A Fluorescent Dye Method Based on Changes in Membrane Potential for Detecting PSP Toxins in Shellfish
GAO Li-juan1,YANG Wei-dong1, 2*,LIU Jie-sheng1, 2
1. Department of Biotechnology, Jinan University, Guangzhou 510632, China 2. Guangdong Province’s Higher Education Key Lab of Eutrophication and Red Tide Control, Guangzhou 510632, China
Abstract：We developed a method to screen paralytic shellfish poisoning (PSP) toxins based on their functional activity. The assay was a fluorimetric assay by detecting changes in the membrane potential of transitional cell carcinoma of the bladder cells T24 and involved several steps: stain of T24 cells with fluorescent dye bis-oxonol, cell depolarization with veratridine, and inhibition of depolarization with PSP toxins GTX2,3 or shellfish samples containing PSP toxins. Toxic potency of the samples was evaluated by measuring toxin-induced changes in membrane potential. Within 2-100 nmol·L-1 of GTX2,3, veratridine-induced depolarization was shown to be inhibited by GTX2,3 in a dose-dependent manner. There was a linear correlation between the percentage of inhibition and toxin concentration. The PSP toxin value in shellfish obtained by this fluorescence assay was in concordance with that by the mouse bioassay, and with higher sensitivity. In conclusion, the fluorescent dye method based on changes in membrane potential was a rapid, specific, and reliable method for detecting paralytic shellfish poisoning toxins in shellfish.
[1] LIU Jie-sheng, YANG Wei-dong, CHE Jun, et al(刘洁生, 杨维东, 车 军, 等). Oceanologia et Limnologia Sinica(海洋与湖沼), 2003, 34(5): 566. [2] LIU Jie-sheng, NIE Li-hua, YANG Wei-dong, et al(刘洁生, 聂利华, 杨维东, 等). Chinese Journal of Public Health(中国公共卫生), 2006, 22(11): 1394. [3] LIU Jie-sheng, LI Yu-rong, NIE Li-hua, et al(刘洁生, 刘玉荣, 聂利华, 等). Chinese Journal of Pathophysiology(中国病理生理杂志), 2005, 21(7): 1368. [4] Hayashi R, Saito H, Okumura M, et al. Journal of Agricultural and Food Chemistry, 2006, 54: 269. [5] YANG Shi-ming, JIANG Si-chang, YANG Wei-yan, et al(杨仕明, 姜泗长, 杨伟炎, 等). Progress in Physiological Sciences(生理科学进展), 2002, 33(2): 106. [6] XU Zheng-hong, ZHANG Zhen-xi, WANG Jing, et al(徐正红, 张镇西, 王 晶, 等). Spectroscopy and Spectral Analysis(光谱学与光谱分析), 2007, 27(7): 1359. [7] Vale C, Alfonso A, Vieytes M R, et al. Analytical Chemistry, 2008, 80(5): 1770. [8] Plasek J, Sigler K A. Journal of Photochemistry and Photobiology B: Biology, 1996, 33: 101. [9] Andrew C, Benniston A H, Iain E. Journal of Photochemistry and Photobiology A: Chemistry, 2004, 163(1-2): 61. [10] PAN Ya-ping, XU Xiang-hua, WANG Xiao-liang(潘雅萍, 徐向华, 王晓良). Acta Pharmaceutica Sinica(药学学报), 2004, 39(2): 85. [11] Russell A N, Li Guo-hua, Enrico B, et al. Toxicon, 2002, 40(6): 831. [12] Louzao M C, Vieytes M R, Cabado A G, et al. Chemical Research in Toxicology, 2003, 16(4): 433. [13] Louzao M C, Vieytes M R, Sousa J M V B, et al. Analytical Biochemistry, 2001, 289: 246. [14] Christian B, Luckas B. Analytical and Bioanalytical Chemistry, 2008, 391: 117. [15] YANG Li, YANG Wei-dong, LIU Jie-sheng, et al(杨 莉, 杨维东, 刘洁生, 等). Journal of Hygiene Research(卫生研究), 2006, 35(4): 435.
