Development of fluorescent polymerization-based signal amplification for sensitive and non-enzymatic biodetection in antibody microarrays.
Development of fluorescent polymerization-based signal amplification for sensitive and non-enzymatic biodetection in antibody microarrays.
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DOI：
10.1016/j.actbio.2009.06.008
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发表时间：
2010-01
影响因子：
9.7
通讯作者：
Bowman, Christopher N.
中科院分区：
文献类型：
--
作者：
Avens, Heather J.;Bowman, Christopher N.
关键词：
抗体微阵列是蛋白质组学的关键工具，需要对大量低丰度生物标志物进行广泛、高灵敏的检测。基于荧光聚合的扩增(FPBA)是一种新型的非酶信号放大方法，它利用自由基聚合的链式反应性质来实现高度放大的荧光响应。链霉亲和素-曙红偶联物将曙红光引发剂定位在生物素化目标蛋白结合的芯片上进行聚合。该芯片与丙烯酰胺作为单体接触，N-甲基二乙醇胺作为共引发剂，以及黄色/绿色荧光纳米颗粒(NPs)，一旦引发，它们结合在一起，形成宏观可见的高荧光薄膜。快速的聚合动力学和交联剂的存在有利于荧光纳米颗粒在聚合物中的包埋，从而实现高灵敏度的荧光生物检测。该方法被证明适用于抗体微阵列，并与使用链霉亲和素-FITC(SA-FITC)和链霉亲和素标记的黄色/绿色纳米粒(SA-NPs)的检测方法进行了比较。结果发现，FBA能检测到0.16(+/−)生物素抗体/µm~2(或40个卓托莫尔表面结合的靶分子)，而SA-FITC的检测限为31(+/−1)生物素抗体/µm~2，SA-NPs在所评价的条件下无法获得任何显著的信号。此外，与使用更昂贵的微阵列扫描仪的SA-曙红的荧光检测相比，FPBA与荧光立体显微镜的结合产生了相同或更好的灵敏度。通过促进高灵敏的检测，FPBA有望实现对低丰度抗原的检测，并使向更便宜的荧光检测仪器过渡成为可能。
Antibody microarrays are a critical tool for proteomics, requiring broad, highly sensitive detection of numerous low abundance biomarkers. Fluorescent polymerization-based amplification (FPBA) is presented as a novel, non-enzymatic signal amplification method that takes advantage of the chain-reaction nature of radical polymerization to achieve a highly amplified fluorescent response. A streptavidin-eosin conjugate localizes eosin photoinitiators for polymerization on the chip where biotinylated target protein is bound. The chip is contacted with acrylamide as a monomer, N-methyldiethanolamine as a coinitiator and yellow/green fluorescent nanoparticles (NPs) which, upon initiation, combine to form a macroscopically visible and highly fluorescent film. The rapid polymerization kinetics and the presence of cross-linker favor entrapment of the fluorescent NPs in the polymer, enabling highly sensitive fluorescent biodetection. This method is demonstrated as being appropriate for antibody microarrays and is compared to detection approaches which utilize streptavidin-FITC (SA-FITC) and streptavidin-labeled yellow/green NPs (SA-NPs). It is found that FPBA is able to detect 0.16 (+/− 0.01) biotin-antibody/µm2 (or 40 zeptomole surface-bound target molecules), while SA-FITC has a limit of detection of 31 (+/− 1) biotin-antibody/µm2 and SA-NPs fail to achieve any significant signal under the conditions evaluated here. Further, FPBA in conjunction with fluorescent stereomicroscopy yields equal or better sensitivity compared to fluorescent detection of SA-eosin using a much more costly microarray scanner. By facilitating highly sensitive detection, FPBA is expected to enable detection of low abundance antigens and also make possible a transition towards less expensive fluorescence detection instrumentation.
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影响因子：
3.3
作者：
Wu, Jing Qin;Dyer, Wayne B.;Chrisp, Jeremy;Belov, Larissa;Wang, Bin;Saksena, Nitin K.
通讯作者：
Saksena, Nitin K.
影响因子：
6.2
作者：
Hansen, Ryan R.;Sikes, Hadley D.;Bowman, Christopher N.
通讯作者：
Bowman, Christopher N.
影响因子：
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作者：
Sikes, Hadley D.;Hansen, Ryan R.;Bowman, Christopher N.
通讯作者：
Bowman, Christopher N.
影响因子：
2
作者：
Brown, Angus;Lattimore, Jo-Dee;dos Remedios, Cristobal
通讯作者：
dos Remedios, Cristobal
影响因子：
2.9
作者：
Wiese, R
通讯作者：
Wiese, R