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.

项目摘要：用于快速生物学的纳秒时间成像的量子点探针 过程 许多已知的生物学功能 - 毫无疑问，尚未认识到更多 过程 - 由小分子，离子和蛋白质的运动进行或以其他方式进行。 在纳秒（NS，10-9 s）中改变几何形状或弥漫性的残基，A 时间尺度可以通过一套光学方法访问。由于光学探针或检测器的局限性 （或两者都），但是，几乎所有不断发展的生物系统的测量都以MS（10-3 s）时间记录事件 解决。那是令人兴奋的问题：我们缺少什么？如何寻找药理学 通过不断发展的生物系统的高空测量来改善目标？许多例子 快速构象变化，结合事件，氧化还原事件和离子流对生物功能至关重要 至少有一个共同点：它们与质子（H+）通量耦合，并且可以原则上通过高 - 局部H+浓度的时间分辨率跟踪。拟议的研究计划将开发 从根本上讲，新的荧光量子点（QD） - 配体问题，以实现全光测量 使用H+作为分析物在活细胞中快速生物过程，并分辨率分辨率。在 在2年的项目期间，我们的目标是通过探索策略来评估超快H+探针的可行性 优化此探针的亮度，灵敏度和响应时间，并评估这些探针的鲁棒性 模拟生物环境中的性质。该技术的长期愿景是使用 在衍射限制的，有时甚至是超分辨率的内部，显微镜设置为空间中的图像过程和 时间有前所未有的细节水平，从而将各种疾病的病理联系起来 基本的分子级机制。我们提出的QD-rigand传感器是可见的光或近红外光 - 发射QD，涂有有机配体，将数十个QD中数以千的酸性位点引入 表面。这些位点的PKA值在pH的各种物理相关范围内可调。 QD的照片兴奋状态（或“激子”）本身就是电偶极子本身，因此当它“看到”电场时产生 例如，通过表面上充电的分子，QD发出的照片的波长变化 在电场旅行的时间尺度上（〜10-15 s）。因此，QD排放的颜色对 H+的局部浓度通过可逆质子化和其配体的去质子化。重要的是，因为 在基于电场的灵敏度机制中，QD H+传感器发射波长的变化应应 相反，由于构象 最先进的GFP-的变化，氧化还原过程，质子转移或能量转移所需的变化 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QD传感器的目标响应时间慢105-106倍。