Fluorescence Lifetime Correlation Spectroscopy for Metal Ion Quantitation

后过渡金属离子在生命科学中的作用一直不明确。一方面，其中一些是蛋白质的基本组成部分。另一方面，相同元素的过量供应通常与阿尔茨海默氏症等严重疾病有关。实时监测任何活生物体中的离子浓度都具有挑战性，因为大多数已建立的金属选择性方法都需要破坏样品。然而，敏感的方法是强制性的研究金属离子的稳态，和新的方法必须区分一次性离子池和强结合的酶components.Recently，我们可以表明，在两个出版物中，二价铜离子导致荧光蛋白的荧光寿命的减少。这种方法也可用于绘制吸收的铜离子在植物根细胞的荧光寿命成像显微镜。所观察到的可逆变化服从滴定行为，中点在微摩尔浓度。尽管取得了这些令人鼓舞的成就，离子稳态的研究需要进一步改进：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视，必须被量化，例如通过荧光相关光谱（FCS）。因此，我们建议将联合收割机寿命测量和FCS结合在所谓的荧光寿命相关光谱（FLCS）中，以评估生命系统中自然发生的离子浓度。在资助期间，我们将在我们的实验室中使用已经描述的铜敏感系统和现有的设置建立FLCS。之后，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级。一旦FLCS将已经建立了低于微摩尔浓度的离子浓度，我们将筛选几个描述，铜结合肽与FLCS的兼容性。这些调查的目的是提出一个检测方案，以及研究铜稳态的肽序列。如果有时间，我们将解决这样一个问题：在这些浓度水平下，如何在其他更丰富的过渡金属离子中定量测定铜。

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.