钠离子荧光探针用于检测雪卡毒素的细胞毒性

摘要

Objective To establish a cell-based detection method of ciguatoxin using fluorescence assay. Methods Mouse neuroblastoma N-2A cells were exposed to ouabain and veratridine and different concentrations of standard ciguatoxin samples (P-CTX-1) to establish the curvilinear relationship between the toxin dosage and fluorescence intensity using the sodium fluorescence probe CoroNa? Green. The toxicity curvilinear relationship was also generated between the toxin dosage and cell survival using CCK-8 method. Based on these standard curves, the presence of ciguatoxin was detected in 33 samples of deep-sea coral fish. Results A correlation was found between the detection results of cell-based fluorescence assay and cytotoxicity assay, whose detection limit reached 10"13 g/ml and 10-12 g/ml, respectively. The cell-based fluorescent assay sensitivity showed a higher sensitivity than cytotoxicity assay with a 2-4 h reduction of the detection time. Conclusions The cell-based fluorescent assay can quickly and sensitively detect ciguatoxin and may serve as a good option for preliminary screening of the toxin.%目的 研究和建立以细胞为基础的结合钠离子荧光探针检测雪卡毒素的方法,为快速、敏感检测雪卡毒素提供科学的实验依据.方法 采用雪卡毒素标准品(p-CTX-1)结合乌苯苷和黎芦定处理小鼠脑神经瘤细胞(N-2A),用钠离子荧光探针(CoroNaTM Green)标记细胞,利用多功能酶标仪检测荧光强度,建立雪卡毒素浓度与荧光强度的标准曲线关系式;同时,采用CCK-8法和酶标仪检测毒素处理组细胞的存活率,建立细胞毒性实验的标准曲线关系式;分析比较两种方法的灵敏度.以两种方法分别对采集的33份深海珊瑚鱼样品进行毒素检测,对检测结果作分析比较.结果 以细胞为基础的荧光检测法检测样品中毒素的检出限为10-13g/ml,而细胞毒性试验检测法检测样品中毒素的检出限为10-12g/ml,其结果具有一定的相关性,且荧光检测法灵敏度比细胞毒性试验高;同时钠离子荧光探针检测法的实验终点比CCK-8法缩短2～4 h.结论 以细胞为基础的结合钠离子荧光探针检测法能快速、敏感地检测雪卡毒素,可作为雪卡毒素检测初筛方法推广应用.