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.

中文翻译：

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ProxyPhos传感器，用于检测带负电的膜

膜包埋的带负电的磷脂（MENCP）可用作一系列生物学过程的生物标志物，包括早期检测动物细胞中的细胞凋亡，药物诱导的磷脂中毒以及对动物细胞中细菌的选择性检测。当前，几种检测凋亡和细菌细胞的技术都是基于对MENCP的识别，包括AnnexinV染色剂和PSVue™探针。作为探针，这些技术有局限性，其中最重要的是需要洗去未结合的探针以获得最佳信号。相比之下，对MENCP具有选择性的开启式化学传感器将解决这一缺点，并允许更快速的方案检测细胞凋亡，细菌和其他相关应用。在这项工作中，目的是探索ProxyPhos化学传感器，我们小组先前报道的用于检测近端磷酸化的肽和蛋白质的方法，可以重新用于检测MENCP。筛选了六种主要的ProxyPhos传感器，以检测含有生物相关负电荷磷脂的合成囊泡，这些磷脂包括磷脂酸（PA），磷脂酰甘油（PG），心磷脂（CL）和磷脂酰丝氨酸（PS）。通过这些屏幕，鉴定出对两性离子脂质表现出对MENCP的检测具有高选择性的ProxyPhos传感器。对于PA和CL观察到特别的选择性。铅传感器对MENCPs的敏感性适合检测细胞凋亡：ProxyPhos在流式细胞术实验中检测到哺乳动物细胞中的PS含量低至2.5％，并检测了喜树碱诱导的细胞凋亡。