Field Lauren D, Walper Scott A, Susumu Kimihiro, Oh Eunkeu, Medintz Igor L, Delehanty James B
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, 4555 Overlook Ave, S.W., Washington, DC 20375, USA.
Sotera Defense Solutions, Inc., 7230 Lee DeForest Drive, Columbia, MD 21046, USA.
Sensors (Basel). 2015 Dec 4;15(12):30457-68. doi: 10.3390/s151229810.
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor-acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing.
了解如何可控地调节基于Förster共振能量转移（FRET）的组装体中的能量转移效率，对于将其用作传感模式至关重要。对于用作细胞内过程和事件实时细胞内传感基础的传感组装体来说尤其如此。我们使用一种量子点（QD）供体-mCherry受体平台，该平台经过工程设计可原位自组装，其中蛋白质受体通过瞬时转染表达，而QD供体则显微注射到细胞中。QD-蛋白质组装由金属亲和相互作用驱动，蛋白质上的末端多组氨酸标签与QD表面结合。使用该系统，我们展示了通过可控地改变QD表面的配体涂层或QD-蛋白质组装过程发生的精确位置来调节供体-受体能量转移过程效率的能力。在细胞内，与基于增溶聚合物聚乙二醇的较长配体相比，一种短的两性离子配体介导更有效的FRET。我们进一步表明，与mCherry受体表达固定在质膜内小叶上相比，当QD-蛋白质组装在细胞质中自由发生时，可实现更高的FRET效率。在后一种情况下，较低的FRET效率可能归因于膜上mCherry受体的表达水平较低以及空间位阻。我们的工作指出了在开发用于细胞内传感的基于FRET的传感方案时必须考虑的一些设计因素。
