Chemburu Sireesha, Ji Eunkyung, Casana Yosune, Wu Yang, Buranda Tione, Schanze Kirk S, Lopez Gabriel P, Whitten David G
Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico 87131, USA.
J Phys Chem B. 2008 Nov 20;112(46):14492-9. doi: 10.1021/jp803358j. Epub 2008 Sep 20.
A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range.
已开发出一种基于荧光的人血清衍生磷脂酶活性检测方法，其中阳离子共轭聚电解质负载在二氧化硅微球上。聚合物包被的珠子再用阴离子磷脂（1,2 - 二肉豆蔻酰 - sn - 甘油 - 3 - [磷酸 - rac - (1 - 甘油)]）（DMPG）包被，以提供作为PLA2传感器的“脂质微球”。该脂质起到双重作用，既是PLA2的底物，又是一种减弱外部电子转移猝灭剂9,10 - 蒽醌 - 2,6 - 二磺酸（AQS）对聚合物荧光猝灭的试剂。在这种情况下，随着PLA2消化脂质，AQS对聚合物荧光的猝灭增加。通过在DMPG中使用少量能量转移猝灭剂取代的脂质，脂质自身也可作为猝灭剂和底物。在这种情况下，当脂质层完整时聚合物的荧光被猝灭；随着酶消化脂质，聚合物的荧光恢复。已通过在多孔板读数器中监测荧光变化以及通过流式细胞术研究了PLA2活性的传感。该检测方法具有良好的灵敏度，EC50值在纳摩尔范围内。
