Heterostructured Quantum Dots as Molecular Probes: Chemistry and Photophysics

描述（由申请人提供）：本项目的首要目标是开发新的生物相容性荧光探针，能够方便地检测活细胞中的单分子事件。为了实现这一目标，将合成尚未开发的新型半导体纳米晶体量子点（NQD）探针，对其光物理、结构和化学性质进行表征，并对其进行筛选以确定生物相容性。nqd具有高信号输出、窄带宽、更好的光漂白稳定性、易于激发的宽带吸收、合理的小尺寸以及表面化学的灵活性，可以潜在地实现可传递性和生理中性。这里将开发的两个基于NQD的系统是(1)近红外发射NQDs和(2)镧系元素（Ln）掺杂NQDs，其中NQD作为Ln发射的敏化剂。我们将以提供600 - 1400纳米发射的系统为目标。1000 nm以下的光谱区域具有通过生物组织的高透射率，值得进一步扩展到1400 nm的红外波段进行细胞研究，因为只有在该波长以上，干扰水的吸收率才会显著增加。尽管基于nqd的材料具有光学成像应用的所有固有优势，但仍存在一些障碍。首先，细胞中单个nqd的长期跟踪受到nqd特征的荧光间歇性（闪烁）的阻碍。其次，对于含重金属的非量子点或表面钝化不当的非量子点，生物相容性是一个值得关注的问题。对于第一个不足，虽然已经假设眨眼的起源与NQD表面的电荷转移过程有关，但实验证据有限，缺乏对眨眼与NQD充电之间关系的定量理解。如果没有经过实验验证的对这一基本过程的理解，设计和合成非闪烁nqd的努力就会受到阻碍。我们将进行稳态和超快光谱研究来阐明充电机制，并将这些结果与单nqd闪烁研究相关联。光谱学研究的结果将为光化学稳定结构的设计提供指导，这些结构预计依赖于无机异质结构（例如，复杂的核/壳结构）。我们将首次研究红外发射nqd中的闪烁，甚至缺乏基本的研究，目的是了解其潜在机制并制定消除其的综合策略。我们还将研究新的Ln- nqd耦合系统，其中发光源自Ln掺杂剂，因此不会出现闪烁。这里的目的是优化从吸收体NQD到发射体Ln的能量传递过程，从而优化组合系统的信号输出。在这些研究的同时，我们将通过研究我们基于nqd的探针在各种人类细胞系中的毒性和定位来解决基于nqd的荧光团的第二个已知缺陷——生物相容性不足。与眨眼研究类似，生物相容性研究将为设计具有适当组成、表面钝化和表面功能的探针提供有价值的反馈。对活细胞内生物分子的位置、活性和反应性进行实时成像的能力是进一步推进生物医学科学的基础，包括药物发现，但目前可用的荧光分子探针无法提供分子和分子事件的常规研究。我们建议通过基础物理、化学和生物学研究的结合来开发先进的基于量子点的分子探针，这将为实现所需的光学分子成像能力提供必要的进步。

DESCRIPTION (provided by applicant): The overriding objective that will be pursued in this project is to develop new biocompatible fluorescent probes capable of providing facile detection of single-molecule events in living cells. In pursuit of this goal, under-explored and novel semiconductor nanocrystal quantum dot (NQD)-based probes will be synthesized, characterized with respect to their photophysical, structural and chemical properties, and screened to ascertain biocompatibility. NQDs offer high signal output, narrow bandwidth, improved stability with respect to photobleaching, broadband absorption for facile excitation, reasonably small size, and flexibility in surface chemistry for potentially achieving deliverability and physiological neutrality. The two NQD-based systems that will be developed here are (1) Near-infrared-emitting NQDs and (2) Lanthanide (Ln) doped NQDs, where the NQD serves as a sensitizer for Ln emission. We will target systems that provide emission from 600 - 1400 nm. This spectral region below 1000 nm is distinguished by a high transmittance through biological tissue and is worth extending farther into the infrared to 1400 nm for cellular studies, as interfering water absorption increases significantly only above this wavelength. Despite all the inherent advantages of NQD-based materials for optical imaging applications, several obstacles remain. Firstly, long-term single-NQD tracking in cells is hindered by fluorescence intermittency (blinking) that is characteristic of NQDs. Secondly, NQD biocompatibility is a concern for heavy-metal-containing NQDs or for NQDs that are improperly surface passivated. With respect to the first deficiency, though it has been postulated that the origin of blinking is related to charge transfer processes at the NQD surface, the experimental evidence is limited and the quantitative understanding of the connection between blinking and NQD charging is lacking. Without an experimentally validated understanding of this fundamental process, efforts to design and synthesize non-blinking NQDs are inherently impeded. We will perform steady-state and ultrafast spectroscopic studies to elucidate the mechanism of charging and correlate these results with single-NQD blinking studies. Results of spectroscopic studies will provide guidance for the design of photochemically stable structures that is anticipated to rely on inorganic heterostructuring (e.g., complex core/shell architectures). We will for the first time investigate blinking in infrared-emitting NQDs for which even rudimentary studies are lacking with the objective to understand the underlying mechanism and to develop synthetic strategies for its elimination. We will also investigate novel Ln-NQD coupled systems, in which the luminescence originates in the Ln dopant and is therefore not expected to exhibit blinking. The aim here will be to optimize the energy transfer process from the absorber NQD to the emitting Ln and, thereby, the signal output of the combined system. In parallel with these studies, we will address the second perceived deficiency of NQD-based fluorophores - insufficient biocompatibility - by investigating the toxicity and localization of our NQD-based probes in a variety of human cell lines. Similar to the blinking studies, the biocompatibility studies will provide valuable feedback in the design of probes possessing appropriate composition, surface passivation, and surface functionality. The ability to image real-time the location, activity and reactivity of biomolecules as they occur within living cells is fundamental to furthering biomedical science, including drug discovery, but currently available fluorescent molecular probes are not capable of providing for the routine study of molecules and molecular events. The advanced quantum dot based molecular probes that we propose to develop through a combination of fundamental physical, chemical and biological studies will enable the advances necessary for achieving the required optical molecular imaging capability.