Name: real-time monitoring of ligand-receptor interactions with fluorescence resonance energy transfer
Uploaded: 2012-08-20T12:00:00
Description: Watch this Scientific Journal Video about Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer at JoVE.com

Økonomisk støtte til dette arbejde blev tilvejebragt gennem National Science Foundation, National Institute of Health (NIH), Materials Technology Center (MTC) og Orda på SIUC. Vi takker NSF om tilskud (CHE-0.959.568) for indkøb af en FE-SEM. Vi vil gerne takke professor Matthew McCarroll for personer diskussioner. Julia Reyes vil gerne takke COLCIENCIAS, Colombiansk Agency og Universidad Pedagogica y Tecnologica de Colombia for hendes stipendium og finansiel støtte.

A. Syntese og karakterisering af PDA liposomer 4,5,6 Note 1: Beskyt PDA-løsning fra lys ved hjælp af aluminiumsfolie indpakning på hver beholder i alle de eksperimentelle trin. Note 2: To forskellige sæt liposomopløsning (B og C) blev fremstillet følgende procedure A (Syntese og karakterisering af PDA liposomer). 1. Syntese af N-hydroxysuccinimid Diacteylene (NHS-PCDA) <ol><li> Til fremstilling af liposomer, er en vigt...

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	<li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. , (1999).</li><li>Charych, D. H., Nagy, J. O., Spevak, W., Bednarski, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Colorimetric+Detection+of+a+Receptor-Ligand+Interaction+by+a+Polymerized+Bilayer+Assembly.">Direct Colorimetric Detection of a Receptor-Ligand Interaction by a Polymerized Bilayer Assembly.</a> Science. 261, 585-588 (1993).</li><li>Yoon, B., Lee, S., Kim, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Conceptual+and+Technological+Advances+in+Polydiacetylene-based+Supramolecular+Chemosensors.">Recent Conceptual and Technological Advances in Polydiacetylene-based Supramolecular Chemosensors.</a> Chem. Soc. Rev. 38, 1958-1968 (2009).</li><li>Xuelian, L. i., Kohli, P. u. n. i. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+Molecular+Interactions+in+Biosensors+Based+on+Fluorescence+Resonance+Energy+Transfer.">Investigating Molecular Interactions in Biosensors Based on Fluorescence Resonance Energy Transfer.</a> J. Phys. Chem. C. 114, 6255-6264 (2010).</li><li>Li, X., Matthews, S., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Resonance+Energy+Transfer+in+Polydiacetylene+Liposomes.">Fluorescence Resonance Energy Transfer in Polydiacetylene Liposomes.</a> J. Phys. Chem. B. 112, 13263-13272 (2008).</li><li>Li, X., McCarroll, M., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+Fluorescence+Resonance+Energy+Transfer+in+Conjugated+Liposomes.">Modulating Fluorescence Resonance Energy Transfer in Conjugated Liposomes.</a> Langmuir. 22, (2006).</li><li>New, R. R. C., New, R. R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Liposomes: A Practical Approach. , 33-104 (1990).</li><li>Chen, X., Lee, J., Jou, M. J., Kim, J. -. M., Yoon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+and+Fluorometric+Detection+of+Cationic+Surfactants+Based+on+Conjugated+Polydiacetylene+Supramolecules.">Colorimetric and Fluorometric Detection of Cationic Surfactants Based on Conjugated Polydiacetylene Supramolecules.</a> Chem. Commun. , 3434-3436 (2009).</li><li>Yarimaga, O., Im, M., Choi, Y. -. K., Kim, T. W., Jung, Y. K., Park, H. G., Lee, S., Kim, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Color+Display+System+Based+on+Thermochromic+Conjugated+Polydiacetylene.">A Color Display System Based on Thermochromic Conjugated Polydiacetylene.</a> Macromolecular Research. 18, 404-407 (2010).</li></ol>

Vi har udført selektiv binding af lysinrest af protein på liposomoverfladen med NHS-amin reaktion. Dette FRET metode er i stand til at gøre Realtidsovervågning af biotin-streptavidin-binding og protein (BSA) binding til liposomoverfladen. Tilsvarende procedure kan anvendes til at studere de bindende dynamikken i forskellige protein-interaktioner med deres selektive receptorer. Der er fleksibilitet til at vælge fluoroforer, der vil give ændringer i J-værdier afhængig af de spektrale karakteristika for de...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> 10,12-pentacosadiynoic acid (PCDA) </td><td> GFS kemikalier </td><td> 3261 </td><td> Lysfølsomme </td></tr><tr><td> N-hydroxysuccinimid (NHS) </td><td> Acros Organics </td><td> 157270250 </td><td> Fugtfølsomme </td></tr><tr><td> 1 - (3 - (dimethylamino) propyl)-3-ethylcarbodiimidhydrochlorid (EDC) </td><td> Chem-Impex International </td><td> 00.050 </td><td></td></tr><tr><td> 1,2-dimyristoyl-sn-glycero-3-phosphocholin (DMPC) </td><td> Avanti Polar Lipids </td><td> 850345P </td><td></td></tr><tr><td> Rhodamin-mærket bovin Serum Albumin (BSA-Rh) </td><td> Sigma Aldrich </td><td> A4537 </td><td></td></tr><tr><td> (1,2-dioleoyl-sn-glycero-3-phosphoethanolamin-N-(biotinyl) (biotin-DOPE) </td><td> Avanti Polar Lipids </td><td> 870.282 </td><td></td></tr></tbody></table>

real-time monitoring of ligand-receptor interactions with fluorescence resonance energy transfer

FRET er en proces, hvor energi er ikke-radiativt overført fra en exciteret donormolekyle til en ground state acceptor molekyle gennem langtrækkende dipol-dipol interaktioner 1. I det foreliggende sensing assay, benytter vi en interessant egenskab ved PDA: blå-forskydning i det UV-synlige elektroniske absorptionsspektrum for PDA (figur 1) efter en analyt vekselvirker med receptorer knyttet til PDA 2,3,4,7. Denne ændring i PDA absorptionsspektrum giver ændringer i spektral overlapning (J) mellem PDA (acceptor) og rhodamin (donor), der fører til ændringer i FRET effektivitet. Således er vekselvirkningen mellem analyt (ligand) og receptorer detekteres via FRET mellem donor fluoroforer og PDA. Især viser vi affølingen af ​​en model proteinmolekyle streptavidin. Vi viser også den kovalente-binding af bovint serumalbumin (BSA) til liposomoverfladen med FRET mekanisme. Disse interaktioner mellem than dobbeltlag liposomer og proteinmolekyler kan sanses i real-tid. Den foreslåede fremgangsmåde er en generel metode til at føle små kemiske og store biokemiske molekyler. Da fluorescens er i sig selv mere følsom end kolorimetri, kan detektionsgrænsen for assayet være i sub-nanomolære område eller lavere 8. Endvidere kan PDA fungere som en universal acceptor i FRET, hvilket betyder, at flere sensorer kan udvikles med PDA (acceptor) funktionaliseret med donorer og forskellige receptorer bundet på overfladen af ​​PDA liposomer.

Watch this Scientific Journal Video about Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer at JoVE.com

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

Vi viser FRET mellem konjugeret polymer polydiacetylene (PDA) og fluorofor bundet til overfladen af ​​PDA liposomer til affølingen af ​​biomolekyler. PDA liposomer indeholdt også receptormolekyler på deres overflader for biomolekyler, der skal anvendes som prober. Ligand-receptor-interaktioner fører til ændringer i FRET effektivitet mellem fluoroforen og PDA som er grundlaget for følemekanisme.

Real-time overvågning af ligand-receptor interaktioner med Fluorescence Resonance Energy Transfer

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

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