Detection of polychlorinated biphenyls using an antibody column in tandem with a fluorescent liposome column: Effect of albumin on phospholipase A2-catalyzed membrane leakage
- 作者：Xue-Ying Liu ; Chikashi Nakamura ; Noriyuki Nakamura ; Takashi Hirano ; Toshio Shinbo and Jun Miyake
- 关键词：PCBs detection ; Fluorescent ; Liposome column ; PLA2 ; Albumin ; Anionic liposomes ; Immunoassay
- 刊名：Journal of Chromatography A
- 出版年：2005
- 期刊代码：47_00219673
- 类别：ch
- 出版时间：2005
- 卷：1087
- 期：1-2
- 页码：229-235
- 文件大小：126 K
摘要
Phospholipase A2 (PLA2)-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, 3,3-bis[N,N-di(carboxymethyl)aminomethyl]fluorescein (calcein). This enzymatic reaction was applied as signal amplification for biosensor detection of low concentrations of polychlorinated biphenyls (PCBs). In order to increase the fluorescent signal for improvement of PCBs detection, the effect of BSA on optimal lipid composition for PLA2-catalyzed membrane leakage from fluorescent liposomes has been investigated in this report. Various kinds of calcein-entrapped liposomes were immobilized in Sephacryl S1000 gel beads using avidin–biotin binding. In a contrast, free calcein was removed by size exclusion chromatography on Sephacryl S300 for free liposome suspensions. The PLA2-catalyzed membrane leakage was detected both in these gel-bead-immobilized liposomes and in free liposome suspensions. In both systems, the fluorescent release from the liposomes by PLA2 hydrolytic action significantly increased with increasing albumin concentration. The most rapid and greatest membrane leakage by PLA2 hydrolysis was found in anionic liposomes in the presence of albumin, both in free liposome suspensions and gel-bead-immobilized liposomes. Finally, the stabilities of various free liposomes and gel-bead-immobilized liposomes were monitored. Immobilized 1-palmitoy-2-oleoylphosphatidylcholine (POPC)/1-palmitoy-2-oleoylphosphatidylglycerol (POPG) liposome gel was chosen due to its excellent stability and large dye leakage by PLA2. A concentration of PCBs as low as 0.1 ng/mL was detectable using this tandem column system.