电压敏感荧光染料在鱼贝类毒素检测中的应用初探
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摘要
目的建立快速、简便、能反映样品整体毒性的鱼贝类毒素筛查方法。方法以神经母细胞瘤细胞为实验材料,采用电压敏感荧光染料bis-oxonol,根据膝沟藻毒素2,3(GTX2,3)、短裸甲藻毒素(BTX)和河豚鱼毒(TTX)的毒理特点,观察不同浓度毒素作用下细胞荧光的变化。结果 GTX2,3的浓度在2～200ng/ml范围内,TTX的浓度在20～600ng/ml范围内,BTX的浓度在15～400ng/ml范围内,培养体系荧光强度与毒素的浓度之间存在很好的线性关系。结论利用电压敏感荧光染料法可实现对GTX2,3、TTX和BTX的快速检测。
Objective To develop a rapid and sensitive assay for detecting the common sea food toxins including paralytic shellfish poisoning toxin(PST),tetrodotoxin(TTX) and neurotoxic shellfish poisoning toxin(NST) based on their toxicological character.Methods Neuroblastoma cells were incubated with the uorescent dye bis-oxonol,whose distribution across the membrane was potential-dependent.Changes in membrane potential of the cells induced by gonyautoxins(GTX2,3),brevetoxin(BTX) and TTX were observed respectively,using bis-oxonol.Results Within 2-200nmol/L of GTX2,3 or 20-600nmol/L of TTX,veratridine-induced depolarization was shown to be inhibited by GTX2,3 or TTX in dose-dependent manner.Within 15-400ng/ml,there was a dose-dependent relationship between the NSP-induced depolarization and toxin concentration.Conclusion It was likely to find a rapid,specific,and reliable method with bis-oxonol for detecting GTX2,3,TTX and BTX in sea food.
Objective To develop a rapid and sensitive assay for detecting the common sea food toxins including paralytic shellfish poisoning toxin(PST),tetrodotoxin(TTX) and neurotoxic shellfish poisoning toxin(NST) based on their toxicological character.Methods Neuroblastoma cells were incubated with the uorescent dye bis-oxonol,whose distribution across the membrane was potential-dependent.Changes in membrane potential of the cells induced by gonyautoxins(GTX2,3),brevetoxin(BTX) and TTX were observed respectively,using bis-oxonol.Results Within 2-200nmol/L of GTX2,3 or 20-600nmol/L of TTX,veratridine-induced depolarization was shown to be inhibited by GTX2,3 or TTX in dose-dependent manner.Within 15-400ng/ml,there was a dose-dependent relationship between the NSP-induced depolarization and toxin concentration.Conclusion It was likely to find a rapid,specific,and reliable method with bis-oxonol for detecting GTX2,3,TTX and BTX in sea food.
引文
1王初升,唐森铭,宋普庆.我国赤潮灾害的经济损失评估[J].海洋环境科学,2011,30(3):428-431.
2崔伟民,杨维东,刘洁生.双壳贝类麻痹性贝毒抗性机制的研究[J].卫生研究,2008,37(3):377-380.
3 OKUMURA M,TSUZUKI H,TOMITA B.A rapiddetection method for paralytic shellfish poisoning toxinsby cell bioassay[J].Toxicon,2005,46(1):93-98.
4杨莉,杨维东,刘洁生,等.广州市售贝类腹泻性贝毒和麻痹性贝毒污染状况分析[J].卫生研究,2006,35(4):435-437.
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10 LOUZAO M C,VIEYTES M R,CABADO A G,et al.Afluorimetric microplate assay for detection and quantitationof toxins causing paralytic shellfish poisoning[J].ChemRes Toxicol,2003,16(4):433-438.
11 ANDERSON D M,ANDERSEN P,BRICELJ V M,et al.Monitoring and management strategies for harmful algalblooms in coastal waters[Z].Paris:APEC,2001:31-40.
12 MORTON S L,TINDALL D R.Determination of okadaicacid content of dinoflagellate cells:a comparison of theHPLC-fluorescent method and two monoclonal antibodyELISA test kits[J].Toxicon,1996,34(8):947-954.
13 USLEBER E,DONALD M,STRAKA M,et al.Comparison of enzyme immunoassay and mouse bioassayfor determining paralytic shellfish poisoning toxins inshellfish[J].Food Addit Contam,1997,14(2):193-198.
2崔伟民,杨维东,刘洁生.双壳贝类麻痹性贝毒抗性机制的研究[J].卫生研究,2008,37(3):377-380.
3 OKUMURA M,TSUZUKI H,TOMITA B.A rapiddetection method for paralytic shellfish poisoning toxinsby cell bioassay[J].Toxicon,2005,46(1):93-98.
4杨莉,杨维东,刘洁生,等.广州市售贝类腹泻性贝毒和麻痹性贝毒污染状况分析[J].卫生研究,2006,35(4):435-437.
5 NICHOLSON R A,LI G H,BUENAVENTURA E,et al.Arapid and sensitive assay for paralytic shellfish poison(PSP)toxins using mouse brain synaptoneurosomes[J].Toxicon,2002,40:831-838.
6 KANAI T,NEMOTO T,YANAGITA T,et al.Nav1.7sodium channel-induced Ca2+influx decreases tauphosphorylation via glycogen synthase kinase-3βinadrenal chromaffin cells[J].Neurochem Int,2009,54(8):497-505.
7 ZHOU Yu,PAN Fengguang,LI Yansong,et al.Colloidalgold probe-based immunochromatographic assay for therapid detection of brevetoxins in fishery product samples[J].Biosens Bioelect,2009,24(8):2744-2747.
8 BADEN D G,BOURDELAIS A J,JACOCKS H,et al.Natural and derivative brevetoxins:historicalbackground,multiplicity,and effects[J].Environ HealthPersp,2005,113(5):621-625.
9高利娟,杨维东,刘洁生.利用电压敏感荧光染料测定贝体中的麻痹性贝毒[J].光谱学与光谱分析,2009,29(4):1032-1035.
10 LOUZAO M C,VIEYTES M R,CABADO A G,et al.Afluorimetric microplate assay for detection and quantitationof toxins causing paralytic shellfish poisoning[J].ChemRes Toxicol,2003,16(4):433-438.
11 ANDERSON D M,ANDERSEN P,BRICELJ V M,et al.Monitoring and management strategies for harmful algalblooms in coastal waters[Z].Paris:APEC,2001:31-40.
12 MORTON S L,TINDALL D R.Determination of okadaicacid content of dinoflagellate cells:a comparison of theHPLC-fluorescent method and two monoclonal antibodyELISA test kits[J].Toxicon,1996,34(8):947-954.
13 USLEBER E,DONALD M,STRAKA M,et al.Comparison of enzyme immunoassay and mouse bioassayfor determining paralytic shellfish poisoning toxins inshellfish[J].Food Addit Contam,1997,14(2):193-198.