Ion Selective Quantum Dots for Intracellular Mapping of Sodium Sparks in Cardiac

描述(申请人提供)：离子选择性量子点(ISQD)是离子选择性聚合物为基础的光学纳米传感器，它将量子点(QD)结合到传感器的核心中。钠选择性ISQD测量钠的范围为1 mm到1 M，选择性是钾的100倍，分辨率为80 5M。离子选择性量子点由量子点、pH敏感染料和离子选择性聚合物组成。选择性离子萃取进入聚合物基质会引起基质内部的pH变化，从而改变对pH敏感的染料的吸收特性。吸光度的变化通过直接吸收其荧光发射来衰减量子点的强度。我们的假设是，使用ISQD来绘制细胞内钠的空间分布将揭示心肌细胞动作电位期间离子活性的不同分布。我们的假设建立在以下基础上：首先，ISQD是唯一一种对生理水平的钾具有选择性、光稳定和生物相容的钠探针。其次，已经证明，离子通道打开时的离子流量会产生高离子浓度的局部区域，即钙火花。由于钠离子通道的性质，钠离子离子应该出现在钠离子通道的开口处，然而文献中记录在案的病例很少。我们相信，使用更好的钠成像工具，如ISQD，将提供关于这一鲜为人知的过程的丰富信息。本申请期的具体目标是：1.量身定做ISQD，以适应细胞内环境中钠离子测量的分析要求。一个坚固的传感器必须在以下几个方面表现出最佳的结果：生理上相关的动态范围、传感器的浸出/寿命和尺寸。2.验证细胞内环境中ISQds对钠离子的响应。ISQD对细胞内环境中钠离子变化的响应必须与在特定目标1的溶液研究中达到的响应相当。验证将在定义良好的细胞系统中使用同步膜片钳和光学记录进行。此外，还将与膜片钳(无ISQD)和电晕染料进行比较。对已知的通道阻滞剂的作用也将进行剂量反应。3.绘制心肌细胞内钠离子的空间分布图。通过外膜离子通道的钠通量导致细胞内钠浓度的不均匀分布，至少在开放通道的持续时间内是这样。钠火花将在心肌细胞中被识别，并将被评估对钠通道阻滞剂的影响。 公共卫生相关性：这项应用的最终目标是开发和使用一种新的细胞内成像工具--离子选择性量子点来绘制心肌细胞中的钠微域。这些探测器将提供有关离子通道分布的关键信息，而目前的工具无法提供这些信息。最终，这一工具将提供心脏动作电位的新知识，并可能导致在长QT综合征等疾病中预防致命的心律失常。

DESCRIPTION (provided by applicant): Ion-selective quantum dots (ISQDs) are ion-selective polymer-based optical nanosensors that incorporate quantum dots (QDs) into the core of the sensor. A sodium-selective ISQD measures sodium over the range of 1mM to 1 M with 100 fold selectivity over potassium and a resolution of 80 5M. Ion-selective quantum dots consist of a quantum dot, a pH sensitive dye, and an ion-selective polymer. Selective ion extraction into the polymer matrix causes a pH change inside the matrix therefore changing the absorbance properties of the pH sensitive dye. The change of absorbance attenuates the intensity of the quantum dot by directly absorbing its fluorescence emission. Our hypothesis is that using ISQDs to map the spatial distribution of intracellular sodium will reveal a heterogeneous distribution of ion activity during the action potential of a cardiac cell. We base our hypothesis on the following: First, ISQDs are the only sodium probes available that are selective over physiological levels of potassium, photostable, and biocompatible. Second, it has been shown that fluxes of ions at the opening of an ion channel create localized regions of high ion concentrations, or calcium sparks . Because of the nature of the channel sodium sparks should be present at the opening of sodium channels, however there are very few documented cases in the literature. We believe that using better tools for sodium imaging, such as ISQDs, will provide a wealth of information on this little known process. The specific aims of this application period are: 1. To tailor ISQDs to be compatible with the analytical requirements of measuring sodium in an intracellular environment. A robust sensor must demonstrate optimal results in the following categories: physiologically relevant dynamic range, leaching/lifetime of sensors, and size. 2. To validate the response of ISQDs to sodium in the intracellular environment. ISQDs must show a response to changes in sodium in the intracellular environment that are comparable to those achieved in solution studies in Specific Aim 1. Validation will be performed using simultaneous patch clamp and optical recording in a well-defined cell system. Additionally, a comparison to patch-clamp alone (no ISQDs) and CoroNa dyes will be performed. A dose response to the effects of known channel blockers will also be carried out. 3. To map the spatial distribution of sodium in cardiac myocytes. Sodium fluxes through ion channels in the outer membrane lead to inhomogeneous distributions of sodium concentration in the cell, at least during the duration of the open channel. Sodium sparks will be identified in cardiac myocytes, and will be evaluated for effects to sodium channel blockers. PUBLIC HEALTH RELEVANCE: The ultimate goal of this application is to develop and use a new intracellular imaging tool, Ion- Selective Quantum Dots to map sodium microdomains in cardiac cells. These probes will provide crucial information on ion channel distribution that is not available with current tools. Ultimately, this tool will provide new knowledge of cardiac action potentials and possibly lead to the prevention of fatal arrhythmias in diseases such as Long QT syndrome.