Multiplexed single molecule immunoassays.
Multiplexed single molecule immunoassays.
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DOI：
10.1039/c3lc50416f
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发表时间：
2013-08-07
期刊：
影响因子：
6.1
通讯作者：
Duffy, David C.
中科院分区：
文献类型：
--
作者：
Rissin, David M.;Kan, Cheuk W.;Song, Linan;Rivnak, Andrew J.;Fishburn, Matthew W.;Shao, Qichao;Piech, Tomasz;Ferrell, Evan P.;Meyer, Raymond E.;Campbell, Todd G.;Fournier, David R.;Duffy, David C.
我们已经开发出一种方法，使基于计数单个分子的蛋白质的多重检测。顺磁珠用荧光染料标记，形成光学上不同的珠亚群，然后将针对特定蛋白质的抗体固定在单个亚群上。然后用样品孵育珠子亚群的混合物，并在其特定的珠子上捕获特定的蛋白质；然后通过免疫复合物的形成用酶标记这些蛋白质。这些微球悬浮在酶底物中，装入飞升孔阵列或单分子阵列（Simoa），后者被集成到一个微流体装置（Simoa圆盘）中。然后用油密封孔，并用荧光成像来确定：a)飞升孔中单个微球的位置和亚群特征，b)每个微球相关的单一酶的存在或缺失。对图像进行分析，以确定每个头亚群的平均每粒酶（AEB），为确定每种蛋白质的浓度提供定量参数。我们使用这种方法以亚飞摩尔浓度的单分子分辨率同时检测人血浆中的TNF-α、IL-6、IL-1α和IL-1β，即比目前的多重免疫测定方法灵敏度高200至1000倍。使用多重数字ELISA同时、特异和敏感地测量几种蛋白质可以使疾病的诊断更加可靠。
We have developed a method that enables the multiplexed detection of proteins based on counting single molecules. Paramagnetic beads were labeled with fluorescent dyes to create optically distinct subpopulations of beads, and antibodies to specific proteins were then immobilized to individual subpopulations. Mixtures of subpopulations of beads were then incubated with a sample, and specific proteins were captured on their specific beads; these proteins were then labeled with enzymes via immunocomplex formation. The beads were suspended in enzyme substrate, loaded into arrays of femtoliter wells—or Single Molecule Arrays (Simoa)—that were integrated into a microfluidic device (the Simoa disc). The wells were then sealed with oil, and imaged fluorescently to determine: a) the location and subpopulation identity of individual beads in the femtoliter wells, and b) the presence or absence of a single enzyme associated with each bead. The images were analyzed to determine the average enzyme per bead (AEB) for each bead subpopulation that provide a quantitative parameter for determining the concentration of each protein. We used this approach to simultaneously detect TNF-α, IL-6, IL-1α, and IL-1β in human plasma with single molecule resolution at subfemtomolar concentrations, i.e., 200- to 1000-fold more sensitive than current multiplexed immunoassays. The simultaneous, specific, and sensitive measurement of several proteins using multiplexed digital ELISA could enable more reliable diagnoses of disease.
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影响因子：
3.1
作者：
Croft, Heather;Malinowski, Tadeusz;James, Delano
通讯作者：
James, Delano
影响因子：
2.2
作者：
Song, Linan;Hanlon, David W.;Duffy, David C.
通讯作者：
Duffy, David C.
影响因子：
24.5
作者：
Gustot, T;Lemmers, A;Franchimont, D
通讯作者：
Franchimont, D
影响因子：
7.4
作者：
Rissin, David M.;Fournier, David R.;Piech, Tomasz;Kan, Cheuk W.;Campbell, Todd G.;Song, Linan;Chang, Lei;Rivnak, Andrew J.;Patel, Purvish P.;Provuncher, Gail K.;Ferrell, Evan P.;Howes, Stuart C.;Pink, Brian A.;Minnehan, Kaitlin A.;Wilson, David H.;Duffy, David C.
通讯作者：
Duffy, David C.
影响因子：
7.4
作者：
Fichorova, Raina N.;Richardson-Harman, Nicola;Alfano, Massimo;Belec, Laurent;Carbonneil, Cedric;Chen, Silvia;Cosentino, Lisa;Curtis, Kelly;Dezzutti, Charlene S.;Donoval, Betty;Doncel, Gustave F.;Donaghay, Melissa;Grivel, Jean-Charles;Guzman, Esmeralda;Hayes, Madeleine;Herold, Betsy;Hillier, Sharon;Lackman-Smith, Carol;Landay, Alan;Margolis, Leonid;Mayer, Kenneth H.;Pasicznyk, Jenna-Malia;Pallansch-Cokonis, Melanie;Poli, Guido;Reicholderfer, Patricia;Roberts, Paula;Rodriguez, Irma;Saidi, Hela;Sassi, Rosaria Rita;Shattock, Robin;Cummins, James E., Jr.
通讯作者：
Cummins, James E., Jr.