Kulagina Nadezhda V, Mikulski Christina M, Gray Samuel, Ma Wu, Doucette Gregory J, Ramsdell John S, Pancrazio Joseph J
Center for Bio/Molecular Science and Engineering, Code 6900, Naval Research Laboratory, Washington, DC 20375, USA.
Environ Sci Technol. 2006 Jan 15;40(2):578-83. doi: 10.1021/es051272a.
There is a need for assay systems that can detect known and unanticipated neurotoxins associated with harmful algal blooms. The present work describes our attempt to monitor the presence of brevetoxin-3 (PbTx-3) and saxitoxin (STX) in a seawater matrix using the neuronal network biosensor (NNB). The NNB relies on cultured mammalian neurons grown over microelectrode arrays, where the inherent bioelectrical activity of the network manifested as extracellular action potentials can be monitored noninvasively. Spinal cord neuronal networks were prepared from embryonic mice and the mean spike rate across the network was analyzed before and during exposure to the toxins. Extracellular action potentials from the network are highly sensitive not only to purified STX and PbTx-3, but also when in combination with matrixes such as natural seawater and algal growth medium. Detection limits for STX and PbTx-3, respectively, are 0.031 and 0.33 nM in recording buffer and 0.076 and 0.48 nM in the presence of 25-fold-diluted seawater. Our results demonstrated that neuronal networks could be used for analysis of Alexandrium fundyense (STX-producer) and Karenia brevis (PbTx-producer) algal samples lysed directly in the seawater-based growth medium and appropriately diluted with HEPES-buffered recording medium. The cultured network responded by changes in mean spike rate to the presence of STX-or PbTx-producing algae but not to the samples of two non-STX and non-PbTx isolates of the same algal genera. This work provides evidence that the NNB has the capacity to rapidly detect toxins associated with cells of toxic algal species or as dissolved forms present in seawater and hasthe potential for monitoring toxin levels during harmful algal blooms.
需要能够检测与有害藻华相关的已知和意外神经毒素的检测系统。目前的工作描述了我们尝试使用神经网络生物传感器（NNB）监测海水中短裸甲藻毒素-3（PbTx-3）和石房蛤毒素（STX）的存在。NNB依赖于在微电极阵列上生长的培养哺乳动物神经元，其中网络固有的生物电活动表现为细胞外动作电位，可以进行非侵入性监测。从胚胎小鼠制备脊髓神经网络，并在接触毒素之前和期间分析整个网络的平均放电率。网络的细胞外动作电位不仅对纯化的STX和PbTx-3高度敏感，而且在与天然海水和藻类生长培养基等基质结合时也高度敏感。在记录缓冲液中，STX和PbTx-3的检测限分别为0.031和0.33 nM，在存在25倍稀释海水的情况下分别为0.076和0.48 nM。我们的结果表明，神经网络可用于分析直接在海水基生长培养基中裂解并用HEPES缓冲记录培养基适当稀释的芬迪湾亚历山大藻（产STX）和短裸甲藻（产PbTx）藻类样品。培养的网络通过平均放电率的变化对产STX或产PbTx藻类的存在做出反应，但对同一藻类属的两种非产STX和非产PbTx分离株的样品没有反应。这项工作提供了证据，证明NNB有能力快速检测与有毒藻类物种的细胞相关的毒素或海水中存在的溶解形式的毒素，并有可能在有害藻华期间监测毒素水平。
