Name: insights into the interactions of amino acids and peptides with inorganic materials using single-molecule force spectroscopy
Uploaded: 2017-03-06T12:30:00
Description: Watch this Scientific Journal Video about Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy at JoVE.com

This work was supported by the Marie Curie International Reintegration Grant (EP7). P. D. acknowledges the support of the Israel Council for Higher Education.

1. Tip Ændring <ol><li> Køb siliciumnitrid (Si 3 N 4) AFM cantilevere med silicium tips (nominel cantilever radius af ~ 2 nm). </li><li> Rengør hver AFM cantilever ved at dyppe i vandfri ethanol i 20 min. Tørt ved stuetemperatur. Derefter behandle cantilevere ved at udsætte dem for O 2 plasma i 5 min. </li><li> Suspendere de rene tips ovenfor (3 cm) en opløsning indeholdende methyltriethoxysilan og 3- (aminopropyl) triethoxysilan i et forhold på 15: 1 (v / v) i en ekssikkator un...

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...

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<html> 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 &quot;&gt; 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.

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		<tr height="173">
			<td height="173" style="height:173px;width:164px;">Silicon nitride (Si3N4) AFM cantilevers with silicon tips</td>
			<td style="width:147px;">Bruker (Camarilo, CA, USA)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">MSNL10, nominal cantilevers radius ~2 nm&#160;</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:164px;">Methyltriethoxysilane&#160;</td>
			<td style="width:147px;">Acros Organics (New Jersey, USA)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">For Silaylation of the AFM tip&#160;</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:164px;">3-(Aminopropyl) triethoxysilane</td>
			<td style="width:147px;">Sigma-Aldrich (Jerusalem, Israel)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used for tip modification&#160;</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:164px;">Triisopropylsilane</td>
			<td style="width:147px;">Sigma-Aldrich (Jerusalem, Israel)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used for tip modification</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:164px;">N-Ethyldiisopropylamine</td>
			<td style="width:147px;">Alfa-Aesar (Lancashire, UK)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used for tip modification</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:164px;">Triethylamine</td>
			<td style="width:147px;">Alfa-Aesar (Lancashire, UK)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used for tip modification</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:164px;">Piperidine</td>
			<td style="width:147px;">Alfa-Aesar (Lancashire, UK)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used for tip modification</td>
		</tr>
		<tr height="232">
			<td height="232" style="height:232px;width:164px;">Fluorenylmethyloxycarbonyl-PEG-N-hydroxysuccinimide&#160; (Fmoc-PEG-NHS)</td>
			<td style="width:147px;">Iris Biotech GmbH (Deutschland, Germany)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used as the covalent flexible linker&#160; (MW = 5000 Da)</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:164px;">2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU)</td>
			<td style="width:147px;">Alfa Aser (Heysham, England)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used as a coupling reagent.&#160;</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:164px;">N-methyl-2-pyrrolidone (NMP)</td>
			<td style="width:147px;">Acros Organics (New Jersey, USA)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used as Solvent in Tip modification procedure</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:164px;">DMF (dimethylformamide)</td>
			<td style="width:147px;">Merck (Darmstadt, Germany)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">Used as Solvent in Tip modification procedure</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:164px;">Trifluoro acetic acid (TFA)</td>
			<td style="width:147px;">Merck (Darmstadt, Germany)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:164px;">Acetic anhydride</td>
			<td style="width:147px;">Merck (Darmstadt, Germany)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:164px;">Peptides</td>
			<td style="width:147px;">GL Biochem (Shanghai, China).</td>
			<td style="width:64px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:164px;">Phenylalanine and Tyrosine&#160;</td>
			<td style="width:147px;">Biochem (Darmstadt, Germany)&#160;</td>
			<td style="width:64px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="240">
			<td height="361" rowspan="7" style="height:361px;width:164px;">30% TiO2 dispersion in the mixture of solvent 2-(2-Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP)</td>
			<td rowspan="7" style="width:147px;">Applied Vision Laboratories (Jerusalem, Israel)</td>
			<td rowspan="7" style="width:64px;"></td>
			<td rowspan="7" style="width:264px;">(30%) in the mixture of solvent 2-(2 Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP)</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
		</tr>
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		<tr height="21">
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		<tr height="148">
			<td height="148" style="height:148px;width:164px;">Mica substrates</td>
			<td style="width:147px;">TED PELLA, INC. (Redding, California, USA)</td>
			<td style="width:64px;"></td>
			<td style="width:264px;">9.9 mm diameter</td>
		</tr>
	</tbody>
</table>

insights into the interactions of amino acids and peptides with inorganic materials using single-molecule force spectroscopy

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.

Figur 1 udviser tip modifikation procedure. I det første trin, en plasmabehandling ændrer overfladen af ​​siliciumnitrid spids. Spidsen præsenterer OH-grupper. Disse grupper vil derefter reagere med silanerne. Ved afslutningen af dette trin, vil overfladen af spidsen være dækket af frie -NH2-grupper. Disse frie aminer vil derefter reagere med Fmoc -PEG-NHS, en kovalent linker. Fmoc-gruppen af ​​PEG-linker fjernes ved pipyridine, et afbeskyttelsesr...

Watch this Scientific Journal Video about Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy at JoVE.com

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

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.

Indsigt i samspillet mellem aminosyrer og peptider med uorganiske materialer Brug Single-Molecule force spektroskopi

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

The overall goal of the single-molecule force spectroscopy technique is to determine the adhesion force and nature of interactions between amino acids or peptides with different surfaces. This matter can help answer key questions in the area of surface chemistry and be used for the development of new composites and chordates. The main advantage of this technique is that it provides information on interaction between the desired molecule and surfaces at the single molecule level.To begin, purchase silicon nitride AFM cantilevers equipped with silicon tips with a nominal cantilever radius of two nanometers. Mount the tip on the holder. Clean each of the AFM cantilevers by dipping them into ethanol for 20 minutes and then dry them at room temperature.Next, place the tips into a plasma chamber and treat the cantilevers by exposing them to oxygen plasma for five minutes. Prepare a solution containing 15 parts methyltriethoxysilane and one part three amino propyltriethoxysilane and suspend the canitlevers three centimeters above the solution while under an inert atmosphere. It is important that the tips and the holder do not touch the silane mixture.In addition, the silane is highly moisture sensitive, and therefore these tips need to be carried out in an inert atmosphere. Once sealed, connect the desiccator to a vacuum pump and apply a vacuum for two hours to form a mono-layer of the two types of mixed silane compounds on the surface of the cantilevers. Carefully remove the tips and place them onto a hot plate preheated to 70 degrees Celsius.Leave the tips to dry for 10 minutes. Cool the tips at room temperature before immersing them in a solution of Fmoc-PEG-NHS for one hour at room temperature. Next, dip the tips into chloroform for five minutes and then into DMF for an additional five minutes.After incubation in DMF, de-protect the Fmoc groups of the attached PEG molecules by dipping the tips into 20%piperidine solution in DMF for 30 minutes. Again, dip the tips in DMF for four minutes and then in N-Methyl-2-pyrrolidone for an additional four minutes. To couple amino acids onto the tips, immerse the for one and a half hours into an amino acid coupling solution with a total concentration of 30 millimolar and five milliliters of N-Methyl-2-pyrrolidone.To protect the remaining free or unreactant PEG residues by the acetyl group, dip the tips for 30 minutes in a 0.65 molar mixture of four parts acetic anhydride and one part diisopropylethylamine and N-Methyl-2-pyrrolidone. Then sequentially dip the peptide amino acid functionalized tips for four minutes each into NMP, chloroform, 50%ethanol and water before finally drying the tip in air. Attach the desired test surface to a metallic holder of the AFM with commercially available epoxy.Then, place the metallic holder into the glass holder of the AFM, which is shaped as a small petri dish. Fill the glass holder with TRIS buffer. And then place the metallic holder on the AFM stage below the tip holder.Next, mount the AFM tip on the AFM's optic glass. Place the optic glass block in the AFM head. And put the AFM head on the stage.Next, calibrate the AFM cantilevers with spring constants ranging from 10 to 30 piconewtons per nanometer. A typical force measurement results in a force distance curve like those shown here. The first peek indicates non-specific interactions between the tip and the surface and the second peek refers to the specific adhesion event.From several hundred force distance curves, it is possible to construct a histogram by plotting the number of adhesion events versus force. Applying a Gaussian fit on these histograms will determine the most probable force. After watching this video, you should have a good understanding of how to chemically modify AFM tips with amino acids or peptides and how to perform single-molecule force spectroscopy using AFM.

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