Fluorescence Lifetime Correlation Spectroscopy for Metal Ion Quantitation

用于金属离子定量的荧光寿命相关光谱

• 批准号: 228325432
• 负责人: Professor Dr. Gregor Jung
• 金额: --
• 依托单位: Professur für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/228325432?language=en
• 关键词: Fluorescence; Lifetime; Correlation; Spectroscopy; Metal

The late transition-metal ions have an ambiguous role in the life science. On the one hand, some of these are essential constituents of proteins. On the other hand, excess supply of the same elements is often associated with serious ailments like Alzheimer's disease. A real-time monitoring of the ion concentration in any living organism is challenging as most established, metal selective methods entail sample destruction. However, sensitive methods are mandatory for studying metal ion homeostasis, and new methods have to distinguish between the disposable ion pool and the strongly bound enzyme components.Recently, we could show in two publications that bivalent copper ions lead to a reduction of the fluorescence lifetime of fluorescent proteins. This approach could also be used to map the uptake of copper ions in plant root cells by fluorescence lifetime imaging microscopy. The observed reversible change obeys a titration behavior with a midpoint at micromolarity. Despite these promising achievements, the investigation of ion homeostasis demands further improvements: firstly, the sensitivity of the probe must be increased; secondly and more importantly, the concentration of the indicator cannot longer be neglected and must be quantified, e.g. by fluorescence correlation spectroscopy (FCS). We therefore propose to combine lifetime measurements and FCS in the so-called fluorescence lifetime correlation spectroscopy (FLCS) for accessing naturally occurring ion concentration in living systems.During the grant period, we will establish at the beginning FLCS in our laboratory with the already described copper-sensitive system and an existing setup. Afterwards, we'll head for nanomolarity by using synthetic fluorophores with a higher affinity for metal ions. Once FLCS will have been established for lower ion concentrations than micromolarity, we'll screen several described, copper-binding peptides for their compatibility with FLCS. These investigations are aimed at proposing a detection scheme as well as a peptide sequence for studying copper homeostasis. If time is left, we will address the question how copper can be quantified at these concentration levels among other, more abundant transition-metal ions.

后过渡金属离子在生命科学中的作用是模糊的。一方面，其中一些是蛋白质的基本成分。另一方面，相同元素的过量供应往往与阿尔茨海默病等严重疾病有关。实时监测任何生物体内的离子浓度是具有挑战性的，因为大多数成熟的金属选择方法需要销毁样品。然而，对于金属离子动态平衡的研究，敏感的方法是强制性的，新的方法必须区分一次性离子池和强结合的酶组分。最近，我们在两个文献中表明，二价铜离子导致荧光蛋白的荧光寿命缩短。这种方法也可以用来用荧光寿命成像显微镜来描绘植物根细胞对铜离子的吸收。观察到的可逆变化服从以微摩尔为中点的滴定行为。尽管取得了这些有希望的成就，但离子动态平衡的研究还需要进一步的改进：首先，必须提高探针的灵敏度；其次，更重要的是，指示剂的浓度不能再被忽略，必须通过荧光相关光谱(FCS)进行定量。因此，我们建议在所谓的荧光寿命相关光谱(FLCS)中结合寿命测量和FCS来获取生命系统中自然产生的离子浓度。在授权期内，我们将在实验室一开始就建立具有已经描述的铜敏感系统和现有设置的荧光寿命相关光谱。然后，我们将通过使用对金属离子具有更高亲和力的合成荧光团来实现纳米分子。一旦建立了比微摩尔浓度更低的离子浓度，我们将筛选出几种已描述的铜结合多肽，以确定它们与FLCS的兼容性。这些研究旨在提出一种检测方案以及用于研究铜的动态平衡的肽序列。如果剩下时间，我们将解决如何在这些浓度水平上量化铜以及其他更丰富的过渡金属离子的问题。