Name: flow-pattern guided fabrication of high-density barcode antibody microarray
Uploaded: 2016-01-06T15:00:01
Description: Watch this Scientific Journal Video about Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray at JoVE.com

The authors would like to acknowledge the startup fund from SUNY Albany and the access of facilities at the University at Albany Cancer Research Center.

Forsigtig: Flere kemikalier, der anvendes i denne protokol er irritanter og er farlig i tilfælde af kontakt med huden. Consult sikkerhedsdatablade (MSDS) og slid egnede personlige værnemidler før du udfører denne protokol. Den Piranha anvendt i trin (1.1.1) er stærkt ætsende og skal fremstilles ved tilsætning af peroxid langsomt til syren under omrøring. Håndtere denne løsning med ekstrem forsigtighed i et stinkskab. Brug passende øjenbeskyttelse og syre-resistente handsker. Trimethylchlorsilan (TMCS) er en ?...

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Strømningsmønster design er den første kritiske trin ved fremstilling af 2-D microarray. At generere to overlappende DNA mønstre på et glassubstrat, bør den kanal funktioner i det første design være vinkelret på de af den anden (figur 1A-B). De designs også overveje de nedstrøms anvendelser af microarray. I tilfælde af en enkelt celleanalyse, der mikroarray anvendes til at detektere proteiner fra enkelte celler indesluttet i microchambers derfor kanaldimensionerne de gøres foreneli...

Antistof mikroarrays er ofte blevet brugt i proteomiske undersøgelser i årtier for at undersøge tilstedeværelsen af målrettede proteiner, herunder protein biomarkører 1-3. Selvom dette felt i øjeblikket står over for store udfordringer fra andre high-throughput teknologier såsom massespektrometri (MS), er der stadig masser af plads til nytten af ​​antistof microarrays, især fordi disse enheder har råd til simple data fortolkning og nem grænseflade med andre analyser. I de senere år har integrationen af microarrays i microchip stilladser billede antistoffet microarray en ny mulighed for at trives 4-7. For eksempel har stregkoden microarray integreret i en enkeltcelle-mikrochip blevet anvendt i celle kommunikation studier 8,9. Denne teknologi har markante fordele i forhold til andre tilgængelige microarray teknologi. Den er udstyret med array elementer på 10-100 um, meget mindre end den typiske 150 um størrelse, der anvendes i konventionelle microarray elements. Opførelsen af ​​mindre array elementer opnås ved hjælp af systematiske flow-mønsterdannende tilgange, og det giver anledning til kompakte mikroarrays, der kan opdage encellede udskilte proteiner og intracellulære proteiner. En anden fordel er anvendelsen af ​​en enkel, instrument-fri setup. Dette er især vigtigt, fordi de fleste laboratorier og små virksomheder ikke kan være i stand til at få adgang til microarray core faciliteter. En sådan stregkode antistof microarrays funktionen forbedret assay gennemløb og kan bruges til at udføre meget multiplex assays af enkelte celler og samtidig opnå høj følsomhed og specificitet kan sammenlignes med konventionelle sandwich enzymbundet immunosorbent assay (ELISA 8). Denne teknologi har fundet talrige anvendelser i forbindelse med afsløring proteiner fra glioblastom 9-11, T-celler 12, og cirkulerende tumorceller 13. Alternativt stregkode DNA microarrays alene er blevet anvendt i den præcise placering af neuroner og astrocytter til mimicking in vivo samling af hjernevæv 14. Denne protokol fokuserer kun på de eksperimentelle trin og opbygge-up blokke af todimensionale (2-D) stregkode antistof microarray som har potentielle anvendelser i detektion af biomarkører i fluide prøver og i enkelte celler. Teknologien er baseret på et adresserbart enkeltstrenget, endimensional (1-D) DNA microarray konstrueret ved anvendelse af ortogonale oligonukleotider, der er mønstrede rumligt på glassubstrater. Den 1-D mønster dannes, når der anvendes parallelle strømningskanaler i strømningen-mønsterdannelsestrinnet og et sådant mønster vises som adskilte bånd visuelt ligner 1-D Universal Product Code (UPC) stregkoder. Opførelsen af en 2-D (n x m) antistof array - minder om en 2-D Quick Response (QR) matrixkode - brug for mere komplekse mønsterruller strategier, men giver mulighed for immobilisering af antistoffer på en højere densitet 8,15. Fabrikationenkræver to DNA mønsterdannende trin, med det første mønster vinkelret på den anden. Skæringspunkterne af disse to mønstre udgør n x m elementer i arrayet. Ved strategisk at vælge sekvenserne af enkeltstrenget DNA (ssDNA) anvendes i flow-mønsterdannelse, er hvert element i et bestemt opstilling tildelt en bestemt adresse. Denne rumlige henvisning er nødvendig at skelne mellem fluorescenssignaler på microarray dias. SsDNA matrix omdannes til et antistof matrix gennem inkorporering af komplementære DNA-antistof-konjugater, der danner en platform kaldet DNA-kodede antistof bibliotek (DEAL 16). Denne video protokol beskriver de vigtigste skridt i at skabe nxm antistof arrays, som omfatter forberedelse polydimethylsiloxan (PDMS) stregkode forme, flow-mønster ssDNA i to orienteringer, forbereder antistof-oligonukleotid DEAL konjugater, og konvertering af 3 x 3 DNA-array i en 3x 3 antistof array.

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			<td height="21" style="height:21px;width:263px;">Silicon wafers&#160;</td>
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			<td>*Custom-ordered from Integrated DNA Technologies, see table below</td>
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			<td height="21" style="height:21px;width:263px;">Capture antibodies</td>
			<td style="width:403px;">various</td>
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			<td>*Antibody selection depends on application</td>
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			<td style="width:119px;">S318</td>
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			<td height="21" style="height:21px;width:263px;">Amicon Ultra spin filter 10 kDa MWCO</td>
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			<td height="21" style="height:21px;">Biopsy punch with plunger (0.50 mm diameter)</td>
			<td style="width:403px;">Electron Microscopy Sciences</td>
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			<td align="right" style="width:119px;">92361</td>
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flow-pattern guided fabrication of high-density barcode antibody microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

Motiverne for de PDMS forme (figur 1A-1B), er opstillet ved hjælp af et CAD-program (AutoCAD). To designs vist har kanaler for strømning mønster, en vandret og en lodret. De venstre og højre dele af hvert design er symmetriske; en af ​​dem kunne være indløb eller udløb. Hver af 20 kanaler vikling fra den ene ende hele vejen til den anden ende. Hvert design er trykt på en krom fotomaske (figur 1C). Den færdige SU-8 Master på en wafer <strong...

Watch this Scientific Journal Video about Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray at JoVE.com

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Flow-mønster Guidet Fremstilling af High-density Barcode Antibody Microarray

Denne protokol beskriver fremstillingen af ​​en storstilet, multiplex todimensional DNA eller antistof array, med mulige anvendelser i cellesignalering undersøgelser og biomarkør opdagelse.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

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