An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.
An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.
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一种超灵敏无标记生物传感器，用于基于放大荧光共轭聚合物测定序列特异性 DNA 结合蛋白。
DOI：
10.1016/j.bios.2012.08.027
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发表时间：
2013-03
影响因子：
12.6
通讯作者：
Wei Huang
中科院分区：
文献类型：
--
作者：
Xingfen Liu;Lan Ouyang;Xiaohui Cai;Yanqin Huang;Xiaomiao Feng;Quli Fan;Wei Huang
序列特异性DNA结合蛋白（DBP）的灵敏、可靠和简单的检测在蛋白质组学、基因组学和生物医学领域具有极其重要的意义。我们在此描述了一种新的荧光放大策略，用于DBP的超灵敏、视觉、定量和“开启”检测。设计了一种利用阳离子共轭聚合物（CCP）和嵌入染料的Förster共振能量转移（FRET）测定法，用于检测关键转录因子核因子-κ B（NF-κB）（模型靶标）。用一系列带有一个或两个蛋白结合位点（PBS）的无标记DNA探针特异性鉴定目的蛋白。结合DBP保护探针免受核酸外切酶III的消化，由于嵌入染料和双链体DNA之间的高亲和力以及CCP和DNA探针之间的强静电相互作用，导致高效的FRET。采用无标记发夹DNA或双链DNA作为探针，可检测到低至1 pg/μL的NF-κB，比以往报道的方法灵敏10000倍。该方法还允许通过在手持UV灯的帮助下观察溶液的荧光颜色来进行裸眼检测。此外，得到小于10%的相对标准偏差，这提供了一个新的平台，上级精度，低成本，和简单的检测DBP。我们的光学生物传感器的特点显示了良好的潜力，许多疾病的早期诊断和高通量筛选的新药物靶向DNA结合蛋白。
Sensitive, reliable, and simple detection of sequence-specific DNA-binding proteins (DBP) is of paramount importance in the area of proteomics, genomics, and biomedicine. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, visual, quantitative, and “turn-on” detection of DBP. A Förster resonance energy transfer (FRET) assay utilizing a cationic conjugated polymer (CCP) and an intercalating dye was designed to detect a key transcription factor, nuclear factor-kappa B (NF-κB), the model target. A series of label-free DNA probes bearing one or two protein-binding sites (PBS) were used to identify the target protein specifically. The binding DBP protects the probe from digestion by exonuclease III, resulting in high efficient FRET due to the high affinity between the intercalating dye and duplex DNA, as well as strong electrostatic interactions between the CCP and DNA probe. By using label-free hairpin DNA or double-stranded DNA containing two PBS as probe, we could detect as low as 1pg/μL of NF-κB in HeLa nuclear extracts, which is 10000-fold more sensitive than the previously reported methods. The approach also allows naked-eye detection by observing fluorescent color of solutions with the assistance of a hand-held UV lamp. Additionally, a less than 10% relative standard deviation was obtained, which offers a new platform for superior precision, low-cost, and simple detection of DBP. The features of our optical biosensor shows promising potential for early diagnosis of many diseases and high-throughput screening of new drugs targeted to DNA-binding proteins.
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调整阳离子共轭聚合物的主链和侧链，用于荧光 DNA 检测的光学信号放大。
影响因子：
12.6
作者：
通讯作者：
--
影响因子：
--
作者：
P. Selvin
通讯作者：
P. Selvin
DOI：
10.1002/asia.201000363
发表时间：
2010-12
期刊：
Chemistry, an Asian journal
影响因子：
--
作者：
Qingling Xu;Chunxian Wu;Chunlei Zhu;Xinrui Duan;Libing Liu;Yuchun Han;Yilin Wang;Shu Wang
通讯作者：
Qingling Xu;Chunxian Wu;Chunlei Zhu;Xinrui Duan;Libing Liu;Yuchun Han;Yilin Wang;Shu Wang
影响因子：
14.9
作者：
Wang J;Li T;Guo X;Lu Z
通讯作者：
Lu Z
影响因子：
7.4
作者：
Yong Zhang;Yanyan Wang;B. Liu
通讯作者：
Yong Zhang;Yanyan Wang;B. Liu