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 erkjenner midler fra NIH (NHLBI kontrakt nummer HHSN268201000044C til RW, SH og SYS).

En. Utarbeidelse av GST og nanopartikler (NP) Konjugater <ol><li> GST-TCO forberedelse: <ol><li> Tilsett 8 mL av TCO-NHS-løsning (50 mM i DMSO) til 100 mL av GST (1 mg / ml i PBS) og ryste blandingen ved RT i 1 time. </li><li> Fjern overflødig reagens ved hjelp av en Zeba spin avsalting kolonne. Det utvunnede filtratet inneholdende GST-TCO konjugatet lagres ved 4 ° C før bruk. </li></ol></li><li> GST-Tz forberedelse: <ol><li> Tilsett 6 mL av Tz-NHS-løsning (25 mM i DMF) i 75 mL av GST (1 mg / ml i PBS) og ryste b...

<ol>
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Fangst-cycloaddition metoden beskrevet her muliggjør rask, reversibel immobilisering av modifiserte nanopartikler og små molekyler for label-fri chip-basert samhandling og kinetiske studier. Immobiliseringen protokollen kan utføres på få minutter som krever &lt;10 uM konsentrasjon av små-molekyl ligander. Ved modulering av ligandkonsentrasjon og kontakttid immobilisering tettheter kan reguleres nøye. Våre data viser at on-chip bioorthogonal reaksjoner bevare muligheten for in situ functionalized nanopar...

Effektiv konjugeringsreaksjoner er verdifulle verktøy for å feste bioaktive molekyler til overflater for en rekke av bioteknologi. Nylig har meget rask bioorthogonal [4 2] cycloaddition reaksjon mellom trans-cyclooctene (TCO) og 1,2,4,5-tetrazine (TZ) blitt brukt til å merke celleoverflater, subcellulære strukturer, antistoffer og nanopartikler en. - 7 Her bruker vi [4 +2] cycloaddition reaksjon i kombinasjon med antigen / antistoff fangst (GST / anti-GST) for reversibel on-chip syntese av flerkomponent strukturer for Surface Plasmon Resonans (SPR) interaksjonsanalyse og overvåke Prosessen i sanntid (figur 1). 8,9 Spesielt muliggjør fangst-cycloaddition strategi overflate regenerering ved hjelp av en etablert protokoll. 8. Som følge av monteringen av stabile sensoroverflater med kontroll over ligand retning og tettheten for forskjellige nye assay formatene er nå mulig. Brukedenne strategien vi demonstrere immobilisering av TCO / Tz-derivatized små molekyler og karakterisere cycloaddition priser i en rekke bufferbetingelser. Vi valgte velkjente interaksjon mellom FKBP12 og et molekyl som binder AP1497 FKBP12 10-12 som et eksempel for å verifisere at fangst-cycloaddition strategi bevarer evnen av den lille molekylet for å interagere med dens target når enten direkte festet til immobilisert GST antigen eller til immobilisert nanopartikler (NPS). Denne metoden gir flere fordeler. For det første er det nå mulig den reversible immobiliseringen av små molekyler på sensorbrikker. Sekund, TCO / Tz immobilisering av små molekyler kan også label-free interaksjonsstudier som reverserer retningen på kanoniske SPR studier, og kan gi en utfyllende visning av en forpliktende samhandling. For det tredje gir denne metoden microfluidic syntese av målrettede nanopartikler, og øyeblikkelig vurdering av deres binding egenskaper. Dette lover å forbedre effektiviteten av å evaluere eller screening målrettede nanopartikler, og også redusere mengden av nanopartikler som kreves. 13-15 Fjerde, kan denne tilnærmingen måle reaksjonskinetikken bioorthogonal cycloaddition reaksjoner i sanntid under kontinuerlig flyt. Endelig er den TCO / Tz immobilisering kjemi robust i nærvær av serum. Til sammen forventer vi at denne allsidige tilnærming vil grovt rette for bygging av stabile sensoroverflater for et bredt utvalg av microfluidic studier med relevans til in vitro og in vivo cellulære applikasjoner.

<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 for rask overflate immobilisering av bioaktive små molekyler med kontroll over retningen og immobilisering tetthet er svært ønskelig for biosensor og microarray-applikasjoner. I denne studien bruker vi en svært effektiv kovalent bioorthogonal [4 2] cycloaddition reaksjon mellom trans-cyclooctene (TCO) og 1,2,4,5-tetrazine (TZ) for å aktivere microfluidic immobilisering av TCO / Tz-derivatized molekyler . Vi overvåke prosessen i sanntid i henhold til kontinuerlige strømningsbetingelser ved å bruke overflate-plasmonresonans (SPR). For å muliggjøre reversibel immobilisering og forlenge forsøksområdet til sensoroverflaten, vi kombinerer en ikke-kovalent antigen-antistoff-fangst komponent med cycloaddition reaksjonen. Ved vekselvis å presentere TCO eller Tz rester til sensoroverflaten, flere fangst-cykloaddisjon prosesser er det nå mulig på en sensorflaten for on-chip monterings-og interaksjons studier av en rekke flerkomponent-strukturer. Vi illustrate denne metoden med to forskjellige immobilisering eksperimenter på en biosensor chip, et lite molekyl, AP1497 som binder FK506-bindende protein 12 (FKBP12), og den samme lille molekyl som en del av en immobilisert, og in situ-funksjonalisert nanopartiklene.

Data og figurer har blitt tilpasset fra referanse 8. Effektiv reversibel immobilisering av bioaktive små molekyler med kontroll over retningen og tetthet spiller en nøkkelrolle i utviklingen av nye biosensor applikasjoner. Ved hjelp av den raske bioorthogonal reaksjon mellom TCO og Tz, beskriver vi en metode for trinnvis montering og regenerering av ligand overflater med oppbevaring av biologisk aktivitet. Figur 2 viser sanntids overvåking av Tz-BnNH to immobil...

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 presenterer en fremgangsmåte for hurtig, reversible immobiliseringen av små molekyler og funksjonnanopartikkel sammensetninger for overflate-plasmonresonans (SPR) undersøkelser, ved hjelp av sekvensiell on-chip bioorthogonal cycloaddition kjemi og antistoff-antigen fangst.

Microfluidic On-chip Capture-cycloaddition Reaksjon på reversibelt Immobilize små molekyler eller flerkomponent Profiler for Biosensor Applications

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

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