A Quantum Dot Probe for Nanosecond-Timescale Imaging of Fast Biological Processes

用于快速生物过程纳秒级成像的量子点探针

• 批准号: 9502603
• 负责人: Emily Allyn Weiss
• 金额: $ 22.83万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9502603
• 关键词: Achievement; Binding; Biological; Biological Process; Buffers; Cations; Cell Culture Techniques; Cells; Charge; Color; Coupled; Culture Media; Detergents; Diffuse; Diffusion; Disease; Electrons; Elements; Energy Transfer; Environment; Equilibrium; Event; Exciton; Fluorescent Probes; Geometry; Goals; Image; Ions; Ligands; Light; Link; Liposomes; Measurement; Measures; Mediating; Mental Depression; Methods; Microscopy; Molecular; Molecular Conformation; Monitor; Motion; NMR Spectroscopy; Optical Methods; Optics; Oxidation-Reduction; Oxygen; Pathology; Pharmacology; Photons; Physiological; Precipitation; Probability; Procedures; Process; Property; Proteins; Protons; Quantum Dots; Reaction Time; Research; Resolution; Semiconductors; Shapes; Signal Transduction; Site; Spectrum Analysis; Structure; Sulfhydryl Compounds; Surface; System; Technology; Thinness; Time; Travel; Visible Radiation; Vision; Work; absorption; aqueous; base; biological systems; density; deprotonation; detector; divalent metal; electric dipole; electric field; improved; migration; monolayer; nanosecond; programs; protonation; quantum; response; sensor; small molecule; sugar

PROJECT SUMMARY: A Quantum Dot Probe for Nanosecond-Timescale Imaging of Fast Biological Processes A great number of known biological functions – and undoubtedly a much larger number of as-yet unrecognized processes – are performed by, gated by, or otherwise linked to the motions of small molecules, ions, and protein residues that change geometry or diffuse over biologically relevant distances in nanoseconds (ns, 10-9 s), a timescale readily accessible by a suite of optical methods. Due to limitations of the optical probe or the detector (or both), however, nearly all measurements of evolving biological systems record events with ms (10-3 s) time resolution. The exciting questions are then: What are we missing? How could the search for pharmacological targets be improved by high-time resolution measurements of evolving biological systems? Many examples of fast conformational changes, binding events, redox events, and ion flows critical for biological functions have at least one thing in common: they are coupled to proton (H+) fluxes, and can, in principle, be monitored via high- time resolution tracking of local H+ concentrations. The proposed research program will develop a fundamentally new class of fluorescent quantum dot (QD)-ligand probes to enable all-optical measurements of fast biological processes in live cells using H+’s as an analyte, with nanosecond time resolution. At the end of the 2-yr project period, we aim to have evaluated the feasibility of our ultrafast H+ probe, by exploring strategies to optimize the brightness, sensitivity, and response time of this probe and evaluating the robustness of these properties in simulated biological environments. The longer-term vision for this technology is that it be used within diffraction-limited, and eventually super-resolution, microscopy setups to image processes in space and time with an unprecedented level of detail, and thereby connect pathologies of a vast array of diseases with their underlying molecular-level mechanisms. Our proposed QD-ligand sensor is a visible light- or near-infrared light- emitting QD, coated in organic ligands that introduce tens to hundreds of acidic sites within angstroms of the QD surface. The pKa values at these sites are tunable within various physiologically relevant ranges of pH. The photo-excited state (or “exciton”) of the QD is an electric dipole itself, so when it “sees” electric fields generated by, for instance, charged molecules on the surface, the wavelength of the photons that the QD emits changes on the timescale of travel of the electric field (~10-15 s). The color of the QD’s emission is therefore sensitive to the local concentration of H+’s via reversible protonation and deprotonation of its ligands. Importantly, because of the electric field-based sensing mechanism, the change in emission wavelength of the QD H+ sensor should occur effectively instantaneously with a change in local H+ concentration. In contrast, due to the conformational changes, redox processes, proton transfer, or energy transfer required for emission shifts in state-of-the-art GFP- based pH sensors, these sensors have response times of ~20 ms (with an estimated lower limit of 0.5 ms), at least a factor of 105-106 slower than the targeted response time of our QD sensor.

项目概要：用于快速生物成像的纳秒时间尺度量子点探针 过程 大量已知的生物学功能--无疑还有更多尚未被认识的功能 过程-由小分子、离子和蛋白质的运动执行、门控或以其他方式与之相关联 改变几何形状或在纳秒（ns，10-9 s）生物相关距离上扩散的残基，a 一套光学方法很容易获得的时间尺度。由于光学探头或检测器的限制， (or然而，几乎所有对进化生物系统的测量都以ms（10-3 s）时间记录事件 分辨率那么令人兴奋的问题是：我们错过了什么？药物研究怎么可能 通过对进化中的生物系统进行高时间分辨率的测量来改进目标？的许多实例 快速的构象变化、结合事件、氧化还原事件和对生物学功能至关重要的离子流， 至少有一个共同点：它们与质子（H+）通量耦合，原则上可以通过高通量监测。 局部H+浓度的时间分辨率跟踪。该研究计划将开发一个 从根本上说，新型荧光量子点（QD）-配体探针能够实现全光学测量， 使用H+作为分析物在活细胞中进行快速生物过程，具有纳秒时间分辨率。结束时 在为期2年的项目期间，我们的目标是通过探索策略来评估我们的超快H+探测器的可行性 优化该探头的亮度、灵敏度和响应时间，并评估这些探头的鲁棒性。 在模拟生物环境中的性能。这项技术的长期愿景是， 在衍射有限，最终超分辨率，显微镜设置图像的空间过程， 时间与前所未有的详细程度，从而将大量疾病的病理与他们的疾病联系起来。 潜在的分子水平机制。我们提出的量子点配体传感器是一种可见光-或近红外光- 发射QD，涂覆在有机配体中，所述有机配体在QD的埃内引入数十至数百个酸性位点 面这些位点处的pKa值在各种生理学相关的pH范围内是可调的。 量子点的光激发态（或“激子”）本身就是一个电偶极子，所以当它“看到”产生的电场时， 例如，通过表面上的带电分子，量子点发射的光子的波长改变 在电场传播的时间尺度上（~10-15 s）。因此，QD的发射的颜色对 H+通过其配体的可逆质子化和去质子化的局部浓度。重要的是因为 在基于电场的感测机制中，QD H+传感器的发射波长的变化应 随着局部H+浓度的变化而有效地瞬时发生。相反，由于构象 变化，氧化还原过程，质子转移，或能量转移所需的发射位移在国家的最先进的GFP- 基于pH传感器，这些传感器的响应时间约为20 ms（估计下限为0.5 ms），在 比我们的QD传感器的目标响应时间慢至少105-106倍。