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

描述（由申请人提供）：离子选择性量子点（ISQDs）是基于离子选择性聚合物的光学纳米传感器，它将量子点（QDs）集成到传感器的核心中。钠选择性ISQD在1mM至1m范围内测量钠，选择性比钾高100倍，分辨率为805m。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。选择性离子萃取到聚合物基质中会引起基质内部的pH值变化，从而改变pH敏感染料的吸光度特性。吸光度的变化通过直接吸收量子点的荧光发射来减弱量子点的强度。我们的假设是，使用ISQDs来绘制细胞内钠的空间分布将揭示心脏细胞动作电位期间离子活性的异质性分布。我们基于以下假设：首先，isqd是唯一可用的钠探针，对钾的生理水平具有选择性，光稳定性和生物相容性。其次，已经证明，离子通道开口处的离子通量会产生高浓度离子的局部区域，或钙火花。由于通道的性质，钠火花应该出现在钠通道的打开处，然而文献中很少有记录的病例。我们相信，使用更好的钠成像工具，如isqd，将为这一鲜为人知的过程提供丰富的信息。本申请期的具体目的是：1.申请期限；调整isqd，使其与细胞内环境中测量钠的分析要求相兼容。一个健壮的传感器必须在以下类别中展示最佳结果：生理学相关的动态范围，传感器的浸出/寿命和尺寸。2. 验证细胞内环境下ISQDs对钠的响应。isqd必须表现出对细胞内环境中钠变化的反应，这与在特异性目标1中的溶液研究中取得的结果相当。验证将在定义良好的细胞系统中使用膜片钳和光学记录同时进行。此外，将进行单独膜片钳（无isqd）和CoroNa染料的比较。还将进行已知通道阻滞剂效应的剂量反应。3. 绘制钠在心肌细胞中的空间分布。钠通过外膜离子通道的通量导致细胞内钠浓度分布不均匀，至少在开放通道期间是如此。钠火花将在心肌细胞中被识别，并将评估对钠通道阻滞剂的影响。

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.