Name: multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles
Uploaded: 2013-10-10T21:00:00
Description: Watch this Scientific Journal Video about Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles at JoVE.com

Dette arbejde blev støttet af National Institutes of Health (Grant 08UDE017788-05). EBP anerkender også støtte fra den spanske Foundation for Videnskab og Teknologi (FECYT). Forfatterne takker Shonda T. Gaylord og Pratyusha Mogalisetti for kritisk læsning af manuskriptet.

<img alt="Figur 1" width="600px" fo:content-width="5.5in" src="/files/ftp_upload/50726/50726fig1.jpg" /> Figur 1. . Arbejdsgang for anvendelse fiberoptisk mikrosfære antistof array til spyt profilering (1) Mikrosfærer internt kodet med to fluorescerende farvestoffer, (2) de kodede mikrosfærer eksternt modificeret med protein-specifikke monoklonale antistoffer, (3) de multipleksede mikrosfærer blandes, og (4) tilfældigt deponeret i mikrobrønde ætset ved d...

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Assoc. 137, 313-321 (2006).</li><li>Segal, A., Wong, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+diagnostics:+enhancing+disease+detection+and+making+medicine+better.">Salivary diagnostics: enhancing disease detection and making medicine better.</a> Eur. J. Dent. Educ. 12, 22-29 (2008).</li><li>St John, M. A. R., 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=Interleukin+6+and+interleukin+8+as+potential+biomarkers+for+oral+cavity+and+oropharyngeal+squamous+cell+carcinoma.">Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma.</a> Arch. Otolaryngol. Head Neck Surg. 130, 929-935 (2004).</li><li>Herr, A. E., 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=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 5268-5273 (2007).</li><li>Pantano, P., Walt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+nanowell+arrays.">Ordered nanowell arrays.</a> Chem. Mater. 8, 2832-2835 (1996).</li><li>Schena, 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> Protein Microarrays. , (2005).</li></ol>

Forskerne bør være ekstra opmærksom på de følgende trin: for bedre afkodning nøjagtighed, er det nødvendigt at kontrollere de mikrosfærer blev homogent suspenderet i al inkubation og vask skridt i løbet af mikrosfærer kodning procedure. Desuden skal beskyttes mod lys i hele eksperimentet kodede mikrosfærer. Efter korrekt kodning og opbevaring procedurer, fandt vi, at den samlede dekodning nøjagtighed var over 99%. De kodede mikrosfærer skal opbevares ved 4 ° C. Undgå nedfrysning og beskytte mod ly...

Siden den første microarray rapporteret af Mark Schena og kolleger i midten af 1990&#39;erne, har denne kraftfulde værktøj er blevet brugt i mange områder af biologisk forskning 1. Antistof microarrays i stand til samtidigt at detektere multiple proteiner i diagnostiske fluider, såsom blod, har vigtige anvendelser i klinisk diagnostik og biomarkør screening 2-10. Spyt, som indeholder mange af de samme analytter såsom blod, er blevet betragtet som et foretrukket alternativ til blod, fordi spyt samling er sikker, ikke-invasiv og kan udføres ved minimalt uddannet medicinsk personale 11-13. I øjeblikket er multiplekset proteinanalyse ved hjælp af spytprøver begrænset af flere vigtige faktorer, herunder den lave koncentration af målanalyt 14 og bredt koncentrationsområde af forskellige biomarkører 15. . Heri påviser vi, at analysen af ​​seks proteiner: human vaskulær endotel vækstfaktor (VEGF), interferon gamma-inducerede protein 10 (IP-10), interleukin-8 (IL-8), epidermal vækstfaktor (EGF), matrix metallopeptidase 9 (MMP-9) og interleukin-1 beta (IL-1β) . Udførelsen af ​​metoden blev oprindeligt kontrolleres ved hjælp af standardløsninger, der udgør rekombinante analyt proteiner og blokerende buffer. Rigtige spytprøver indsamlet fra patienter med forskellige kroniske luftvejssygdomme samt raske kontrolpersoner blev også testet med tilfredsstillende præstation. Protokollen skal være gældende for andre protein analytter og andre mikrosfære-baserede analyser. Denne platform giver betydelige fordele for Analytisk Kemi banen, da det giver mulighed for hurtig, præcis og reproducerbar samtidig analyse af lave koncentrationer af flere proteiner med et bredt dynamikområde, minimale ikke-specifikke interaktioner, nedsat forbrug prøve, og lave omkostninger i forhold til en analog enzymmaerket Assay (ELISA).

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Eu-TTA dye</td>
			<td>Fisher Scientific</td>
			<td>AC42319-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>THF</td>
			<td>Sigma-Aldrich</td>
			<td>34865-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber glass vial</td>
			<td>Fisher Scientific</td>
			<td>03-339-23B</td>
			<td></td>
		</tr>
		<tr>
			<td>Coumarin 30 dye</td>
			<td>Sigma-Aldrich</td>
			<td>546127-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspheres</td>
			<td>Bangslabs</td>
			<td>PC05N/6698</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 10x concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma-Aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tw-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-100 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>BupH MES buffered saline</td>
			<td>Thermo Scientific</td>
			<td>28390</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich</td>
			<td>05030-500ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution</td>
			<td>Fisher Scientific</td>
			<td>SS256-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-lock microcentrifuge tube</td>
			<td>VWR labshop</td>
			<td>53511-997</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC</td>
			<td>Thermo Scientific</td>
			<td>22980</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS</td>
			<td>Thermo Scientific</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB293</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IP-10 capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB266</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB208</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB636</td>
			<td></td>
		</tr>
		<tr>
			<td>Human MMP-9 capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB936</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-1&#946; capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB601</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 isotype control antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB002</td>
			<td></td>
		</tr>
		<tr>
			<td>StartingBlock (TBS) buffer</td>
			<td>Thermo Scientific</td>
			<td>37542</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl standard solution 1.0 N</td>
			<td>Sigma-Aldrich</td>
			<td>318949-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein-free (PBS) buffer</td>
			<td>Thermo Scientific</td>
			<td>37572</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human VEGF 165</td>
			<td>R&#38;D Systems</td>
			<td>293-VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IP-10</td>
			<td>R&#38;D Systems</td>
			<td>266-IP</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human MMP-9</td>
			<td>R&#38;D Systems</td>
			<td>911-MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-1&#946;</td>
			<td>R&#38;D Systems</td>
			<td>201-LB</td>
			<td></td>
		</tr>
		<tr>
			<td>StartingBlock T20 (PBS) buffer</td>
			<td>Thermo Scientific</td>
			<td>37539</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocker BSA in PBS</td>
			<td>Thermo Scientific</td>
			<td>37525</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated VEGF detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF293</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated IP-10 detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF266</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated IL-8 detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF208</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated EGF detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF236</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated MMP-9 detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF911</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated IL-1&#946; detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF201</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin, R-phycoerythrin</td>
			<td>Invitrogen</td>
			<td>S-21388</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (200 proof)</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles

Heri beskriver vi en protokol for samtidig måling seks proteiner i spyt ved hjælp af et fiberoptisk mikrosfære-baserede antistof array. Immuno-array-teknologi anvendes kombinerer fordelene ved mikrosfære-baseret suspension matrix fabrikation med anvendelse af fluorescensmikroskopi. Som beskrevet i videoen protokol blev kommercielt tilgængelige 4,5 um polymermikrokugler kodet i syv forskellige typer, differentieret ved koncentrationen af ​​to fluorescerende farvestoffer fysisk fanget inde mikrosfærerne. De kodede mikrosfærer indeholdende overfladeaktive carboxylgrupper blev ændret med monoklonale fangantistoffer via EDC / NHS koblingskemi. For at samle protein microarray, de forskellige typer af kodede og funktionaliserede mikrosfærer blev blandet og tilfældigt deponeret i 4,5 um mikrobrønde, som blev kemisk ætset ved den proximale ende af et fiberoptisk bundt. Det fiberoptiske bundt blev anvendt som både bærer og til afbildning af microspheres. Når den er samlet, blev microarray bruges til at indfange proteiner i spyttet supernatanten opsamles fra klinikken. Påvisningen var baseret på en sandwich-immunassay under anvendelse af en blanding af biotinylerede antistoffer til diagnosticering af forskellige analytter med streptavidin-konjugeret fluorescerende probe, R-phycoerythrin. Microarray blev afbildet ved fluorescensmikroskopi i tre forskellige kanaler, to til mikrosfære registrering og én for assaysignalet. Fluorescens-mikrografer blev derefter afkodes og analyseret under anvendelse af et hjemmelavet algoritme i MATLAB.

Fluorescens billeder fra tre kanaler, der viser en lille del af det fiberoptiske bundt er vist i figur 2A-C. Disse billeder blev analyseret under anvendelse af en algoritme, som er skrevet i Matlab (som beskrevet mere detaljeret i diskussionen afsnit). Analysen beskæftiger både information fra EU-TTA kodning billede (figur 2A) og C30 kodning billedet (Figur 2B) til at afkode mikrokugler, og fluorescensintensiteterne forskellige mikrokugler i signalet billedet ...

Watch this Scientific Journal Video about Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles at JoVE.com

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Multiplexed Fluorescent Microarray for Human spytprotein analyse ved hjælp polymermikrosfærer og fiber-optisk Bundter

Vi beskriver en procedure for profilering spytproteiner hjælp multiplexede mikrosfære-baserede antistof arrays. Monoklonale antistoffer blev kovalent bundet til fluorescerende farvestof-kodet 4,5 um polymermikrokugler hjælp carbodiimidkemi. De modificerede mikrosfærer blev deponeret i fiberoptiske microwells&#39;ne måle protein niveauer i spyt ved hjælp af fluorescens sandwichimmunassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

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Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Tidsopløst Photophysical karakterisering af Triplet-høst organiske forbindelser på en ilt-frit miljø ved hjælp af en iCCD kamera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Ved hjælp af cyklisk Voltammetry, UV-Vis-NIR og EPR Spectroelectrochemistry til at analysere organiske forbindelser

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...