Name: automated lipid bilayer membrane formation using a polydimethylsiloxane thin film
Uploaded: 2016-07-10T13:00:00
Description: Watch this Scientific Journal Video about Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film at JoVE.com

This work was supported by the Pioneer Research Center Program (NRF-2012-0009575) and National Research Foundation Grants (NRF-2012R1A1B4002413, NRF-2014R1A1A2059341) from the National Research Foundation of Korea. This work was also partially supported by the Inha University Research Grant.

1. Fremstilling af opløsning <ol><li> Fremstilling af pufferopløsning: <ol><li> Til formulering pufferopløsning, opløses 1 M KCl (kaliumchlorid), 10 mM Tris-HCI (Tris-hydrochlorid), og 1 mM EDTA (ethylendiamintetraeddikesyre) i destilleret vand og justere pH til 8,0. </li><li> Opløsningen filtreres under anvendelse af et 0,20 um filter. At sterilisere, autoklaveres opløsningen ved 121 ° C i 15 min. </li></ol></li><li> Fremstilling af lipidopløsning for pre-maleri: <ol><li> At formulere lipidopløsning for pre-...

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Our BLM formation technique provides a powerful tool for cell membrane and ion channel studies, in contrast to conventional techniques that have limited potential for industrial use. We developed a membrane precursor using a PDMS thin film, and devised a frozen membrane precursor with expedited self-assembly.
As opposed to conventional membrane formation methods with hydrophobic films, where membrane formation only occurs via surface interactions between the film and the lipid solution,20...

Kunstig lipiddobbeltlagsmembran, eller sort lipidmembranen (BLM), er et vigtigt redskab til at belyse mekanismerne i cellemembraner og ionkanaler, samt for at forstå samspillet mellem ionkanaler og ioner / molekyler. 1-7 Skønt patch-clamp-metoden er ofte betragtes som den gyldne standard for cellemembran undersøgelser, det er besværligt og kræver dygtige operatører for ionkanal-målinger. 8. Mens kunstigt rekonstituerede lipiddobbeltlagsmembraner er dukket op som alternative værktøjer til ionkanal-studier, 9,10 de er også forbundet med besværlige processer og særlig ekspertise. Desuden membraner er modtagelige for mekaniske forstyrrelser. Derfor har lipiddobbeltlag teknologier, der indføres til dato begrænsede praktiske anvendelser. 11 For at øge robustheden og levetiden af lipiddobbeltlagsmembraner, Costello et al. 12, og Ide og Yanagida <sup&gt; 13 har udtænkt en fritstående lipiddobbeltlaget understøttet af hydrogeler. Trods forbedret levetid dog (&lt;24 timer), blev dobbeltlag robusthed ikke forbedret. Jeon et al. 14 udtænkt en hydrogel indkapslet membran (HEM) med intim hydrogel-lipiddobbeltlag kontakt, hvilket resulterer i forøget levetid (op til flere dage). For yderligere at forøge levetiden af HEM, Malmstadt og Jeon et al. Skabte en hydrogel-indkapslet membran med hydrogel-lipid binding via in-situ kovalent konjugering (cgHEM). 15 I begge systemer, membran levetider forbedret betydeligt (&gt; 10 dage) . Men dannelse membran systemer var ikke tilstrækkeligt robuste, og kunne ikke opbevares eller leveret, hvis det kræves for at befri ekspertise til brug af lipid dobbeltlag. Udviklingen af ​​et lipiddobbeltlag platform har primært drejet sig stigende robusthed og levetiden af ​​BLMs. Selv levetiden af ​​BLMs har været substantially forbedret nylig har deres ansøgninger været begrænset på grund af mangel på transporterbart og lagerholdbarhed. For at overvinde disse problemer, Jeon et al. Skabt et lagres membransystem og introducerede en membran forløber (MP). 16 at konstruere et parlamentsmedlem, de forberedt en blanding af n-dekan og hexadecan indeholder 3% DPhPC (1,2-diphytanoyl- sn-glycero-3-phosphatidylcholin) til styring af frysepunkt lipidopløsning sådanne, at det fryser ved ~ 14 ° C (under stuetemperatur, over typiske køleskabstemperatur). I dette forsøg blev MP spredt over en lille åbning på en polytetrafluorethylen (PTFE) film og efterfølgende frosset i et køleskab ved 4 ° C. Når MP blev bragt til stuetemperatur, MP optøet og et lipiddobbeltlag blev automatisk dannet, hvilket eliminerer ekspertise typisk er forbundet med dannelse membran. Men succesraten for BLM lavet fra MP var så lav som ~ 27%, og membran GERn gang var inkonsekvent (30 min til 24 timer), hvilket begrænser den praktiske anvendelse. I denne undersøgelse er en polydimethylsiloxan (PDMS) tynd film anvendes i stedet for en konventionel hydrofobe tynde film (PTFE, polyoxymethylen, polystyren) til (a) kontrol fabrikation tid og (b) øge succesraten af ​​BLM dannelse som tidligere rapporteret af Ryu et al. 17. Heri dannelse membran blev lettet ved ekstraktion af opløsningsmidler på grund af den porøse beskaffenhed af PDMS, og den tid, der kræves til dannelse membran blev med held styret ved denne undersøgelse. I dette system, som det lipidopløsning blev absorberet i PDMS tynd film, blev en konsistent membrandannelse tid opnået. Desuden blev membran levetid forlænget på grund af langsom absorption af opløsningsmidler i PDMS tyndfilm, et resultat af tilsætning af squalen til lipid løsning. Vi har udført optiske og elektriske målinger for at kontrollere, at membraner dannet ved anvendelse af denne teknik er egnet til ipå kanaler studier.

<table border="0" cellpadding="0" cellspacing="0" width="1448">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Potassium Chloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">P9333</td>
			<td style="width:743px;">For buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tris-hydrochloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">1185-53-1</td>
			<td>For buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">60-00-4</td>
			<td>For buffer solution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:335px;">n-decane</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">44074-U</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Hexadecane</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">544-76-3</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Squalene</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">S3626</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gramicidin A</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">11029-61-1</td>
			<td>Membrane protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1,2-diphytanoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:172px;">Avanti Polar Lipids, Inc.</td>
			<td style="width:199px;">850356</td>
			<td>For membrae formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Sylgard 184a and 184b elastromer kit</td>
			<td style="width:172px;">Dow Corning Asia</td>
			<td style="width:199px;"></td>
			<td>To produce PDMS thin film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">0.2 &#956;m filter</td>
			<td style="width:172px;">Satorius stedim</td>
			<td style="width:199px;">16534----------K</td>
			<td>To filter buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rotator</td>
			<td style="width:172px;">FinePCR</td>
			<td style="width:199px;">AG</td>
			<td>To dissolve lipid homogeneously</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Autoclave</td>
			<td style="width:172px;">Biofree</td>
			<td style="width:199px;">BF-60AC</td>
			<td>To sterilize buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Spin coater</td>
			<td style="width:172px;">Shinu Mst</td>
			<td style="width:199px;">SP-60P</td>
			<td>To spread PDMS prepolymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Vaccum dessiccator</td>
			<td style="width:172px;">Welch</td>
			<td style="width:199px;">2042-22</td>
			<td>To remove air bubble in PDMS prepolymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">500 &#956;m&#160; punch</td>
			<td style="width:172px;">Harris Uni-Core</td>
			<td style="width:199px;">0.5</td>
			<td>To create an aperture on the PDMS thin film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">CNC machine</td>
			<td style="width:172px;">SME trading</td>
			<td style="width:199px;">SME 2518</td>
			<td>To fabricate membrane formation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Halogen fiber optic illuminator</td>
			<td style="width:172px;">Motic</td>
			<td style="width:199px;">MLC-150C</td>
			<td>To illuminate the aperture of PDMS thin film for optical observation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Digital microscope</td>
			<td style="width:172px;">Digital blue</td>
			<td style="width:199px;">QX-5</td>
			<td>To optically observe lipid bilayer membrane formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Electrode</td>
			<td style="width:172px;">A-M Systems</td>
			<td style="width:199px;"></td>
			<td>To electrically observe membrane formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microelectrode amplifier (Axopatch amplifier)</td>
			<td style="width:172px;">Axon Instruments</td>
			<td>Axopatch 200B Amplifier</td>
			<td>To measure capacitance of the membrane (described as microelectrode amplifier in the manuscript)</td>
		</tr>
	</tbody>
</table>

automated lipid bilayer membrane formation using a polydimethylsiloxane thin film

En kunstig lipiddobbeltlag eller sorte lipidmembranen (BLM), er et stærkt værktøj til at studere ionkanaler og protein-interaktioner, samt til biosensoranvendelser. Men konventionelle BLM dannelse teknikker har flere ulemper og de ofte kræver særlig ekspertise og arbejdskrævende processer. Især lider konventionelle BLMs fra lave dannelse succesrate og inkonsekvent dannelse membran tid. Her demonstrerer vi en oplagres og transportabelt BLM dannelse systemet med styret udtynding-out tid og forbedret BLM dannelse sats ved at erstatte konventionelt anvendte film (polytetrafluorethylen, polyoxymethylen, polystyren) til polydimethylsiloxan (PDMS). I dette forsøg er en porøs struktureret polymer, såsom PDMS tynd film anvendes. Derudover, i modsætning til konventionelt anvendte opløsningsmidler med lav viskositet, anvendelse af squalen tilladt en styret udtynding-ud via langsom absorption opløsningsmiddel ved PDMS, forlænger membran levetid. I annoncenbetingelse, ved anvendelse af en blanding af squalen og hexadecan, frysepunktet af lipidopløsning blev forøget (~ 16 ° C), blev desuden fremstillet membran forstadier, der kan ubestemt tid opbevares og let transporteres. Disse membran forstadier har reduceret BLM dannelse tid &lt;1 time og opnåede en BLM dannelse på ~ 80%. Endvidere ionkanal-eksperimenter med gramicidin A demonstrerede gennemførligheden af ​​membransystemet.

Optimering af maksimalt tilladelige fejl Solution Sammensætning Forskellige sammensætninger af lipider og opløsningsmidler blev testet med succes rekonstituere lipiddobbeltlagsmembraner fra maksimalt tilladelige fejl. MP-system med en blanding af N- decan og hexadecan indeholdende 3% DPhPC 14 udviste en lav succesrate for dannelse membran (~ 27%). Hertil kommer, som PDMS filmen kontinuerligt ekstraheret lipidopløsning, var det nødvendigt a...

Watch this Scientific Journal Video about Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film at JoVE.com

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

Vi demonstrerer en kan lagres, transportabelt lipiddobbeltlag dannelse system. En lipiddobbeltlagsmembranen kan dannes inden for 1 time med over 80% succesrate når en frossen membran precursor er bragt til omgivelsestemperatur. Dette system vil reducere besværlige processer og ekspertise, der er forbundet med ionkanaler.

Automatiseret lipiddobbeltlagsmembranen Formation Ved hjælp af en Polydimethylsiloxan Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

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Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Dobbelt Emulsion Generation Ved hjælp af en Polydimethylsiloxan (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Ligand Nano-klynge Arrays i et understøttet lipid-dobbeltlag

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

Spontane dannelse og omlægning af kunstige Lipid nanorør netværk som en Bottom-Up-Model for endoplasmatiske Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Immobilisering af levende Caenorhabditis elegans enkeltpersoner ved hjælp af en ultra-tynd Polydimethylsiloxan mikrofluid Chip med væskeophobning

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Materiale dannelse af rekombinante Spider Silks gennem vandig Solvation ved hjælp af varme og tryk

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

En automatiseret metode til udførelse af in vitro-Mikronukleusanalysen ved hjælp af multispektral billedbehandlings flow cytometri

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...