Liu Xue-Ying, Nakamura Chikashi, Nakamura Noriyuki, Hirano Takashi, Shinbo Toshio, Miyake Jun
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Center 6th, 111 Higashi, Tsukuba 305-8566, Japan.
J Chromatogr A. 2005 Sep 16;1087(1-2):229-35. doi: 10.1016/j.chroma.2005.03.013.
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.
磷脂酶A2（PLA2）催化的膜渗漏可以通过含有自猝灭荧光染料3,3-双[N,N-二（羧甲基）氨甲基]荧光素（钙黄绿素）的固定化脂质体来检测。这种酶促反应被用作生物传感器检测低浓度多氯联苯（PCBs）的信号放大。为了增加荧光信号以改善PCBs的检测，本报告研究了牛血清白蛋白（BSA）对PLA2催化荧光脂质体膜渗漏的最佳脂质组成的影响。使用抗生物素蛋白-生物素结合将各种包载钙黄绿素的脂质体固定在Sephacryl S1000凝胶珠中。相比之下，通过在Sephacryl S300上进行尺寸排阻色谱法去除游离脂质体悬浮液中的游离钙黄绿素。在这些凝胶珠固定化脂质体和游离脂质体悬浮液中均检测到了PLA2催化的膜渗漏。在这两种体系中，随着白蛋白浓度的增加，PLA2水解作用导致脂质体的荧光释放显著增加。在游离脂质体悬浮液和凝胶珠固定化脂质体中，在白蛋白存在下，PLA2水解导致的最快速和最大程度的膜渗漏发生在阴离子脂质体中。最后，监测了各种游离脂质体和凝胶珠固定化脂质体的稳定性。由于其优异的稳定性和PLA2导致的大量染料渗漏，选择了固定化的1-棕榈酰-2-油酰磷脂酰胆碱（POPC）/1-棕榈酰-2-油酰磷脂酰甘油（POPG）脂质体凝胶。使用这种串联柱系统可检测低至0.1 ng/mL的PCBs浓度。
