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.

Forsiktig: Flere kjemikalier som brukes i denne protokollen er irriterende og er farlig ved ved hudkontakt. Konsultere sikkerhetsdatablad (MSDS) og bruke egnet verneutstyr før du utfører denne protokollen. Piraja oppløsning som anvendes i trinn (1.1.1) er sterkt etsende, og må fremstilles ved tilsetning av peroksyd sakte til syren under omrøring. Håndtere denne løsningen med ekstrem forsiktighet hos en avtrekkshette. Bruk egnet øyebeskyttelse og syrefaste hansker. Trimetylklorsilan (TMCS) er et etsende, brannfar...

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Strømningsmønsteret utforming er den første kritiske trinn i fremstilling av 2-D microarray. For å generere to overlappende DNA-mønster på et glass-substrat, bør kanal trekk ved den første konstruksjon være vinkelrett på de i den andre (Figur 1A-B). Designene også vurdere nedstrøms anvendelser av microarray. I tilfelle av enkeltcelleanalyse, blir brukt til å detektere mikromatriseproteiner fra enkeltceller innesluttet i microchambers derfor dimensjonene kanal gjøres forenlig med d...

Antistoff mikromatriser har blitt mye brukt i proteomikk studier i flere tiår for å undersøke tilstedeværelse av målrettede proteiner, inkludert protein biomarkører 1-3. Selv om dette feltet er i dag overfor store utfordringer fra andre high-throughput teknologier som massespektrometri (MS), er det fortsatt god plass for nytten av antistoff mikromatriser, hovedsakelig fordi disse enhetene råd enkel tolking og enkelt grensesnitt med andre analyser. I de senere årene har integreringen av mikromatriser inn microchip stillaser gitt antistoffet microarray en ny mulighet til å trives 4-7. For eksempel har strekkode microarray integrert i en encellet mikrobrikke blitt brukt i celle kommunikasjon studier 8,9. Denne teknologien har særegne fordeler fremfor andre tilgjengelige microarray teknologi. Det har array elementer på 10-100 mikrometer, mye mindre enn den typiske 150 mikrometer størrelsen som brukes i konvensjonelle microarray elets. Konstruksjonen av mindre oppstillingselementene oppnås ved hjelp av systematiske strømningsmønster nærmer seg, og dette gir opphav til kompakte mikromatriser som kan oppdage encellet utskilte proteiner og intracellulære proteiner. En annen fordel er bruken av en enkel, instrument-fri oppsett. Dette er spesielt viktig, fordi de fleste laboratorier og små selskaper ikke kan være i stand til å få tilgang microarray kjernefasiliteter. En slik strek antistoff mikromatriser funksjonen forbedret assay gjennomstrømming, og kan brukes til å utføre analyser på multipleksede meget enkle celler samtidig oppnå høy sensitivitet og spesifisitet sammenlignes med den for konvensjonell sandwich-enzymbundet immunosorbent assay (ELISA 8). Denne teknologien har funnet en rekke programmer for å påvise proteiner fra glioblastom 9-11, T-celler 12, og sirkulerende tumorceller 13. Alternativt strekkode DNA-mikromatriser alene er blitt anvendt i nøyaktig posisjonering av neuroner og astrocytter for mimicking in vivo sammenstillingen av hjernevev 14. Denne protokollen fokuserer bare på de eksperimentelle skritt og bygge opp blokker av to-dimensjonale (2D) strekkode antistoff microarray som har potensielle bruksområder i påvisning av biomarkører i fluidic prøver og i enkeltceller. Teknologien er basert på et adresserenkeltrådet, endimensjonale (1-D) DNA microarray konstruert ved hjelp av ortogonale oligonukleotider som er mønstret romlig på glass underlag. Den 1-D mønsteret er dannet når parallelle strømningskanaler blir brukt i strømningsmønstertrinn, og et slikt mønster fremstår som adskilte bånd visuelt ligner på en D-Universal Product Code (UPC) strekkoder. Konstruksjonen av et 2-D (n x m) antistoff-gruppe - som minner om en 2-D Rask respons (QR) matrise-kode - behov for mer kompliserte mønster strategier, men gjør det mulig for immobilisering av antistoffer på en høyere tetthet 8,15. Fabrikasjonkrever to DNA mønstringstrinn, med det første mønsteret vinkelrett på den andre. Krysningspunkt mellom disse to mønstrene danner de n x m elementene i matrisen. Ved strategisk å velge sekvensene av enkelt-trådet DNA (ssDNA) anvendes i flow-mønster, blir hvert element i en gitt matrise tilordnes en bestemt adresse. Denne romlige referanse er nødvendig å skille mellom fluorescenssignaler på microarray lysbildet. SsDNA matrisen omdannes til et antistoff matrise gjennom inkorporering av komplementære DNA-antistoff-konjugater, som danner en plattform som kalles DNA-kodede antistoff-bibliotek (DEAL 16). Denne videoen protokollen beskriver de viktigste trinnene i å skape NxM antistoff arrays som inkluderer forbereder polydimethylsiloxane (PDMS) strekkode muggsopp, flow-mønster ssDNA i to retninger, forbereder antistoff-oligonukleotid DEAL konjugater, og konvertere den 3 x 3 DNA array i et trex 3 antistoff array.

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			<td height="21" style="height:21px;width:263px;">SU-8 2025 photoresist</td>
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			<td height="21" style="height:21px;width:263px;">Silicon wafers&#160;</td>
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			<td style="width:119px;">452</td>
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			<td height="21" style="height:21px;width:263px;">Poly-L-lysine coated glass slides</td>
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			<td style="width:119px;">C40-5257-M20</td>
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			<td height="21" style="height:21px;width:263px;">Oligonucleotides</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;">1x Phosphate buffered saline, pH 7.4</td>
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			<td height="40" style="height:40px;width:263px;">&#196;kta Explorer 100 Fast Protein Liquid Chromatography (FPLC) System</td>
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			<td>18-1112-41</td>
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			<td>17-5172-01</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 height="40" style="height:40px;width:263px;">Succinimidyl-6-hydrazino-nicotinamide (S-HyNic)</td>
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			<td style="width:119px;">S-1004</td>
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			<td height="21" style="height:21px;width:263px;">N,N-dimethylformamide</td>
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			<td style="width:119px;">227056</td>
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			<td height="21" style="height:21px;width:263px;">Sodium hydroxide</td>
			<td style="width:403px;">Fisher Scientific</td>
			<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>UFC201024</td>
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			<td height="21" style="height:21px;width:263px;">Spin coater</td>
			<td style="width:403px;">Laurell Technologies</td>
			<td>WS-650-MZ</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 height="21" style="height:21px;">Diamond scribe (Style 60)</td>
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			<td height="21" style="height:21px;">Trimethylchlorosilane</td>
			<td style="width:403px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">92361</td>
			<td></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.

Motivene for PDMS muggsopp (figur 1A-1B) ble trukket ved hjelp av et CAD-program (AutoCAD). To design vist har kanaler for flyt mønster, en horisontal og en vertikal. Til venstre og høyre del av hver design er symmetrisk; noen av dem kan være innløp eller utløp. Hver av kanalene 20 er viklingen fra den ene ende helt til den andre enden. Hvert design er trykt på en krom fotomaske (figur 1C). Den fremstilte SU-8 Hoved på en skive er vist i f...

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 Fabrikasjon av høy tetthet Barcode Antistoff Microarray

Denne protokollen skisserer fabrikasjon av en storstilt, multiplekset todimensjonal DNA eller antistoff array, med potensielle bruksområder i cellesignale studier og biomarkør gjenkjenning.

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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A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

En Guidet Materialer Screening tilnærming for å utvikle kvantitative Sol-gel produsert Protein Mikromatriser

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

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

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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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This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films.  Typically, ultrathin films are prepared ...

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Chemo-enzymatisk syntese av N- glykaner for matrisen utvikling og HIV antistoff profilering

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

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This paper aims to present a protocol to form smooth and well-controlled films of silver/silver chloride (Ag/AgCl) with designated coverage on top of thin ...