Name: tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes
Uploaded: 2020-12-08T13:00:00
Description: Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Dette arbejde blev støttet af Australian Research Council (ARC) Discovery Program (DP150101065) og ARC Research Hub for Integrated Device for End-user Analysis at Low-levels (IDEAL) (IH150100028).

1. tBLMs elektroder forberedelse
<ol><li>Forberedelse af første monolagsbelægning<ol>
<li>Fordyb en frisk sputtered guld mønstret elektrode mikroskop dias i en ethanolisk opløsning bestående af en 3 mM 1:9 forholdet mellem benzyl-disulfid-tetra-ethylenglycol-OH "spacer" molekyler (benzyl disulfid bestående af en fire ilt-ethylenglycol spacer, afsluttet med en OH gruppe) og benzyl-disulfid (tetra-ethylenglycol) n =2 C20-phytanyl "tethered" molekyler. Dette skaber det første lag belægning, som en...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurements+of+heating+by+electromagnetically+trapped+gold+nanoparticles+on+supported+lipid+bilayers.">Direct measurements of heating by electromagnetically trapped gold nanoparticles on supported lipid bilayers.</a> ACS Nano. 4 (4), 2256-2262 (2010).</li><li>Plaksin, M., Shapira, E., Kimmel, E., Shoham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+transients+excite+neurons+through+universal+intramembrane+mechanoelectrical+effects.">Thermal transients excite neurons through universal intramembrane mechanoelectrical effects.</a> Physical Review X. 8 (1), 011043 (2018).</li><li>Alghalayini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+heat+transfer+between+gold+nanoparticles+and+tethered+bilayer+lipid+membranes.">Real-time monitoring of heat transfer between gold nanoparticles and tethered bilayer lipid membranes.</a> Biochimica et Biophysica Acta (BBA)-Biomembranes. , 183334 (2020).</li><li>Moradi-Monfared, S., Krishnamurthy, V., Cornell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+machine+biosensor:+construction,+predictive+models+and+experimental+studies.">A molecular machine biosensor: construction, predictive models and experimental studies.</a> Biosensors and Bioelectronics. 34 (1), 261-266 (2012).</li><li>Hoiles, W., Gupta, R., Cornell, B., Cranfield, C., Krishnamurthy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+tethers+on+artificial+cell+membranes:+A+coarse-grained+molecular+dynamics+study.">The effect of tethers on artificial cell membranes: A coarse-grained molecular dynamics study.</a> PloS One. 11 (10), 0162790 (2016).</li><li>Cranfield, C. 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Denne protokol beskriver brugen af tBLM-model med et coplanar elektrodesubstrat i forbindelse med en vandret laserjustering, der er oprettet, der muliggør realtids elektrisk impedansregistrering som reaktion på laserbestråling af guldnanopartikler. Metoden til EIS-optagelse, der præsenteres her, konstruerer en minimal liste over eksperimenter, der er nødvendige for at give registrering af ionstrømsændringer på tværs af membranen, hvilket svarer til den varme, der genereres af den...

Den hypertermiske ydeevne af bestrålede guld nanomaterialer tilbyder en ny klasse af minimalt invasiv, selektiv, målrettet behandling for infektioner og tumorer1. Ansættelsen af nanopartikler, der kan opvarmes af en laser, er blevet brugt til selektivt at ødelægge syge celler samt give et middel til selektiv lægemiddellevering2,3. En konsekvens af fototermikfænomenerne af opvarmede plasmonic nanopartikler er skader på cellemembranerne. Væsken lipid bilayer membran betragtes som et særligt sårbart sted for celler, der gennemgår sådanne behandlinger, fordi denaturering af iboende membranproteiner samt membranskader også kan føre til celledød4, da mange proteiner er der for at opretholde den ioniske potentielle gradient på tværs af cellemembraner. Selv om evnen til at bestemme og overvåge varmeoverførsel på nanoskalaen er af afgørende interesse for undersøgelse og anvendelse af bestrålede GNP'er1,5,6,7, vurdering og forståelse af de molekylære interaktioner mellem GNP'er og biomembraner samt de direkte konsekvenser af de laserinducerede opvarmningsfænomener af indlejrede GNP'er i biologisk væv, er endnu ikke fuldt belyst8. Derfor er en grundig forståelse af hypertermiprocessen med bestrålede GNP'er fortsat en udfordring. Som sådan kan udviklingen af en nanomaterialeelektrodegrænseflade, der efterligner cellernes naturlige omgivelser, være et middel til at foretage en tilbundsgående undersøgelse af varmeoverførselsegenskaberne for bestrålede guldnanopartikler i biologiske systemer.
Kompleksiteten af indfødte cellemembraner er en af de betydelige udfordringer i forståelsen af de bestrålede GNPs interaktioner i celler. Der har været forskellige kunstige membran platforme udviklet til at give tætte enkle bio-mimetiske versioner af naturlige lipid membran arkitektur og funktionalitet, herunder, men ikke begrænset til, sorte lipid membraner9, understøttet planar bilayer membraner10, hybrid bilayer membraner11, polymer-polstret lipid bilayer membraner12 og tøjret bilayer lipid membraner13. Hver kunstig lipidmembranmodel har klare fordele og begrænsninger med hensyn til at efterligne de naturlige lipidmembraner14.
Denne undersøgelse beskriver anvendelsen af lipidmembranbelagte elektroder som en sensor til vurdering af guldnanopartikler og lipidmembraninteraktioner ved hjælp af tBLM-modellen. Den tBLM-baserede biosensordetekteringsordning giver iboende stabilitet og følsomhed13, da tøjrede membraner kan selvreparere, i modsætning til andre systemer (såsom membraner dannet af patch-clamp eller liposomer), hvor kun en lille mængde membranskader resulterer i deres sammenbrud15,16,17,18. Endvidere, fordi tBLMs er af mm2 dimensioner, baggrunden impedans er størrelsesordener lavere end patch-clamp optagelse teknikker, som muliggør en registrering af ændringer i basal membran ioniske flux på grund af nanopartikler interaktioner. Som et resultat af dette kan den nuværende protokol kontrastændringer i membranledningsevnen ved bundne GNP'er, der er ophidset af lasere, hvis kræfter er så lave som 135 nW /μm2.
Systemet præsenteres her giver en følsom og reproducerbar metode til bestemmelse af præcise laser parametre, partikelstørrelse, partikelbelægninger og sammensætning er nødvendige for at designe og udvikle termiske behandlinger. Dette er afgørende for forfinelsen af nye fototermiske behandlinger samt for at give værdifulde oplysninger til detaljerede mekanismer for varmeoverførsel inden for biologiske systemer. Den præsenterede protokol er baseret på tidligere offentliggjort arbejde19. En oversigt over protokollen er som følger: det første afsnit definerer tBLM-formationen; I det andet afsnit beskrives, hvordan du konstruerer opsætningen og justerer excitationslaserkilden. Det sidste afsnit illustrerer, hvordan du kan udtrække oplysninger fra de elektriske impedansspektroskopidata.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 nm diameter streptavidin-conjugated gold nanoparticles</td>
			<td>Cytodiagnostics</td>
			<td>AC-30-04-05</td>
			<td>This is a streptavidin-conjugated GNPs product ready for use</td>
		</tr>
		<tr>
			<td>30 nm diameter bare gold nanoparticles</td>
			<td>Sigma-Aldrich</td>
			<td>753629</td>
			<td>This is a bare GNPs product ready for use</td>
		</tr>
		<tr>
			<td>Cholesterol-PEG-Biotin (MW1000)</td>
			<td>NANOCS</td>
			<td>PG2-BNCS-10k</td>
			<td>Dissolved in highly pure ethanol</td>
		</tr>
		<tr>
			<td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S1</td>
			<td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Spacer molecules</td>
		</tr>
		<tr>
			<td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&#160;</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-S2</td>
			<td>Tethered molecules</td>
		</tr>
		<tr>
			<td>532 nm green laser continuous light</td>
			<td>OBIS LS/OBIS CORE LS, China</td>
			<td>ND-1000</td>
			<td>The power of this laser was ~135&#160;mW&#160;</td>
		</tr>
		<tr>
			<td>tethaPod EIS reader</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-R1</td>
			<td>A reader of conductance and capacitance on six channels simultaneously</td>
		</tr>
		<tr>
			<td>tethaPlate cartridge assembly</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-BG</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Clamp and slide assembly jig</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-A1</td>
			<td>Materials to attach the slide with electrodes to the flow cell cartridge</td>
		</tr>
		<tr>
			<td>Lipid coated coplanar gold electrodes</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-T10</td>
			<td>Coplanar&#160; gold electrodes are made from 25 mm x 75&#160;mm x&#160;1&#160;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&#160;</td>
		</tr>
		<tr>
			<td>tethaQuick software</td>
			<td>SDx Tethered Membranes Pty. Ltd.</td>
			<td>SDx-B1</td>
			<td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td>
		</tr>
		<tr>
			<td>&#160;99.9% Pure ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34963</td>
			<td>Absolute,&#160; 99.9%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>pH 7</td>
		</tr>
	</tbody>
</table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

Her rapporterer vi en protokol til at undersøge varmeoverførslen mellem bestrålede guld nanopartikler (GNPs) og bilayer lipid membraner ved elektrokemi ved hjælp af tøjret bilayer lipid membraner (tBLMs) samlet på guld elektroder. Bestrålede modificerede GNP'er, såsom streptavidin-konjugerede GNP'er, er indlejret i tBLMs, der indeholder målmolekyler, såsom biotin. Ved at bruge denne tilgang medieres varmeoverførselsprocesserne mellem bestrålede GNP'er og model bilayer lipidmembran med interesseenheder af en vandret fokuseret laserstråle. Den termiske prædiktive beregningsmodel bruges til at bekræfte de elektrokemisk inducerede ledningsændringer i tBLMs. Under de særlige forhold, der blev anvendt, krævede detektering af varmeimpulser specifik fastgørelse af guldnanopartiklerne til membranoverfladen, mens ubundne guldnanopartikler ikke kunne fremkalde et målbart svar. Denne teknik fungerer som en kraftfuld detektion biosensor, som kan udnyttes direkte til design og udvikling af strategier for termiske behandlinger, der tillader optimering af laser parametre, partikelstørrelse, partikelbelægninger og sammensætning.

Det guldsubstrat, som tBLMs kan oprettes på, er vist i figur 1. Et skema over forsøgsopsætningen vises i figur 2.
Coplanar guldelektroder, som vist i figur 1A, er lavet af 25 mm x 75 mm x 1 mm polycarbonatbasesubstrat med mønstrede guld arrays. Et gennemsigtigt klæbelag definerer de seks individuelle målekamre. Coplanar guld elektroden tillader direkte eksponering af laserlys til tBLMs membran. Hver...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Dette arbejde skitserer en protokol for at opnå dynamisk, ikke-invasiv overvågning af varmeoverførsel fra laserbestrålede guld nanopartikler til tBLMs. Systemet kombinerer impedansspektroskopi til realtidsmåling af ledningsændringer på tværs af tBLMs, med en vandret fokuseret laserstråle, der driver guld nanopartikler belysning, til varmeproduktion.

Tøjrede Bilayer Lipid Membraner til at overvåge varmeoverførsel mellem Gold Nanopartikler og Lipid Membraner

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Research

JoVE Journal

Biology

Method for the Isolation of Francisella tularensis Outer Membranes

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Rekonstituering af et Kv Channel i lipidmembraner for strukturelle og funktionelle studier

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Brug af Caco-2 celler til at studere Lipid Transport af tarmen

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Isolering og analyse af sammensætningen af ​​Plant neglebånd Lipid Polyester Monomerer

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Kvantificering af Lipid overflod og evaluering af Lipid Distribution i Caenorhabditis elegans af Nilen rød og olie røde O farvning

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

Måling af energimetabolisme i eksplanterede retinale væv ved hjælp af ekstracellulære Flux analyse

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Dissektion og lipid dråbe farvning af Oenocytter i Drosophila larver

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...