Department of Chemical and Biological Engineering, ECCH 111, UCB 424, University of Colorado, Boulder, CO 80309, USA.
Acta Biomater. 2010 Jan;6(1):83-9. doi: 10.1016/j.actbio.2009.06.008. Epub 2009 Jun 7.
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-fluorescein isothiocyanate (SA-FITC) and streptavidin-labeled yellow/green NPs (SA-NPs). It is found that FPBA is able to detect 0.16 + or - 0.01 biotin-antibody microm(-2) (or 40 zmol surface-bound target molecules), while SA-FITC has a limit of detection of 31 + or - 1 biotin-antibody microm(-2) 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.
抗体微阵列是蛋白质组学的重要工具，需要广泛、高度敏感地检测许多低丰度生物标志物。荧光聚合放大（FPBA）被提出作为一种新颖的、非酶信号放大方法，利用自由基聚合的链式反应性质实现高度放大的荧光响应。亲和素-曙红缀合物将曙红光引发剂定位在芯片上，生物素化的靶蛋白结合在芯片上。芯片与丙烯酰胺接触作为单体，N-甲基二乙醇胺作为共引发剂和黄/绿荧光纳米粒子（NPs），引发后，它们结合形成宏观可见和高度荧光的膜。快速聚合动力学和交联剂的存在有利于将荧光 NPs 困在聚合物中，从而实现高灵敏度的荧光生物检测。该方法适用于抗体微阵列，并与利用亲和素-荧光素异硫氰酸酯（SA-FITC）和亲和素标记的黄/绿 NPs（SA-NPs）的检测方法进行了比较。结果发现，FPBA 能够检测到 0.16 ± 0.01 生物素-抗体 μm(-2)（或 40 zmol 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)，并且在评估的条件下，SA-NPs 无法获得任何显著信号。此外，FPBA 结合荧光立体显微镜，与使用成本更高的微阵列扫描仪荧光检测 SA-曙红相比，具有同等或更高的灵敏度。通过促进高度敏感的检测，FPBA 有望实现对低丰度抗原的检测，并且还可能实现向更廉价的荧光检测仪器的转变。
