DRESSA: biosensing of dioxin and dioxin-like chemicals using secreted alkaline phosphatase
DRESSA: biosensing of dioxin and dioxin-like chemicals using secreted alkaline phosphatase
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DOI：
10.1016/j.ab.2004.08.017
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发表时间：
2004-12-01
影响因子：
2.9
通讯作者：
Kitamura, M
中科院分区：
文献类型：
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作者：
Kasai, A;Hiramatsu, N;Kitamura, M
在这篇文章中，我们描述了一个高灵敏度的生物传感系统，DRESSA，检测二恶英和二恶英类化学品。串联拷贝的二恶英反应元件（DRE）融合到一个最小的病毒启动子亚克隆到表达质粒上游的分泌型碱性磷酸酶（SEAP）基因。当小鼠肝癌细胞系Hepa-1c 1c 7稳定转染这种结构，建立传感器克隆分泌SEAP刺激后，与2，3，7，8-四氯二苯并-p-二恶英（TCDD）。克隆HeDS 49被认为是非常敏感的，它分泌SEAP响应TCDD的剂量和时间依赖性的方式，和最低检测限为100 fM。为了检测超过6 pM的TCDD，整个检测时间（从接种细胞到测量SEAP活性）可以缩短至4 h。SEAP的分泌被DRE的其他激活剂（3-甲基胆蒽、苯并[a]芘和β-萘酮）选择性地诱导，但不被不相关的响应元件的激活剂诱导。这些数据表明，由于快速，简单，特异性和高灵敏度的DRESSA，它是更适合于目前可用的二恶英和二恶英类化学品的检测系统，并将有很大的优势，高通量筛选这些污染物在环境样品中。(C)2004年爱思唯尔公司All rights reserved.
In this article, we describe a highly sensitive biosensing system, DRESSA, for detection of dioxin and dioxin-like chemicals. Tandem copies of the dioxin-responsive element (DRE) fused to a minimal viral promoter were subcloned into an expression plasmid upstream of a secreted alkaline phosphatase (SEAP) gene. When murine hepatoma cell line Hepa-1c1c7 was stably transfected with this construct, established sensor clones secreted SEAP following stimulation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A clone HeDS49 was found to be extremely sensitive; it secreted SEAP in response to TCDD in dose- and time-dependent manners, and the minimal detection limit was 100 fM. To detect more than 6 pM of TCDD, the whole assay time (from cell seeding to measurement of SEAP activity) could be reduced to 4 h. Secretion of SEAP was induced selectively by other activators of DRE (3-methylcholanthrene, benzo[a]pyrene, and beta-naphthoflavone) but not by activators of unrelated responsive elements. These data suggested that because of the rapidity, easiness, specificity, and high sensitivity of DRESSA, it is more suitable than currently available detection systems for dioxin and dioxin-like chemicals and would be of great advantage to high-throughput screening of these pollutants in environmental samples. (C) 2004 Elsevier Inc. All rights reserved.