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首页> 外文期刊>Acta biomaterialia >Development of fluorescent polymerization-based signal amplification for sensitive and non-enzymatic biodetection in antibody microarrays.
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Development of fluorescent polymerization-based signal amplification for sensitive and non-enzymatic biodetection in antibody microarrays.

机译：基于荧光聚合的信号放大技术的开发，用于抗体微阵列中的灵敏和非酶促生物检测。

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摘要

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.
机译：抗体微阵列是蛋白质组学的关键工具，需要广泛，高度灵敏地检测许多低丰度生物标志物。基于荧光聚合的扩增（FPBA）是一种新颖的非酶信号放大方法，它利用了自由基聚合的链反应性质来实现高度放大的荧光响应。链霉亲和素-曙红结合物将曙红光引发剂定位在芯片上的聚合反应上，在该芯片上结合了生物素化的靶蛋白。使芯片与作为单体的丙烯酰胺，作为共引发剂的N-甲基二乙醇胺和黄/绿荧光纳米颗粒（NP）接触，在引发时，它们结合在一起形成宏观可见的高荧光膜。快速的聚合动力学和交联剂的存在有利于荧光NP截留在聚合物中，从而实现了高度灵敏的荧光生物检测。已证明该方法适用于抗体微阵列，并与使用链霉亲和素-异硫氰酸荧光素（SA-FITC）和链霉亲和素标记的黄色/绿色NP（SA-NP）的检测方法进行了比较。发现FPBA能够检测到0.16 +或-0.01生物素抗体microm（-2）（或40 zmol表面结合的靶分子），而SA-FITC的检测极限是31 +或-1生物素-抗体microm（-2）和SA-NPs在此处评估的条件下无法获得任何明显的信号。此外，与使用成本更高的微阵列扫描仪进行的SA-曙红的荧光检测相比，结合荧光立体显微镜的FPBA产生的灵敏度等于或更好。通过促进高度灵敏的检测，FPBA有望实现低丰度抗原的检测，并且还可能向廉价的荧光检测仪器过渡。

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