Cell-free bioassay for measurement of dioxins based on fluorescence enhancement of fluorescein isothiocyanate-labeled DNA probe
Cell-free bioassay for measurement of dioxins based on fluorescence enhancement of fluorescein isothiocyanate-labeled DNA probe
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基于异硫氰酸荧光素标记 DNA 探针荧光增强的无细胞生物测定法测量二恶英
DOI：
10.1021/ac060442e
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发表时间：
2006-10-15
影响因子：
7.4
通讯作者：
Zheng, Ming-Hui
中科院分区：
文献类型：
--
作者：
You, Fan;Zhou, Ya-Feng;Zheng, Ming-Hui
本研究旨在建立一种快速、灵敏的二恶英无细胞生物检测方法。已知二恶英配体可以结合异二聚体芳烃受体（AhR）并触发二恶英-AhR、AhR核转运子（ARNT）和DNA的二恶英响应元件（DRE）区域的复合物的形成。所提出的方法的假设是，如果在DRE序列处标记FITC，则当复合物形成时，其荧光强度将增强，因为结合组分（AhR、ARNT和DRE）的相互作用界面产生有利于FITC发射的相当疏水的条件。分别通过表面等离子体共振和荧光分析研究了FITC修饰位点对复合物组分之间结合效率和荧光发射增强的影响。结果表明，DRE 5'端第二个碱基（5'-TNGCGTG-3 '）的标记位点对DNA探针与二恶英-AhR/ARNT杂交体的结合无明显干扰，但对荧光发射有明显增强作用。以2，3，7，8-四氯二苯并-对-二恶英（TCDD）为典型毒素。该方法的线性范围为1- 100 pM，检测限为0.1 pM（0.64 fg/assay），变异系数为5.6%（n = 10，50 pM TCDD在转化胞质溶胶）。整个检测周期约为4 h。该方法还用于估算1，2，3，7，8-五氯二苯并对二恶英（PeCDD）和1，2，3，4，7，8-六氯二苯并对二恶英（HxCDD）的毒性当量（TEQ）。TCDD、PeCDD和HxCDD混合物的毒性当量测量结果与预测值高度一致。使用粉煤灰提取物的平均回收率接近93%。
This study aims to develop a rapid and sensitive cell-free bioassay of dioxins. It is known that dioxin ligand can bind heterodimeric aryl hydrocarbon receptor (AhR) and triggers the formation of the complex of dioxin-AhR, AhR nuclear translocator (ARNT), and dioxin-responsive element (DRE) region of the DNA. The hypothesis of the proposed method is that if FITC were labeled at the DRE sequence, its fluorescence intensity would be enhanced when the complex forms because the interaction interface of the binding components (AhR, ARNT, and DRE) creates a rather hydrophobic condition that is in favor of FITC emission. Effects of modification site of FITC on the DNA probes on binding efficiency between the complex components and fluorescence emission enhancement were evaluated by surface plasmon resonance and fluorescence analysis, respectively. Results showed that the labeling site at the second base at the 5' end apart from the core region (5'-TNGCGTG-3') of DRE did not obviously interfere with the binding between the DNA probe and dioxin-AhR/ARNT hybrid but presented significant fluorescence emission enhancement. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the typical toxin in this study. The method had a linear range of 1- 100 pM, with detection limit of 0.1 pM (0.64 fg/assay) and coefficient of variation of 5.6% (n = 10, 50 pM TCDD in transformed cytosol). The whole detection cycle was similar to 4 h. The method was also used to estimate the toxic equivalents (TEQ) of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin ( HxCDD). Measurement of TEQs of the mixture of TCDD, PeCDD, and HxCDD were highly consistent with the predicted data. The average recovery using fly ash extract was similar to 93%.