我们感谢 Dr. 抢 Sladek 的使用喷墨观察员。我们承认加拿大卫生研究所 (研究院)、加拿大自然科学和工程研究理事会 (NSERC)、加拿大癌症协会研究所和加拿大创新基金会 (CFI) 的最后支持。感谢来自加拿大研究主席的支持。

1. 快照芯片的制作和存储 <ol> <li> 单传输方法 ( 图 1a ) <ol> <li> 现场 cAb 解决方案, 包含400和 #181; 在磷酸盐缓冲盐水中的 g/毫升抗体和20% 甘油(PBS) 在硝化纤维 (或功能化玻璃) 上使用喷墨微阵列检测幻灯片, 在相对湿度为 60% (每点 1.2 nL) 的情况下使用800和 #181; m 中心间距。确保幻灯片是固定在观察甲板根据一个角落 (这里, 左下角使用). </li>
		<li> 在室温下孵育 1 h 的斑点化验幻灯片, 相对湿度为 60%. </li>
		<li> 在化验幻灯片上夹住16隔间的滑动模块垫圈, 将其分成16口井。通过添加80和 #181 的 PBS, 冲洗三次幻灯片; 在每个井中都有 0.1% Tween-20 (PBST), 每次在摇床上晃动 450 rpm, 每一次都有5分钟. </li>
		<li> 添加80和 #181; L 对每个井的堵塞解决方案, 并在 450 rpm 时抖动1小时. </li>
		<li> 取下垫片, 用氮气流干燥化验滑块. </li>
		<li> 在观察甲板上固定一张传送幻灯片, 并将幻灯片的左下角按下左下角的探测甲板。喷墨点一组对准标记 (聚苯乙烯微珠的解决方案) 与驾驶室的布局相同. </li>
		<li> 让校准标记为干燥。翻转转移幻灯片, 并把它放回观察员甲板上, 在左下角固定. </li>
		<li> 准备民建联的解决方案, 包括20和 #181; g/毫升抗体, 20% 甘油和 1% BSA. </li>
		<li> 使用监视器和 #39 的摄像头, 拍摄对齐标记的图片。把这张图片作为一个基准来实现, 并得到喷墨观测器的图像识别系统, 以确定最左上标记。使用它的坐标作为第一个点的民建联阵列。点 8 nL 每液滴与中心间距800和 #181; m. </li>
	</ol> </li> <li> 双传输方法 ( 图 1b ) <ol> <li> 将传输幻灯片1放在底部左下角的喷墨检测甲板上。包含400和 #181 的现场 cab 解决方案; PBS 中的 g/毫升抗体, 20% 甘油和 1% BSA, 在相对湿度为60% 的喷墨微阵列检测 (每个点 0.4 nL, 直径为200和 #181) 的幻灯片上。中心间距为400和 #181; m. </li>
		<li> 使用一台 snap 设备 ( 图 2 ) 将转印的小水滴转到化验幻灯片上, 用一个硝化棉涂层 (或功能化的玻璃) 检测幻灯片来对传送幻灯片进行捕捉。
		<ol> <li> 将 snap 设备一侧的所有六按钮按90度打开, 并将所有活塞在打开的位置上锁定。将传送滑块插入其插座中的滑动架上, 并将所修剪的拐角朝向固定的弹簧针。打开第一组三按钮, 以释放活塞与金色的弹簧针, 并确保幻灯片是推对齐针. </li>
			<li> 在4弹簧脚上倒置的装置上插入一个硝化纤维素涂层的滑动器。打开第二组三按钮, 释放活塞与银针, 并确保幻灯片是推对齐针脚. </li>
			<li> 通过将顶部壳体放置在滑动架上, 使用对齐柱和孔进行精确定位, 从而关闭 snap 装置. </li>
			<li> 将闭合的 snap 装置插入其保持架中, 并将关闭标签完全向下推送, 以便在应用适当压力时面对面地进行微阵列。保持关闭1分钟. </li>
		</ol> </li> <li> 分离幻灯片。在室温下孵育 1 h 的化验幻灯片, 相对湿度为60%。如步骤 1.1.3-1.1.5 所述, 清洗、阻挡和干燥化验幻灯片. </li>
		<li> 在喷墨探测甲板上放置另一个传送幻灯片, 然后按下左下角。斑点民建联解决方案包含50或100和 #181; 抗体的 g/毫升 (见表 1), 20% 甘油, 和 1% BSA 在 PBS。确保每个点都是 0.8 nL, 点之间的中心间距是400和 #181; m. </li>
		<li> 存储检测和转移幻灯片。在含有干燥剂的密闭袋中密封化验和转移幻灯片, 并放入-20 和 #176; C 冷冻. </li>
	</ol> </li> </ol> 2。多路免疫与 snap 芯片 <ol> <li> 从冰箱中检索化验幻灯片。把袋子密封30分钟, 直到幻灯片到室温. </li>
	<li> 在含有 0.05% Tween-20 的 PBS 中通过峰值蛋白质制备7点系列稀释样品溶液. 
	注: 在这里, 使用5倍的稀释因子。表1列出了每个蛋白质的起始浓度. </li>
	<li> 根据需要准备示例解决方案。例如, 在 PBST 缓冲液中稀释人血清4倍. </li>
	<li> 在化验幻灯片上夹住一个16室垫片. </li>
	<li> 用7蛋白稀释溶液和无蛋白质的 PBST 缓冲液, 用移填充一列8口井。在同一张幻灯片的其他8口井中抽取样品溶液. 
	注:80 和 #181; L 解决方案可以适合每一个井。必要时可以使用更多的幻灯片来测量其他示例. </li>
	<li> 在室温下以 450 rpm 将样品孵化为1小时, 或在4和 #176 过夜; c. 在振动筛上用 PBST 冲洗三次, 每次450分钟。取下垫片, 在氮气流下烘干滑块. </li>
	<li> 从冷冻库中取出带有轻的传输幻灯片。在室温下将袋子密封30分钟。然后, 在关闭的腔室中孵育幻灯片 ( 例如 空提示框), 其中包含60% 湿度稳定珠, 用于20分钟的补液. </li>
	<li> 使用 snap 设备 ( 图 2 ) 将传输幻灯片与分析幻灯片对齐, 以传输 "民建联" 液滴。有关 snap 设备的操作, 请参见1.2.2 节. </li>
	<li> 将幻灯片分开。在包含60% 湿度稳定珠的密闭腔中孵育 1 h. 用16室垫片夹住化验幻灯片, 用 PBST 在 450 rpm、5分钟的振动筛上冲洗4次幻灯片. </li>
	<li> 吸管80和 #181; 包含2.5 和 #181 的溶液的 L; 亲和荧光在 PBS 中的每一个井。在 450 rpm 的振动筛上孵育20分钟. </li>
	<li> 用 PBST 冲洗幻灯片3次, 在 450 rpm 的情况下用蒸馏水在振动筛上清洗一次。取下垫片, 用氮气将滑块擦干. </li>
</ol> 3。幻灯片扫描和数据分析 <ol> <li> 使用 635 nm 激光扫描带有荧光微阵列扫描的检测幻灯片. </li>
	<li> 使用分析软件 (如阵列 pro 分析器) 16 提取每个点的净强度. </li>
	<li> 使用统计软件计算每个蛋白质的检测限度 (LOD), 并确定样本中的蛋白质浓度. 
	注意: LOD 被定义为标准 cu 的 Y 截距rve 增加了三倍的标准偏差三独立的化验. </li>
</ol>

<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>

麦吉尔大学在这项工作的一些方面提出了专利申请, 杨惠妍和大卫. 容克作为发明者。

在这项工作中, 我们提出了一个 snap 芯片技术, 使交叉免费复免疫广泛适用于研究人员的基本实验设置。与现有的抗体微阵列不同, 不需要 end-users。单和双传输方法都证明, 双转移提供了优越的对准精度下降到〜40μ m 为98% 点, 与最大的失调63µm14。开发了一种新颖的 snap 装置, 可以方便地将两张幻灯片保持一致的压力, 使这项技术能够被研究人员使用。可靠的试剂传递不仅在硝化棉涂层的幻灯片上实现, 而且在普通的玻片上也有, 在不需要进行广泛的培训的情况下, 大大扩展了这项技术的应用。为了演示快照芯片的可用性, 使用 pre-spotted 和存储的幻灯片进行了50丛免疫分析, 70% 的蛋白质在 pg/mL14范围内实现了检测。与现有的复合免疫, 应用轻作为一个混合物, 一个重要的好处的 ACM 和 snap 芯片技术是, 在芯片上的化验是相互独立的, 从而允许添加或删除任何抗体对没有干扰任何其他对。数十到数百个蛋白质可以同时用一个短切芯片来测量, 从而缩短了测试时间, 与其他方法, 如连续孵化自动串行测量, 要求每抗体17的串行应用。只有 sub-nanoliter 的每个抗体的芯片制造, 使其更经济比传统的免疫。
抗体浓度的使用和抗原孵化步骤是至关重要的, 以达到高的检测灵敏度。在芯片的制作中, 抗体浓度可以根据抗体对的亲和性和化验滑块 (硝化纤维或 non-nitrocellulose) 的类型来进行优化。根据检测能力和对准精度, 可以对每个抗体点的体积进行调整, 并据此改变斑点之间的中心间距。在将驾驶室转移到化验幻灯片后, 我们在室温下孵育了1小时的化验幻灯片, 在4° c 的夜间孵化可能会给予更好的抗体结合能力。BSA 自由稳定剂解决方案 (见材料表) 被用来阻止化验幻灯片后, 孵化驾驶室。其他阻断试剂, 如动物血清或牛奶也可用于本应用。
在多路复用免疫中, 可以开发和优化具有不同表面化学特性的 non-nitrocellulose 幻灯片的协议。不同的稀释因子可用于使标准曲线取决于目标蛋白质。不同的荧光可用于标记轻, 并且可以使用不同波长的激光扫描该幻灯片以进行信号采集。
目前, 通过双传输方法14实现了625点/cm2的密度。为了进一步提高快照芯片的复用能力, 我们提供了几种策略。首先, 较小的体积可以用来减少斑点的大小, 从而允许更短的中心距离之间的斑点和增加的阵列密度。其次, 可以选择一个更疏水表面的转移幻灯片, 以减少光斑大小。第三, 通过对喷墨观测机进行微调, 进一步优化了喷嘴与滑动面之间的距离, 从而实现了两种微阵列之间的更好的对准。
对于所有的抗体免疫, 芯片免疫检测的性能取决于抗体的可用性和质量。snap 芯片使用三明治化验格式, 这是最广泛使用的检测格式, 在 ELISA 由于高灵敏度和特异性提供的双, 顺序结合的一对抗体针对不同的表位, 但它取决于用于捕获和检测靶蛋白所需的一对相容抗体的可用性。
芯片技术的价值是建立在免疫, 它可以用于任何化学和生物化学的反应, 需要应用不同的试剂没有 cross-contaminations18,19,20,21,22. 事实上, snap 芯片提供了一个一般的解决方案, 提供各种 pre-spotted 试剂从 microarray-to-microarray, 从而解除芯片制造与化验程序, 从而帮助解决 "macro-to-micro "接口挑战23。作为一个例子, 该 snap 芯片已被用于4丛均匀酶抑制试验分析128条件, 准确的时间, 并可比较的结果大体积实验14。它也被用于多复用组织染色。此外, 它可以有助于药物筛选应用, 以测试在不同的细菌或细胞数以千计的化学品, 为单细胞分析21,24, 或测试不同的条件, 在单一或多步骤的化学反应.

一个由多种蛋白质组成的生物标志物的面板比单一的标志物在诊断复杂疾病如癌症1,2中提供更高的灵敏度和特异性。酶联免疫吸附试验 (ELISA) 是在临床实验室中使用的金标准技术, 在低 pg/毫升的等离子体中达到了检测的极限, 但每个化验值限制为一个目标3,4,5。抗体微阵列已开发, 以容纳数以千计的微型检测并行进行的单一显微镜幻灯片6,7,8。但是, 这种方法的复用能力受到试剂驱动交叉的限制, 这是由轻混合的应用引起的, 并且越来越多的目标9,10,11. 解放军et al。指出, 复合夹层分析结果的脆弱性是 4 n (n-1), 其中 n 是目标12的数目。
为了减少抗体微阵列中的交叉, 在我们的实验室中开发了抗体定位基因芯片 (ACM), 用于多重夹层试验12。捕获抗体 (cab) 被发现在基板上的微阵列的观察员。在表面上应用阻断样品后, 在与驾驶室抗原复合物相同的斑点上发现单个轻。所有交叉方案之间的抗体和抗原可以减轻与 ACM, 并限制检测在 pg/毫升已经实现。然而, 该检测协议要求在实验过程中使用一种高精度的 on-site 微阵列探测器来准备和识别轻, 这是昂贵和耗时的, 这限制了该技术的广泛应用其他实验室。一个手持式 ACM, 命名的 snap 芯片已开发为无交叉和无检举的复合夹层免疫13,14,15。驾驶室和轻是 pre-spotted 到一个化验幻灯片和转移幻灯片分别在微阵列格式和存储。在分析过程中, 通过简单地将两个芯片合在一起, 将检索到幻灯片, 并将轻的微阵列集体转移到化验幻灯片上。一种用于可靠的试剂转移的 snap 装置。具有相对较大的抗体结合能力的硝化纤维素切片被用作化验幻灯片, 以吸收液滴, 从而促进试剂的转移, 但是, 幻灯片比普通的玻片和微阵列更昂贵与非透明幻灯片兼容的扫描仪需要进行信号采集。
在这项工作中, 我们演示了执行一个多路三明治免疫分析与快照芯片的协议。为更方便、可靠的试剂从 microarray-to-microarray 中转移, 研制了一种新型的抓拍装置。重要的是, 在这里, 我们已经建立了试剂传输方法到常规玻璃幻灯片与快照芯片。1024斑点成功地被转移了并且被排列了到玻璃幻灯片, 极大地扩展这技术的用途在多数实验室。

<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

多路复用蛋白分析显示, 与单一蛋白相比, 诊断灵敏度和准确度均较高。抗体微阵列允许在单个芯片上同时进行数以千计的微型免疫。三明治化验格式通过检测每个目标的两个抗体, 提高了检测的特异性, 但在试剂之间交叉, 从而限制了它们的复用能力。抗体定位微阵列 (ACM) 已发展为无交叉多路复用蛋白质检测, 但需要一个昂贵的观察员 on-site 芯片制造过程中的化验。在这项工作中, 我们展示了一个 snap 芯片技术, 通过简单地将两个芯片一起从 microarray-to-microarray 转移试剂, 因此在样品孵化和随后应用检测抗体 (轻) 时不需要任何的观测存储 pre-spotted 的幻灯片, 游离的幻灯片准备从化验执行。本文介绍了两种单片和双转印方法, 实现了双微阵列的精确对准, 并描述了这两个方法的滑动制作。结果表明, #60; 40 μ m 对准实现了双转移, 达到了625点/cm2的阵列密度。50-plexed 免疫分析已经进行了演示芯片的可用性, 在多路复用蛋白质分析仪。检测35种蛋白质的限度在 pg/毫升的范围内。

单和双传输方法的检测过程如图 1所示。在单转移, 驾驶室被发现直接在化验幻灯片和轻被转移到化验幻灯片后使用的一个镜像模式的驾驶室 (图 1a)。只有一个转移过程是必需的, 但这种方法是由两个微阵列之间的失调, 主要是由于在幻灯片和喷墨龙门之间的角度失调 (图 2) 造成的。解决这一难题的一种方法是在检测后依次将 cab 和轻传输到分析幻灯片上, 并正确地在 snap 设备中固定幻灯片 (图 1b)。使用这种方法, 两个微阵列都转移到完全相同的位置。双传输方法不需要图像识别系统或对齐标记。98% 点的失调是在41µm, 6 倍的改善相比, 单一的传输方法14。
该 snap 装置已被设计为易于使用, 无需任何培训, 并尽量减少阵列失调的风险。由于对齐引脚共同对分析和传输幻灯片, 使幻灯片与精确定位结合在一起。当被弹簧针悬挂时, 转印滑块稍微小一些, 以适应分析滑动, 直到折断装置关闭。在传输滑块上包括一个间隔, 保持微米间隙, 允许可靠的熔滴转移到玻片上。最后, 笼子和它的关闭标签被设计为在每一个单元中应用需要的有效传输的压力, 以实现可重现、高质量的传输。快照设备的照片如图 3所示。
在图 4中显示了将试剂转移到硝化纤维幻灯片和玻片上的代表性图像。alexa 532 标记 IgGs 作为第一个试剂, 和 alexa 647 标记 IgGs 被用作第二个试剂。在传输后, 幻灯片被扫描 532 nm 和 635 nm 激光器。结果表明, 3136 (对于硝化纤维素滑动) 或 1024 (为玻片) microspots 转移与零故障上的化验幻灯片和没有交叉感染后, 转移第二个试剂。当使用玻片时, 可以通过功能化来创造更多疏水性的表面, 从而减小每个斑点的大小, 减少 spot-to-spot 的距离, 从而转移更多的斑点。这项工作扩展了芯片技术在常用玻璃基板上的应用。
50丛免疫分析靶向乳腺癌相关蛋白的标准曲线在图 5中说明, 对应于迄今没有交叉的最大的复合夹层抗体芯片。本试验采用双转移法, 对滑动进行了扫描, 并对荧光强度进行了量化, 生成了标准曲线。35出50蛋白达到检测在 pg/毫升 (见表 1), 并可通过优化的化验条件进一步改善。定位的试剂有助于在单张幻灯片上使用多个抗体对而不交叉, 并且允许对每对的优化, 而不受其他对12的干扰。这些结果表明, 快照芯片是一种可扩展的技术, 可用于多路复用蛋白质的量化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56230/56230fig1.jpg"> 
图1。比较 (a) 单和 (b) 双重转移方法的化验程序示意图.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56230/56230fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56230/56230fig2.jpg"> 
图2。示意图显示单 double-transfer 之间的差异.(a) 转移试剂的镜像放大了幻灯片和喷墨 XY 工作台之间的角度偏差。(b) double-transfer 方法克服了角偏差。根据权限从引用14改编。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56230/56230fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56230/56230fig3.jpg"> 
图3。快照设备的照片.(a) 打开的设备。(b) 封闭式装置。(c) 设备的全景视图。简要地, (i) 转移幻灯片被安置入幻灯片持有人和推挤反对对准线别针使用 pogopin 安装在活塞。(ii) 化验幻灯片放在 pogopins 的顶部, 并使用第二组活塞对齐销进行推送。(iii) 顶部壳体位于滑动架顶部, 并插入笼中。使用 "关闭" 选项卡压缩 pogopin 并将幻灯片组合在一起时, 将施加压力。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56230/56230fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56230/56230fig4.jpg"> 
图4。使用 snap 芯片来集体转移斑点.荧光扫描使用 532 nm 和 633 nm 激光, 以证明抗体转移到 (a) 硝化纤维素幻灯片 (适应从参考14与许可) 和 (b) 玻璃幻灯片。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56230/56230fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56230/56230fig5.jpg"> 
图5。用双转移法测定50种蛋白质平行测定的荧光图像和标准曲线.(a) 用于生成标准曲线的化验幻灯片的荧光图像。(b) 阵列特写。缩放条 = 2 毫米 (c) 标准曲线为50蛋白质。误差线被计算为标准偏差从三独立实验。根据权限从引用14改编。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56230/56230fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>蛋白质名称</td>
			<td>起始浓度 (ng/毫升)</td>
			<td>LOD (pg/毫升)</td>
			<td>蛋白质名称</td>
			<td>起始浓度 (ng/毫升)</td>
			<td>LOD (pg/毫升)</td>
		</tr>
		<tr>
			<td>ANG2</td>
			<td>500</td>
			<td>1.3 x 104</td>
			<td>IL-6b</td>
			<td>1</td>
			<td>2.1 x 102</td>
		</tr>
		<tr>
		</tr></tbody></table>	<td>bdnf</td>
			<td>500</td>
			<td>1.0 x 102</td>
			<td>IL-5</td>
			<td>50</td>
			<td>77</td>
		
		<tr>
			<td>CA 15-3 *</td>
			<td>1</td>
			<td>4.9 x 103</td>
			<td>IL-4</td>
			<td>1000</td>
			<td>1.5 x 104</td>
		</tr>
		<tr>
			<td>cea</td>
			<td>1000</td>
			<td>5.4 x 103</td>
			<td>IL-2</td>
			<td>50</td>
			<td>76</td>
		</tr>
		<tr>
			<td>CXCL10/IP-10</td>
			<td>50</td>
			<td>1.7 x 102</td>
			<td>lep</td>
			<td>200</td>
			<td>4.0 x 102</td>
		</tr>
		<tr>
			<td>crp</td>
			<td>200</td>
			<td>44</td>
			<td>mig</td>
			<td>500</td>
			<td>11 x 102</td>
		</tr>
		<tr>
			<td>局</td>
			<td>1000</td>
			<td>6.2 x 102</td>
			<td>CCL3/MIP-1α</td>
			<td>50</td>
			<td>3。3</td>
		</tr>
		<tr>
			<td>egf</td>
			<td>50</td>
			<td>1.8 x 102</td>
			<td>CCL4/MIP-1β</td>
			<td>50</td>
			<td>12</td>
		</tr>
		<tr>
			<td>egfr</td>
			<td>200</td>
			<td>1.9 x 102</td>
			<td>MMP-3</td>
			<td>500</td>
			<td>1.0 x 102</td>
		</tr>
		<tr>
			<td>FAS-L</td>
			<td>500</td>
			<td>4.5 x 102</td>
			<td>m-CSF</td>
			<td>500</td>
			<td>8.2 x 103</td>
		</tr>
		<tr>
			<td>fgf</td>
			<td>500</td>
			<td>1.0 x 103</td>
			<td>MMP-9</td>
			<td>200</td>
			<td>3.1 x 102</td>
		</tr>
		<tr>
			<td>g-csf</td>
			<td>500</td>
			<td>39</td>
			<td>CCL2/MCP-1</td>
			<td>50</td>
			<td>55</td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>50</td>
			<td>3。8</td>
			<td>NCAM-1</td>
			<td>500</td>
			<td>1.7 x 103</td>
		</tr>
		<tr>
			<td>α</td>
			<td>50</td>
			<td>3.0 x 102</td>
			<td>β-NGF</td>
			<td>200</td>
			<td>1.4 x 103</td>
		</tr>
		<tr>
			<td>HER2</td>
			<td>500</td>
			<td>3.5 x 103</td>
			<td>NT-3</td>
			<td>200</td>
			<td>5.8 x 102</td>
		</tr>
		<tr>
			<td>BB</td>
			<td>200</td>
			<td>73</td>
			<td>opn</td>
			<td>500</td>
			<td>1.9 x 103</td>
		</tr>
		<tr>
			<td>IL-1β</td>
			<td>500</td>
			<td>7.9 x 102</td>
			<td>RBP4</td>
			<td>200</td>
			<td>1.2 ×102</td>
		</tr>
		<tr>
			<td>IL-1ra</td>
			<td>200</td>
			<td>1.1 x 103</td>
			<td>sparc</td>
			<td>1000</td>
			<td>4.6 ×104</td>
		</tr>
		<tr>
			<td>IL-15</td>
			<td>50</td>
			<td>8.2 x 102</td>
			<td>TNF-α</td>
			<td>50</td>
			<td>4。4</td>
		</tr>
		<tr>
			<td>IL-12</td>
			<td>1</td>
			<td>30</td>
			<td>TNF-RI</td>
			<td>50</td>
			<td>1.3 x 102</td>
		</tr>
		<tr>
			<td>IL-11</td>
			<td>500</td>
			<td>1.2 x 103</td>
			<td>TNF-RII</td>
			<td>50</td>
			<td>12</td>
		</tr>
		<tr>
			<td>IL-10</td>
			<td>500</td>
			<td>6.1 ×102</td>
			<td>FAS/TNFRSF6</td>
			<td>200</td>
			<td>2.8 ×102</td>
		</tr>
		<tr>
			<td>IL-8</td>
			<td>50</td>
			<td>6。6</td>
			<td>upa</td>
			<td>200</td>
			<td>24</td>
		</tr>
		<tr>
			<td>IL-7</td>
			<td>2。5</td>
			<td>26</td>
			<td>uPAR (CD87)</td>
			<td>200</td>
			<td>76</td>
		</tr>
		<tr>
			<td>IL-6a</td>
			<td>1000</td>
			<td>84</td>
			<td>vegf</td>
			<td>200</td>
			<td>6.7 x 102</td>
		</tr>
	

表 1.蛋白质浓度和检测在缓冲区从50丛分析。CA 15-3 的 LOD 位于 U/ml (*) 中。根据权限从引用14改编。

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

我们展示了一个快照技术, 执行无交叉的多路复用三明治免疫简单地捕捉两个幻灯片。一种用于可靠地从 microarray-to-microarray 中转移试剂的 snap 装置。该 snap 芯片可用于任何生化反应需要定位不同的试剂没有交叉污染。

无交叉反应和无免疫多路复用夹芯芯片

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

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.4K 次观看.  McGill University and Génome Québec Innovation Centre. 我们展示了一个快照技术, 执行无交叉的多路复用三明治免疫简单地捕捉两个幻灯片。一种用于可靠地从 microarray-to-microarray 中转移试剂的 snap 装置。该 snap 芯片可用于任何生化反应需要定位不同的试剂没有交叉污染。

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

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

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

科研

生物工程

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

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

对芯片等速电泳离子的分离和纯化核酸

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

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

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

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

芯片上的压力诱导抗生素易感性测试

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

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

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

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

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

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

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

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

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

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

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