Department of Chemistry and Department of Chemical & Physical Sciences, University of Toronto, Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, Canada L5L 1C6.
Analyst. 2017 Nov 20;142(23):4511-4521. doi: 10.1039/c7an00568g.
Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.
膜嵌入带负电荷的磷脂 (MENCP) 可作为多种生物过程的生物标志物，包括动物细胞凋亡的早期检测、药物诱导的磷脂病以及细菌与动物细胞的选择性检测。目前，几种用于检测凋亡和细菌细胞的技术都基于对 MENCP 的识别，包括 AnnexinV 染色和 PSVue™ 探针。作为探针，这些技术存在局限性，其中最显著的是需要洗涤未结合的探针以获得最佳信号。相比之下，对 MENCP 具有选择性的开启型化学传感器将解决这一缺点，并允许更快地检测凋亡、细菌以及其他相关应用。在这项工作中，我们的目的是探索之前我们小组报道的用于检测邻近磷酸化肽和蛋白质的 ProxyPhos 化学传感器是否可以重新用于检测 MENCP。对含有生物相关带负电荷磷脂的合成囊泡（包括磷脂酸 (PA)、磷脂酰甘油 (PG)、心磷脂 (CL) 和磷脂酰丝氨酸 (PS))进行了六种先导 ProxyPhos 传感器的筛选。通过这些筛选，确定了能够对 MENCP 与两性离子脂质进行高选择性检测的 ProxyPhos 传感器。尤其对 PA 和 CL 表现出了高选择性。先导传感器对 MENCP 的敏感性适合于检测凋亡：ProxyPhos 检测到含有低至 2.5% PS 的囊泡，并在流式细胞术实验中检测到喜树碱诱导的哺乳动物细胞凋亡。结果表明，ProxyPhos 传感器可用于检测合成囊泡和活哺乳动物细胞中的 MENCP。
