Supramolecular Tandem Membrane Assays

有机分子的膜转运在小分子药物的发现和递送以及抗菌肽和细胞穿透肽（CPPs）的作用机制中起着至关重要的作用。然而，测量这些化合物在脂质双分子层膜上的实际运输仍然是一个主要的实验挑战。我们最近表明，脂质体封装的超分子报告对通过一种简单的基于荧光的方法非常适合明确地检测有机分子和肽的膜运输。在本项目中，我们希望进一步确立该方法的实用性和适用性。因此，我们将在不同大小和膜组成的脂质体中封装超分子报告对，并将通过吸收和荧光光谱方法研究选定的有机分子和肽的膜转运。这包括光谱表征，时间分辨测量，包括用于快速动力学的停止流动技术，以及具有巨大单层囊泡（GUVs）的荧光显微镜。我们将进一步发展这些“超分子串联膜分析”，应用它们来跟踪小分子，未标记分子和CPPs的膜运输。具体而言，我们计划建立多色方法来研究膜运输过程的机理，测量快速外排动力学，并将串联膜分析与酶促反应相结合，为开发超生物分子传感器系统提供新的途径。

Membrane transport of organic molecules plays a crucial role in the discovery and delivery of small-molecule drugs as well as in the mechanism of action of antimicrobial peptides and cell-penetrating peptides (CPPs). It still remains, however, a major experimental challenge to measure the actual transport of these compounds across the lipid bilayer membrane. We have recently shown that liposome-encapsulated supramolecular reporter pairs excellently qualify to unambiguously detect membrane transport of organic molecules and peptides by a simple and fluorescence-based method. Within this project, we want to further establish the utility and applicability of this method.Therefore, we will encapsulate supramolecular reporter pairs in liposomes of varying size and membrane composition, and we will investigate the membrane transport of selected organic molecules and peptides by absorption and fluorescence spectroscopic methods. This includes spectroscopic characterisations, time-resolved measurements including stopped-flow techniques for rapid kinetics, as well as fluorescence microscopy with giant uni-lamellar vesicles (GUVs).We will further develop these “supramolecular tandem membrane assays” by applying them to follow membrane transport of small, unlabeled molecules and CPPs. In detail, we plan to establish multicolor methods for mechanistic investigation of membrane transport processes, measure fast efflux kinetics, and we will combine tandem membrane assays with enzymatic reactions to provide new avenues for the development of supra-biomolecular sensor systems.