UK) | Used for tip modification | |
| Triethylamine | Alfa-Aesar (Lancashire, UK) | Used for tip modification | |
| Piperidine | Alfa-Aesar (Lancashire, UK) | Used for tip modification | |
| Fluorenylmethyloxycarbonyl-PEG-N-hydroxysuccinimide (Fmoc-PEG-NHS) | Iris Biotech GmbH (Deutschland, Germany) | Used as the covalent flexible linker (MW = 5000 Da) | |
| 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) | Alfa Aser (Heysham, England) | Used as a coupling reagent. | |
| N-methyl-2-pyrrolidone (NMP) | Acros Organics (New Jersey, USA) | Used as Solvent in Tip modification procedure | |
| DMF (dimethylformamide) | Merck (Darmstadt, Germany) | Used as Solvent in Tip modification procedure | |
| Trifluoro acetic acid (TFA) | Merck (Darmstadt, Germany) | ||
| Acetic anhydride | Merck (Darmstadt, Germany) | ||
| Peptides | GL Biochem (Shanghai, China). | ||
| Phenylalanine and Tyrosine | Biochem (Darmstadt, Germany) | ||
| 30% TiO2 dispersion in the mixture of solvent 2-(2-Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP) | Applied Vision Laboratories (Jerusalem, Israel) | (30%) in the mixture of solvent 2-(2 Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP) | |
| Mica substrates | TED PELLA, INC. (Redding, California, USA) | 9.9 mm diameter |
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