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

项目总结：用于快速生物纳秒级成像的量子点探测器 流程 大量已知的生物功能--毫无疑问，还有更多的尚未被认识的功能 过程-由小分子、离子和蛋白质的运动执行，由其控制，或以其他方式与小分子、离子和蛋白质的运动相关联 在纳秒内改变几何结构或在生物相关距离内扩散的残基(纳秒，10-9 S)，a 时间刻度很容易通过一套光学方法获取。由于光学探头或探测器的限制 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 决议。那么，令人兴奋的问题是：我们错过了什么？怎么可能搜索到药理学的 通过进化生物系统的高时间分辨率测量来改进目标？许多例子表明 对生物功能至关重要的快速构象变化、结合事件、氧化还原事件和离子流在 至少有一个共同点：它们与质子(H+)通量耦合，原则上可以通过高密度的 当地H+浓度的时间分辨率跟踪。拟议的研究计划将开发一种 一种全新的荧光量子点(QD)配体探针，可实现全光测量 使用H+‘S作为分析物，以纳秒时间分辨率在活细胞中进行快速生物过程。在…的末尾 在两年的项目期内，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 为了优化该探头的亮度、灵敏度和响应时间，并评估其稳健性 在模拟生物环境中的特性。这项技术的长期愿景是将其用于 在衍射受限，并最终达到超分辨率的显微镜装置内，成像过程在空间和 时间以前所未有的详细程度，从而将大量疾病的病理与它们的 潜在的分子水平的机制。我们提出的QD-配体传感器是可见光-或近红外光- 发射包覆有有机配体的量子点，在量子点的埃内引入数十到数百个酸性位 浮出水面。这些部位的pKa值在各种生理相关的pH范围内是可调的。这个 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，量子点发出的光子的波长会发生变化 关于电场的传播时间尺度(~10-15 S)。因此，量子点发射的颜色对 H~+‘S通过其配体的可逆质子化和去质子化的局域浓度。重要的是，因为 在基于电场的传感机制中，QD H+传感器的发射波长的变化应该 随着局部H+浓度的变化，有效的瞬间发生。相反，由于构象 改变，氧化还原过程，质子转移，或能量转移所需的发射转移在最先进的GFP- 基于pH传感器，这些传感器的响应时间为~20ms(估计下限为0.5ms)，在 比我们QD传感器的目标响应时间慢至少105-106倍。

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.