Name: microfluidic on-chip capture-cycloaddition reaction to reversibly immobilize small molecules or multi-component structures for biosensor applications
Uploaded: 2013-09-23T18:15:00
Description: Watch this Scientific Journal Video about Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications at JoVE.

Vi anerkender midler fra NIH (NHLBI kontrakt nr. HHSN268201000044C til RW, SH og SYS).

1.. Fremstilling af GST og Nanopartikel (NP) Konjugater <ol><li> GST-TCO forberedelse: <ol><li> Tilføj 8 pi TCO-NHS-opløsning (50 mM i DMSO) til 100 pi GST (1 mg / ml i PBS) og omrystes blandingen ved stuetemperatur i 1 time. </li><li> Fjern overskydende reagens ved hjælp af en Zeba spin-afsaltningssøjle. Det udvundne filtrat indeholdende GST-TCO konjugatet opbevaret ved 4 ° C før brug. </li></ol></li><li> GST-Tz forberedelse: <ol><li> Tilsæt 6 pi Tz-NHS-opløsning (25 mM i DMF) til 75 pi GST (1 mg / ml i PBS),...

<ol>
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Den her beskrevne capture-cykloaddition metode muliggør hurtig, reversibel immobilisering af modificerede nanopartikler og små molekyler til label-fri chip-baserede interaktion og kinetiske undersøgelser. Immobiliseringen protokol kan udføres på få minutter kræver &lt;10 uM koncentration af små molekyler ligander. Ved at modulere ligand koncentration og kontakttid immobiliseringstrin tætheder kan nøje kontrolleres. Vore data viser, at on-chip bioorthogonal reaktioner bevare muligheden for in situ-funktiona...

Effektive konjugeringsreaktioner er værdifulde værktøjer til fastgørelse bioaktive molekyler til overflader til forskellige bioteknologiske anvendelser. For nylig har den meget hurtige bioorthogonal [4 +2] cycloaddition reaktion mellem trans-cycloocten (TCO) og 1,2,4,5-tetrazin (Tz) blevet anvendt til at mærke celleoverflader, subcellulære strukturer, antistoffer og nanopartikler 1. - 7 Her bruger vi [4 +2] cycloadditionsreaktion i kombination med antigen / antistof-capture (GST / anti-GST) til reversibel on-chip syntese af multi-komponent strukturer for Surface (SPR) interaktion analyse og overvåge proces i realtid (Figur 1). 8,9 Især capture-cykloaddition strategi gør det muligt overflade regeneration ved hjælp af en fastlagt protokol. 8. Som følge heraf samling af stabile sensor overflader med kontrol over ligand orientering og tæthed for forskellige nye assay formater er nu muligt. Brugdenne strategi vi vise immobilisering af TCO / Tz-derivatiserede små molekyler og karakterisere cycloaddition satser i en række forskellige bufferbetingelser. Vi valgte den velkendte vekselvirkning mellem FKBP12 og et molekyle, AP1497, der binder FKBP12 10-12 som et eksempel for at kontrollere, at indfangning-cykloaddition strategi bevarer evnen af lille molekyle til at interagere med sit mål, når enten direkte knyttet til immobiliserede GST-antigener eller immobiliserede nanopartikler (NPS). Denne metode giver flere fordele. Først reversibel immobilisering af små molekyler på sensorchips er nu muligt. For det andet, TCO / Tz immobilisering af små molekyler også mulighed label-fri interaktionsundersøgelser der vende retningen af ​​kanoniske SPR undersøgelser, og kan give en supplerende visning af en bindende interaktion. For det tredje, denne metode gør det muligt mikrofluid syntese af målrettede nanopartikler, og øjeblikkelig vurdering af deres binding eng egenskaber. Det tegner til at forbedre effektiviteten af evaluering eller screening målrettede nanopartikler, og også mindske mængden af nanopartikler, der kræves. 13-15 fjerde kan denne tilgang måle reaktionskinetik af bioorthogonal cykloaddition reaktioner i realtid under konstant flow. Endelig TCO / Tz immobilisering kemi er robust i tilstedeværelse af serum. Tilsammen forventer vi, at denne alsidige fremgangsmåde stort set vil lette konstruktion af stabile sensor overflader til en lang række mikrofluide undersøgelser med relevans for in vitro og in vivo cellulære applikationer.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Sensor Chip CM5</td>
			<td>GE Healthcare</td>
			<td>BR-1005-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine coupling kit</td>
			<td>GE Healthcare</td>
			<td>BR-1000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>GST capture kit</td>
			<td>GE Healthcare</td>
			<td>BR-1002-23</td>
			<td></td>
		</tr>
		<tr>
			<td>NAP-10 Columns</td>
			<td>GE Healthcare</td>
			<td>17-0854-01</td>
			<td></td>
		</tr>
		<tr>
			<td>GST, lyophilized in 1X PBS</td>
			<td>Genscript</td>
			<td>Z02039</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>rhFKBP12</td>
			<td>R&#38;D Systems</td>
			<td>3777-FK</td>
			<td></td>
		</tr>
		<tr>
			<td>Surfactant P-20</td>
			<td>GE Healthcare</td>
			<td>BR-1000-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine 2.0</td>
			<td>GE Healthcare</td>
			<td>BR-1003-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba spin desalting column</td>
			<td>Thermo</td>
			<td>89882</td>
			<td>7 K MWCO</td>
		</tr>
		<tr>
			<td>Amicon Ultra 4</td>
			<td>Fisher</td>
			<td>UFC810096</td>
			<td>100 K centrifugal filter</td>
		</tr>
		<tr>
			<td>TCO-OH</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>TCO-NHS</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house, *Commercially available from Click Chemistry Tools # 1016-25</td>
		</tr>
		<tr>
			<td>Tz-BnNH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>Tz-NHS</td>
			<td>Ref. 8</td>
			<td>764701</td>
			<td>Synthesized in-house, *Commercially available from Sigma Aldrich # 764701</td>
		</tr>
		<tr>
			<td>NP-NH2 = CLIO-NH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>AP1497, AP1497-Tz</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>SPR Biosensor</td>
			<td>GE Healthcare</td>
			<td>Biacore T100</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic on-chip capture-cycloaddition reaction to reversibly immobilize small molecules or multi-component structures for biosensor applications

Metoder til hurtig overflade immobilisering af bioaktive små molekyler med kontrol over orientering og immobilisering tæthed er særdeles ønskeligt for biosensor og microarray applikationer. I denne undersøgelse, bruger vi en meget effektiv covalent bioorthogonal [4 +2] cykloaddition reaktion mellem trans-cycloocten (TCO) og 1,2,4,5-tetrazin (Tz) for at aktivere mikrofluid immobilisering af TCO / Tz-derivatiserede molekyler . Vi overvåger processen i realtid under kontinuerlig strømning ved hjælp af overflade (SPR). For at aktivere reversibel immobilisering og udvide den eksperimentelle sensorens overflade kombinerer vi en ikke-kovalent antigen-antistof-capture komponent med cycloaddition reaktion. Ved skiftevis at præsentere TCO eller TZ dele til sensoroverfladen, flere capture-cycloaddition processer er nu muligt på en sensor overflade for on-chip montage og interaktion undersøgelser af en række flerkomponent-strukturer. Vi illustrate denne metode med to forskellige immobiliseringsmetoder eksperimenter på en biosensor chip, et lille molekyle, AP1497, der binder FK506-bindende protein 12 (FKBP12), og den samme lille molekyle som del af et immobiliseret, og in situ-funktionaliserede nanopartikler.

Oplysninger og tal er blevet tilpasset fra henvisning 8. Effektiv reversibel immobilisering af bioaktive små molekyler kontrol over orientering og tæthed spiller en central rolle i udviklingen af ​​nye biosensor applikationer. Brug den hurtige bioorthogonal reaktion mellem TCO og Tz beskriver vi en metode til trinvis samling og regenerering af ligand overflader med bibeholdelse af biologisk aktivitet 2 viser real-time overvågning af Tz-BnNH 2 immobilisering...

Watch this Scientific Journal Video about Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications at JoVE.

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Vi præsenterer en metode til hurtig, reversibel immobilisering af små molekyler og funktionaliserede nanopartikler forsamlinger for Surface (SPR) undersøgelser, ved hjælp af sekventiel on-chip bioorthogonal cykloaddition kemi og antistof-antigen capture.

Mikrofluid On-chip Capture-cycloadditionsreaktion reversibelt Immobilisere små molekyler eller Multikomponenttekstilprodukter strukturer biosensoranvendelser

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

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