Name: snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays
Uploaded: 2017-11-13T12:30:00
Description: Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

Biz Dr Rob Sladek mürekkep püskürtmeli gözcü kullanım için teşekkür ederim. Sağlık Araştırma (CIHR), Doğa Bilimleri ve mühendislik Araştırma Konseyi, Kanada (NSERC), Kanada Kanser Derneği Araştırma Enstitüsü ve Kanada Vakfı için son destek için yenilik (CFI) Kanadalı Enstitüleri üzerinden anıyoruz. DJ teşekkürler bir Kanada araştırma sandalyeden destekler.

1. imalat ve depolanmasını snap cips <ol><li>tek transfer yöntemi ( Şekil 1a) <ol><li>400 µg/mL antikorlar içeren Spot taksi çözümleri ve fosfat tamponlu tuz çözeltisi % 20 gliserol (PBS) bir nitroselüloz (ya da functionalized bir cam) üzerine tahlil slayt ile bağıl nem oranı % 60, bir mürekkep püskürtmeli Mikroarray gözcü 13 (1.2 nL her nokta için) 800 µm Merkezden merkeze boşluğu ile. Slaydı bir köşe göre gözcü güvertede sabit old...

<ol>
	<li>Mor, G., 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=Serum+protein+markers+for+early+detection+of+ovarian+cancer.">Serum protein markers for early detection of ovarian cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (21), 7677-7682 (2005).</li><li>Nicolini, A., 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=Intensive+post-operative+follow-up+of+breast+cancer+patients+with+tumour+markers:+CEA,+TPA+or+CA15.3+vs+MCA+and+MCA-CA15.3+vs+CEA-TPA-CA15.3+panel+in+the+early+detection+of+distant+metastases.">Intensive post-operative follow-up of breast cancer patients with tumour markers: CEA, TPA or CA15.3 vs MCA and MCA-CA15.3 vs CEA-TPA-CA15.3 panel in the early detection of distant metastases.</a> BMC Cancer. 6 (1), 1-9 (2006).</li><li>Hnasko, R., Lin, A., McGarvey, J. A., Stanker, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+to+improve+protein+detection+by+indirect+ELISA.">A rapid method to improve protein detection by indirect ELISA.</a> Biochem. Biophys. Res. Commun. 410 (4), 726-731 (2011).</li><li>Percy, A. J., Chambers, A. G., Yang, J., Hardie, D. B., Borchers, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+multiplexed+MRM-based+protein+biomarker+quantitation+toward+clinical+utility.">Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.</a> Biochim. Biophys. Acta - Proteins and Proteomics. 1844 (5), 917-926 (2014).</li><li>Ekins, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-analyte+immunoassay.">Multi-analyte immunoassay.</a> J Pharm Biomed Anal. 7 (2), 155-168 (1989).</li><li>Mahlknecht, P., 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=An+antibody+microarray+analysis+of+serum+cytokines+in+neurodegenerative+Parkinsonian+syndromes.">An antibody microarray analysis of serum cytokines in neurodegenerative Parkinsonian syndromes.</a> Proteome Sci. 10 (1), 71-80 (2012).</li><li>Miller, J. C., 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=Antibody+microarray+profiling+of+human+prostate+cancer+sera:+Antibody+screening+and+identification+of+potential+biomarkers.">Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers.</a> Proteomics. 3 (1), 56-63 (2003).</li><li>Li, H., Leulmi, R. F., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+droplet+microarrays+with+trapped+antibody-functionalized+beads+for+multiplexed+protein+analysis.">Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis.</a> Lab Chip. 11 (3), 528-534 (2011).</li><li>Juncker, D., Bergeron, S., Laforte, V., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-reactivity+in+antibody+microarrays+and+multiplexed+sandwich+assays:+shedding+light+on+the+dark+side+of+multiplexing.">Cross-reactivity in antibody microarrays and multiplexed sandwich assays: shedding light on the dark side of multiplexing.</a> Curr. Opin. Chem. Biol. 18, 29-37 (2014).</li><li>Blank, K., 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=Double-chip+protein+arrays:+force-based+multiplex+sandwich+immunoassays+with+increased+specificity.">Double-chip protein arrays: force-based multiplex sandwich immunoassays with increased specificity.</a> Anal. Bioanal. Chem. 379 (7), 974-981 (2004).</li><li>Albrecht, C., 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=DNA:+A+Programmable+Force+Sensor.">DNA: A Programmable Force Sensor.</a> Science. 301 (5631), 367-370 (2003).</li><li>Pla-Roca, M., 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=Antibody+Colocalization+Microarray:+A+Scalable+Technology+for+Multiplex+Protein+Analysis+in+Complex+Samples.">Antibody Colocalization Microarray: A Scalable Technology for Multiplex Protein Analysis in Complex Samples.</a> Mol. Cell. proteomics. 11 (4), (2012).</li><li>Li, H., Bergeron, S., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-to-Microarray+Transfer+of+Reagents+by+Snapping+of+Two+Chips+for+Cross-Reactivity-Free+Multiplex+Immunoassays.">Microarray-to-Microarray Transfer of Reagents by Snapping of Two Chips for Cross-Reactivity-Free Multiplex Immunoassays.</a> Anal. Chem. 84 (11), 4776-4783 (2012).</li><li>Li, H., Munzar, J. D., Ng, A., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+snap+chip+for+high-density+sub-nanoliter+chip-to-chip+reagent+transfer.">A versatile snap chip for high-density sub-nanoliter chip-to-chip reagent transfer.</a> Sci. Rep. 5, 11688 (2015).</li><li>Li, H., Bergeron, S., Annis, M. G., Siegel, P. M., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+analysis+of+38+proteins+during+the+progression+of+human+breast+tumor+in+mice+using+an+antibody+colocalization+microarray.">Serial analysis of 38 proteins during the progression of human breast tumor in mice using an antibody colocalization microarray.</a> Mol. Cell. Proteomics. 14, 1024-1037 (2015).</li><li>Bergeron, S., Laforte, V., Lo, P. S., Li, H., Juncker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+mixtures+of+14+hygroscopic+additives+to+improve+antibody+microarray+performance.">Evaluating mixtures of 14 hygroscopic additives to improve antibody microarray performance.</a> Anal. Bioanal. Chem. 407 (28), 8451-8462 (2015).</li><li>Whiteaker, J. 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=Sequential+Multiplexed+Analyte+Quantification+Using+Peptide+Immunoaffinity+Enrichment+Coupled+to+Mass+Spectrometry.">Sequential Multiplexed Analyte Quantification Using Peptide Immunoaffinity Enrichment Coupled to Mass Spectrometry.</a> Mol. Cell. Proteomics. 11 (6), (2012).</li><li>Xu, K., Wang, X., Ford, R. M., Landers, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Partitioned+Droplet+Array+on+Laser-Patterned+Superhydrophilic+Glass+Surface+for+Wall-less+Cell+Arrays.">Self-Partitioned Droplet Array on Laser-Patterned Superhydrophilic Glass Surface for Wall-less Cell Arrays.</a> Anal. Chem. 88 (5), 2652-2658 (2016).</li><li>Lee, M. -. Y., Park, C. B., Dordick, J. S., Clark, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolizing+enzyme+toxicology+assay+chip+(MetaChip)+for+high-throughput+microscale+toxicity+analyses.">Metabolizing enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (4), 983-987 (2005).</li><li>Fernandes, T. G., 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=Three-dimensional+cell+culture+microarray+for+high-throughput+studies+of+stem+cell+fate.">Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.</a> Biotechnol. Bioeng. 106 (1), 106-118 (2010).</li><li>Kwon, C. H., 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=Drug-Eluting+Microarrays+for+Cell-Based+Screening+of+Chemical-Induced+Apoptosis.">Drug-Eluting Microarrays for Cell-Based Screening of Chemical-Induced Apoptosis.</a> Anal. Chem. 83 (11), 4118-4125 (2011).</li><li>Jogia, G. E., Tronser, T., Popova, A. A., Levkin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+Microarray+Based+on+Superhydrophobic-Superhydrophilic+Patterns+for+Single+Cell+Analysis.">Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.</a> Microarrays. 5 (4), 28 (2016).</li><li>Schena, M., Shalon, D., Davis, R. W., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Monitoring+of+Gene+Expression+Patterns+with+a+Complementary+DNA+Microarray.">Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray.</a> Science. 270 (5235), 467-470 (1995).</li><li>Wu, J., 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=A+sandwiched+microarray+platform+for+benchtop+cell-based+high+throughput+screening.">A sandwiched microarray platform for benchtop cell-based high throughput screening.</a> Biomaterials. 32 (3), 841-848 (2011).</li></ol>

Bu çalışmada, araştırmacılar ile temel deneysel kurulum için çapraz-reactivity ücretsiz multiplex uzun yaygın olarak kullanılabilir duruma getirir bir ek çip teknolojisi sunulmuştur. Varolan antikor microarrays farklı, hiçbir Mikroarray gözcü son kullanıcılar için gereklidir. Hem tek hem de çift transfer yöntemleri gösterdi ve Çift Kişilik transfer affords üstün hizalama doğruluğu aşağı ~ % 98'i lekeli 63 µm14en büyük kayma için 40 mikron. Bir roman ek aparatı ...

Birden fazla protein oluşan biyolojik bir panel daha yüksek duyarlılık ve özgüllük kanserleri1,2gibi karmaşık hastalıkların tanısında tek bir biyomarker daha sağlayabilir. Enzim bağlı immunosorbent assay (ELISA) klinik laboratuvarlar bir limit düşük pg/mL plazma, algılama, ama sınırları tahlil3,4,5başına bir hedefe ulaşmada kullanılan altın standart teknoloji olmuştur. Paralel bir tek mikroskop slayt6,7,8bin-in küçültülmüş deneyleri accomodating yürütülen için antikor microarrays geliştirilmiştir. Ancak, bu yöntemin çoğullama yeteneği parmak izleri, karışımı uygulamadan kaynaklanan reaktif tahrik olan ile sınırlıdır ve hedefleri9,10 giderek artan sayıda daha sorunlu hale gelir , 11. Pla ve ark. Sonuçta ortaya çıkan güvenlik açığı bir multiplex sandviç testin hedefleri12numarası N olan 4N(N-1) ölçekler belirttiler.
Antikor microarrays, antikor colocalization Mikroarray olan etkisini azaltmak için (ACM) bizim laboratuvar multiplex sandviç tahlil12için geliştirilmiştir. Yakalama antikorlar (taksi) bir substrat Mikroarray gözcü ile tespit. Sonra engelleme örnekleri yüzeye uygulanır ve sonunda bireysel dAbs taksi-antijen kompleksi aynı noktalar üzerinde gördü. Antikorlar antijenleri arasındaki tüm olan senaryoları ACM ile azaltılabilir ve algılama pg/mL, sınırlarını elde ettik. Ancak, tahlil Protokolü hazırlama ve bir yerinde Mikroarray gözcü yüksek hassasiyetle pahalı ve zaman alıcı hizalama amaç için kullanarak, bu teknoloji içinde geniş uygulama sınırlama deneyler sırasında parmak izleri tespit gerekir diğer laboratuvarlar. Ek çip adında bir el ACM için çapraz-reactivity ücretsiz geliştirilmiştir ve gözcü-Alerjik multiplex sandviç uzun13,14,15. Kabinler ve parmak izleri bir tahlil slayt ve sırasıyla Mikroarray biçiminde bir transfer slayt üzerine önceden tespit ve depolanır. Tahlil sırasında slaytlar alınır ve dAbs Mikroarray aktarılır topluca tahlil slayt sadece birlikte iki fiş şaklatarak. Bir ek aparatı güvenilir reaktif aktarımı için kullanılır. Nispeten büyük antikor Bağlama kapasitesi ile kaplı nitroselüloz slaytlar sıvı damlacıkları emmek için tahlil slaytlar olarak kullanılmış ve böylece reaktif transfer kolaylaştırıcı, ancak, slaytları normal cam slaytlar ve Mikroarray daha daha pahalıdır saydam olmayan slaytlar ile uyumlu tarayıcılar sinyal alımı için ihtiyaç vardır.
Bu çalışmada, multiplex sandviç immunoassay ek çip ile gerçekleştirme Protokolü göstermektedir. Bir roman ek aparatı Mikroarray Mikroarray daha rahat ve güvenilir reaktif transferi için geliştirilmiştir. Önemlisi, burada reaktif aktarma yöntemi ek çip ile normal cam slaytlar üzerine kurduk. 1024 noktalar başarıyla transfer ve bu teknolojinin en laboratuarlarında kullanımı önemli ölçüde genişleyen bir cam slayt üzerine hizalanmalıdır.

<table>
	<tbody>
		<tr>
			<td>Phosphate buffered saline tablet</td>
			<td>Fisher Scientific</td>
			<td>5246501EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-conjugated Cy5</td>
			<td>Rockland</td>
			<td>s000-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>p1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Jackson ImmunoResearch Laboratories, Inc</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution: BSA-free StabilGuard Choice Microarray Stabilizer</td>
			<td>SurModics, Inc</td>
			<td>SG02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose coated slides</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>305116</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane coated slides</td>
			<td>Schott North America</td>
			<td>1064875</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap Device</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-SD01</td>
			<td></td>
		</tr>
		<tr>
			<td>Inkjet microarray spotter</td>
			<td>GeSiM</td>
			<td>Nanoplotter 2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide module gasket</td>
			<td>Grace Bio-Laboratories, Inc</td>
			<td>204862</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity Stabilization Beads</td>
			<td>Parallex BioAssays Inc.</td>
			<td>PBA-HU60</td>
			<td></td>
		</tr>
		<tr>
			<td>Array-Pro Analyzer software</td>
			<td>Media Cybernetics</td>
			<td>Version 4.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microarray scanner</td>
			<td>Agilent</td>
			<td>SureScan Microarray Scanner</td>
			<td></td>
		</tr>
		<tr>
			<td>Biostatistics software</td>
			<td>GraphPad Software</td>
			<td>GraphPad Prism 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin capture antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB10972</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin protein</td>
			<td>R&#38;D Systems</td>
			<td>1097-EN</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoglin detection antibody</td>
			<td>R&#38;D Systems</td>
			<td>BAF1097</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6a (see Table 1)</td>
			<td>R&#38;D Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6b (see Table 1)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

snap chip for cross-reactivity-free and spotter-free multiplexed sandwich immunoassays

Çoğaltılmış protein analizi üstün tanılama hassasiyet ve doğruluk için tek proteinler karşılaştırıldığında göstermiştir. Antikor microarrays aynı anda tek bir yonga üzerinde gerçekleştirilen mikro ölçekli uzun binlerce sağlar. Sandviç tahlil biçimi her hedef iki antikorlar ile tespit ederek tahlil özgüllük artırır, ancak böylece çoğullama yeteneklerini sınırlayan reaktifler arasında olan muzdarip. Antikor colocalization Mikroarray (ACM) çoğaltılmış protein çapraz-reactivity ücretsiz algılama için geliştirilmiştir, ancak pahalı bir gözcü yerinde Mikroarray imalat deneyleri sırasında için gerektirir. Bu çalışmada, biz sadece dayama iki fiş birlikte, böylece hiçbir gözcü örnek kuluçka ve algılama antikor (dAbs) sonraki uygulama sırasında gerekli tarafından Mikroarray Mikroarray reaktif aktarır bir ek çip teknolojisi göstermek depolama öncesi benekli slayt, slayt hazırlama tahlil yürütme dissociating. Her iki tek ve Çift Kişilik transfer yöntemleri iki microarrays arasında doğru uyum elde etmek için sunulmaktadır ve slayt imalat yöntemleri açıklanmıştır. Sonuçları gösteriyor ki &#60; 40 mikron hizalama 625 noktalar/cm2bir dizi yoğunluğu ulaşan Çift Kişilik transferi ile elde. 50-plexed immunoassay çoğaltılmış protein analizi ek yongasında kullanılabilirlik göstermek için yapılmıştır. Sınırlar 35 proteinlerin algılama aralığı pg/mL vardır.

Hem tek hem de çift transfer yöntemleri için tahlil yordamı şekil 1' de gösterilen. Tek transfer, kabinler doğrudan tahlil slaytta lekeli ve dAbs kabinler (Şekil 1a) bir ayna model kullanımda üzerine tahlil slayda aktarılır. Tek bir aktarım yordam gereklidir, ancak esas olarak slaytla mürekkep püskürtmeli makas köprüsü (Şekil 2) arasındaki açısal kayma nedeniyle iki microarrays...

Watch this Scientific Journal Video about Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays at JoVE.com

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Biz sadece iki slayt şaklatarak çoğaltılmış sandviç çapraz-reactivity ücretsiz uzun gerçekleştirmek için bir ek çip teknolojisi göstermek. Bir ek aparatı reaktifler Mikroarray Mikroarray güvenilir bir şekilde aktarmak için kullanılır. Ek çip olmadan Çapraz bulaşma farklı reaktifler colocalization gerektiren herhangi bir biyokimyasal reaksiyonlar için kullanılabilir.

Çip için çapraz-reactivity ücretsiz ve gözcü-Alerjik çoğaltılmış sandviç uzun snap

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

The overall goal of this procedure is to simultaneously quantify multiple proteins on the chip using sandwich immunoassays in a spotter-free manner without cross reactivity between reagents. This method can help answer key questions in the quantitative proteomics field, such as the discovery and validation of candidate protein biomarkers in cancer. The main advantages of this technique are that it has cross reactivity between antibodies and antigen in multiplexed sandwich assays and that and users do not need a complex microarray spotter.Demonstrating the procedure will be Heidi Larkin, a scientific director at Parallex BioAssays. To begin preparing slides for the single-transfer method, fix a new transfer slide on the ink jet microarray spotter deck. Using a polystyrene microbead solution, or 30%glycerol, spot an array of alignment marks on the edge of the slide.Using the instrument software and spotter camera, take a picture of the alignment marks for quality control. Next fix a nitrocellulose or functionalized glass assay slide to the left of the transfer slide on an ink jet micro array spotter deck. Using alignment marks, spot 24 arrays of capture antibody solutions with 400 micrometer center-to-center spacing.Incubate the assay slide at room temperature in an atmosphere with 60%relative humidity for one hour. Then clip a 24-well silicone gasket onto the slide, aligned with the arrays. Pipette 80 microliters of a 0.1%solution of polysorbate 20 in PBS into each well.Shake the slide at 450 rpm for five minutes and discard the solution. Wash the slide in this way three times. Next load 80 microliters of BSA free-blocking solution into each rinsed well.Shake the slide at 450 rpm for one hour. Then discard the solution, remove the silicone gasket, and dry the assay slide using either nitrogen gas or a centrifuge. Seal the assay slide in an airtight bag with silica gel or another desiccant and store it at minus 20 degrees Celsius.Using the same alignment marks, spot 24 arrays of detection antibody solutions using 3.2 nanoliters per spot in a center-to-center spacing of 400 micrometers. Store the transfer slide at minus 20 degrees Celsius in a sealed bag with a desiccant. To begin preparing slides for the double-transfer method, fix a transfer slide on an ink jet microarray spotter deck.Spot 24 arrays of capture antibody solutions with the center-to-center spacing of 400 micrometers. Then turn the snap apparatus plunger switches 90 degrees to lock the plungers in the open position. Fit the capture transfer slide into the apparatus with the clipped corner against the fixed pogo pin.Release the pogo pins by turning the corresponding plunger switches back by 90 degrees. Ensure that the transfer slide is firmly held by the pogo pins and is flush with the fixed alignment pins. Then place a new nitrocellulose or a functionalized glass slide on the pogo pins with the coated side face down.Turn the other three switches to release the straight pins. Verify that both slides are held in place against the fixed alignment pins. Then fit the top shell onto the slide holder with the pillar seated in the alignment holes.Insert the closed apparatus into the snap cage and fully depress the closure tab. Leave the apparatus closed for one minute. Then pull up the closure tab to release the apparatus from the cage.Remove the top shell and separate the assay and transfer slides. Incubate the assay slide for one hour. Next clip a 24-well silicone gasket onto the slide and wash the slide three times.Then shake the slide with BSA-free blocking solution for one hour. Remove the gasket and dry the assay slide under a stream of nitrogen gas or a centrifuge. Next fix another transfer slide on the ink jet spotter deck.Spot 24 arrays of detection antibody solutions on the slide with 3.2 nanoliters per spot in a center-to-center spacing of 400 micrometers to make the transfer slide. Store the detection transfer slide at minus 20 degrees Celsius. To begin the immunoassay remove the prepared assay slide from the freezer and allow it to warm to room temperature in its sealed bag for 30 minutes.Prepare seven serially diluted solutions of proteins in PBS with 0.05%polysorbate 20. Prepare appropriately diluted sample solutions in the same buffer. Clamp a 24-well silicone gasket onto the room temperature assay slide.Load the seven protein dilutions into seven of the eight wells in a column. Load protein-free PBS with 0.05%polysorbate 20 into the last well of that column. Load the sample solutions into the remaining wells.Shake the slides at 450 rpm for one hour at room temperature or at 4 degrees Celsius overnight. Then wash the slide three times. Remove the gasket and dry the slide under a stream of nitrogen gas.Next allow the detection antibody transfer slide to warm to room temperature for 30 minutes in its sealed bag. Then incubate the slides for 20 minutes in a closed chamber with the relative humidity of 60%to rehydrate the arrays. Use the pogo pins to fix the detection transfer slide face up in the snap apparatus.Place the assay slide face down on the pogo pins and align the slide with the straight pins. Close the apparatus and snap transfer the detection antibodies to the assay slide. Remove the slides from the apparatus and incubate the assay slide for one hour.Clamp a 24-well silicone gasket to the slide and wash the slide three times. Then load into each well 80 microliters of a 2.5 microgram per milliliter solution of streptavidin fluoro-4 in PBS. Shake the slide at 450 rpm for 20 minutes.Wash the slide three times with a 0.1%solution of polysorbate 20 in PBS and once with distilled water. Then remove the gasket and dry the slide. Scan the slide with a fluorescent scanner and measure the spot intensities.Determine the limits of detection in the protein concentrations. Two fluorescent proteins were sequentially patterned on a nitrocellulose-coated slide using the double-transfer method with a 16-wells template. No cross-contamination was observed upon transferring the second fluorescent protein.Smaller spots with decreased spot-to-spot distance could be transferred to a glass slide with a highly hydrophobic surface. The double-transfer method resulted in minimal angular misalignment of the arrays. The double-transfer method was used to perform a 50-plex immunoassay of breast cancer-related proteins in the sandwich assay format.No cross reactivity was observed, allowing independent optimization of each antibody pair. Standard curves calculated from the assays indicated that 35 of the 50 proteins had picogram per milliliter limits of detection. Once mastered this technique can be completed in approximately three hours.This technique allows scientists to conduct microarray-based assay and to deliver reagents as nano droplets. While attempting this procedure, remember to fix the slides properly on the spotting deck and in the snap apparatus. After watching this video, you should have a good understanding of how to use the snap apparatus for cross-reactivity-free multiplexed sandwich immunoassays.

Research

JoVE Journal

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

Mikroakışkan damlacık platformları için Floresan algılama yöntemleri

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Biyomühendislik

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

İyonların ayrılması ve Nükleik Asit Saflaştırılması için on-chip Isotachophoresis

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Multiplexed Transdermal Elektrokimyasal Algılama Hollow microneedle tabanlı Sensör

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

Çip üzerinde Stres Kaynaklı Antibiyotik Duyarlılık Testi

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Microfluidic Chip for ICPMS Sample Introduction

ICPMS Numune Giriş için mikroakışkan Chip

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Sandviç gibi mikroçevrelerde Hücre / Malzeme Etkileşimleri Harness

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

İmmüno için üç boyutlu Paper-based mikroakışkan Cihazlar İmalat

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Parçacıkların Mekansal Takip için Çoklanmış Elektronik Algılama ile mikroakışkan Platformu

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

İmalat ve Organ-on-chip sistemin doğrudan Transendothelial elektrik direncini ölçmek için entegre elektrotlar ile doğrulama

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Çoğaltılmış yapay hücresel MicroEnvironment dizi imalatı

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...