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

우리는 잉크젯 정찰 병의 사용에 대 한 박사 롭 Sladek을 감사합니다. 우리는 최종 지원 캐나다 기관에서 건강 연구 (CIHR), 자연 과학 및 캐나다 엔지니어링 연구 위원회 (NSERC), 캐나다 암 학회 연구소 및 캐나다 재단에 대 한 혁신 (CFI)에 대 한 인정합니다. Dj가 감사 합니다 캐나다 연구의 자에서 지원합니다.

1. 스냅 칩 제조 및 저장 <ol><li>이전 방법 ( 그림 1a) <ol><li>자리 cAb 솔루션 400 µ g/mL 항 체를 포함 하 고 20% 글리세롤 인산 염 버퍼 염 분에 (PBS)을 nitrocellulose (또는 기능성된 유리)에는 잉크젯 microarray 정찰 병 13 60%의 상대 습도에서 분석 결과 슬라이드 (1.2 각 명소에 대 한 nL) 800 µ m 센터-센터 간격. 슬라이드 한 구석에 따르면 정찰 병 갑판에 고정 되어 있는...

<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>

이 작품에서는, 우리는 기본적인 실험 설치 연구자 멀티플렉스 교차 reactivity 무료 immunoassays 널리 이용할 수 있게 하는 스냅 칩 기술을 제시 했습니다. 다른 기존 항 체 microarrays, 아니 microarray 정찰 병은 최종 사용자를 위해 필요 합니다. 단일 및 이중 전송 방법을 시연 하 고 이중 전송 월급 우수한 정렬 정확도 ~ 40 μ m 63 µ m14의 가장 큰 오차와 98%의 관광 명소에 대 한. 새로운 스냅...

높은 감도 특이성 암1,2등 복잡 한 질병의 진단에 단일 바이오 마커 보다 여러 단백질을 구성 하는 바이오 마커의 패널 제공할 수 있습니다. 효소 연결 된 immunosorbent 분석 결과 (ELISA) 골드 표준 기술 플라즈마, 낮은 pg/mL에 탐지의 제한 하지만 분석 결과3,,45당 하나의 대상에 한계를 달성 하는 임상 실험실에서 사용 되었습니다. 항 체 microarrays는 단일 현미경 슬라이드6,,78에 병렬로 실시 소형된 분석 실험의 수천을 수용에 대 한 개발 되었습니다. 그러나,이 방법의 멀티플렉싱 기능 dAbs, 혼합물의 응용 프로그램에서 발생 하는 시 약 기반 분으로 제한 하 고 대상9,10 의 증가 함께 더 많은 문제가 된다 , 11. Pla 외. 다중 샌드위치 분석 실험의 결과 취약점 확장 4N(N-1)로 N은 대상12의 수 명시 된 있다.
항 체 microarrays 항 체 colocalization microarray에에서 분 완화 (ACM) 멀티플렉스 샌드위치 분석 결과12에 대 한 우리의 실험실에서 개발 되었습니다. 캡처 항 체 (택시) microarray 정찰 병으로 기판에 발견 됩니다. 차단 샘플 표면에 적용 된 다음 개별 dAbs 택시-항 원 복합체와 같은 명소에 발견 후에. 항 체와 항 원 간의 모든 분 시나리오 ACM, 함께 완화 될 수 있습니다 그리고 pg/mL에 탐지의 한계 달성 되었습니다. 그러나 준비 하 고 비싸고 시간이 소모 되는 정렬 목적에 대 한 높은 정밀도 함께 현장 microarray 정찰 병을 사용 하 여,이 넓은 응용 프로그램 제한 실험 동안에 dAbs 안보 분석 결과 프로토콜에서 요구 하는, 다른 실험실입니다. 스냅 칩 라는 휴대용 ACM 개발 되었습니다 교차 reactivity 무료 고 샌드위치 immunoassays13,,1415다중화 정찰 병 무료. 택시와 dAbs는 사전 분석 결과 슬라이드와 microarray 형태로 각각 전송 슬라이드에 발견 하 고 저장. 분석 결과, 동안 슬라이드를 검색 하 고 dAbs microarray 단순히 함께 두 개의 칩을 스냅 하 여 분석 결과 슬라이드에 집단적으로 전송 됩니다. 스냅인 장치는 신뢰할 수 있는 시 약 전송을 위해 사용 됩니다. 그러나 니트로 코팅 슬라이드는 비교적 큰 항 체 바인딩 용량 액체 작은 물방울을 흡수 분석 결과 슬라이드로 사용 되 고 전송 시 약을 촉진,, 슬라이드는 더 비싼 보다 일반 유리 슬라이드와 microarray 비-투명 슬라이드와 호환 스캐너 신호 수집 필요 합니다.
이 작품에서 스냅 칩 다중 샌드위치 immunoassay 수행 프로토콜을 설명 합니다. 새로운 스냅인 장치 microarray microarray에서 보다 편리 하 고 믿을 수 있는 시 약 전송을 위해 개발 되었습니다. 중요 한 것은, 여기 우리가 시 전송 방법 스냅 칩 일반 유리 슬라이드에 설립 했다. 1024 명소 했다 성공적으로 전송 되 고 크게 확대 대부분 실험실에서이 기술 사용 하 여 유리 슬라이드에 정렬 됩니다.

<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

멀티플렉스 단백질 분석 우수한 진단 민감도 단일 단백질에 비해 정확도 보이고 있다. 항 체 microarrays는 단일 칩에 동시에 수행 하는 마이크로 스케일 immunoassays의 수천 수 있습니다. 샌드위치 분석 결과 형식 두 항 체, 각 목표를 감지 하 여 분석 결과 특이성을 향상 하지만 분 따라서 그들의 멀티플렉싱 기능을 제한 하는 시 약 사이에서 겪고 있다. 항 체 colocalization microarray (ACM) 교차 reactivity 무료 다중화 단백질 검출에 대 한 개발 되었습니다 하지만 microarray 제조 분석 실험 중에 비싼 반점을 찍는 현장. 이 작품에서는 단순히 맞춤 2 칩 함께, 따라서 아무 정찰 병 샘플 인큐베이션 및 탐지 항 체 (dAbs) 시의 후속 응용 프로그램 필요에 의해 microarray microarray에서 시 약을 전송 하는 스냅 칩 기술 시연 미리 발견된 슬라이드, 슬라이드 준비 분석 결과 실행에서 해리의 스토리지. 두 메서드 모두 단일 및 이중 전송 두 microarrays 사이 정확한 줄 맞춤을 달성 하기 위해 제공 됩니다 그리고 두 방법에 대 한 슬라이드 제작 설명. 결과 &#60; 40 μ m 맞춤 이중 전송, 625 명소/c m2의 배열 밀도 도달 달성 되었습니다. 50 plexed immunoassay 다중화 단백질 분석에 스냅 칩의 유용성을 설명 하기 위해 실시 되었습니다. 35 단백질의 검출 한계 범위의 pg/ml에서는 이다입니다.

단일 및 이중 전송 방법에 대 한 분석 결과 절차는 그림 1에 표시 됩니다. 단일 전송, 분석 결과 슬라이드에 직접 택시를 발견 하 고는 dAbs 분석 결과 슬라이드 거울 패턴의 택시 (그림 1a)에서 사용 시에 전송 됩니다. 하나의 전송 절차는 필요, 하지만이 방법은 주로 슬라이드와 잉크젯 갠트리 (그림 2) 사이 ?...

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

두 슬라이드를 간단 하 게 스냅 하 여 교차 reactivity 무료 다중화 샌드위치 immunoassays를 수행 하기 위한 스냅 칩 기술을 설명 합니다. 스냅 기구 시 약 microarray microarray에서 안정적으로 전송 하는 데 사용 됩니다. 어떤 생 화 확 적인 반응을 교차 오염 없이 다른 시 약의 colocalization를 요구에 대 한 스냅 칩을 사용할 수 있습니다.

교차 reactivity 무료 및 정찰 병 무료 스냅 칩 샌드위치 Immunoassays 다중화

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

microfluidic 비말 플랫폼 형광 검출 방법

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

연구

생체공학

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

이온의 분리 및 핵산의 정화를위한 온칩 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

다중 Transdermal 전기 감지를위한 빈 Microneedle 기반 센서

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

칩에 스트레스 유발 항생제 감수성 테스트

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 샘플 소개를위한 미세 유체 칩

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

샌드위치 같은 미세 환경은 셀 / 재료의 상호 작용을 활용하는

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

면역 측정법에 대한 3 차원 종이 기반 미세 유체 장치의 제작

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

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

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

제조 및 통합된 전극 Transendothelial 전기 저항을 직접 측정 하는 기관-온-칩 시스템의 유효성 검사

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

멀티플렉스 인공 세포 MicroEnvironment 배열의 제작

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