Center for Bio/Molecular Science and Engineering, Code 6900, U. S. Naval Research Laboratory, Washington, DC 20375 USA.
Nanoscale. 2015 May 7;7(17):7603-14. doi: 10.1039/c5nr00828j.
The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of the energy transfer process(es) along with potential applications of such devices are finally discussed.
在新的纳米尺度结构和设备中控制光能将极大地促进它们的持续发展和最终应用。理想情况下，这种控制应该从产生光本身扩展到设备内的光的空间传播，同时提供定义的发射波长（s），所有这些都在独立的模式下进行。在这里，我们设计并表征了由半导体量子点（QD）、几个不同染料标记的肽和酶荧光素组成的大分子纳米组装体，这些组装体通过多个连续的能量转移步骤累积地展示了许多这些功能。为了创建这些结构，重组表达的荧光素和染料标记的肽被添加了一个末端多组氨酸序列，允许通过金属亲和力配位来控制比率自组装围绕 QD。QD 用于在这些结构中提供多种作用，包括作为中央组装平台或纳米支架，以及作为一种有效的能量收集和转移中继。通过添加腔肠素 H 底物来激活这些设备，该底物被荧光素氧化，产生光能，通过生物发光共振能量转移（BRET）使中央 625nm 发射 QD 受体敏化。敏化的 QD 反过来又作为中继，通过第二次集中的Förster 共振能量转移（FRET）过程将能量传递到其表面显示的第一个肽标记的 Alexa Fluor 647 受体染料。然后，该染料通过第二个同心的Förster 共振能量转移（FRET）过程将能量传递到 QD 表面上的第二个红移肽标记染料受体。Alexa Fluor 700 和 Cy5.5 都被测试作为这个末端 FRET 受体的角色。对由此产生的顺序 BRET-FRET-FRET 过程的光谱轮廓的光物理分析使我们能够估计每个转移步骤的效率。重要的是，通过在 QD 上显示的酶/肽的数量，可以在一定程度上控制这种能量转移级联中的每个步骤的效率。最后讨论了这种能量转移过程的进一步优化及其潜在应用。
