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.
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.
|Ruckh, Timothy T; Clark, Heather A (2014) Implantable nanosensors: toward continuous physiologic monitoring. Anal Chem 86:1314-23|
|Cash, Kevin J; Clark, Heather A (2013) Phosphorescent nanosensors for in vivo tracking of histamine levels. Anal Chem 85:6312-8|
|Balaconis, Mary K; Clark, Heather A (2013) Gel encapsulation of glucose nanosensors for prolonged in vivo lifetime. J Diabetes Sci Technol 7:53-61|
|Balaconis, Mary K; Billingsley, Kelvin; Dubach, Matthew J et al. (2011) The design and development of fluorescent nano-optodes for in vivo glucose monitoring. J Diabetes Sci Technol 5:68-75|
|Ozaydin-Ince, Gozde; Dubach, J Matthew; Gleason, Karen K et al. (2011) Microworm optode sensors limit particle diffusion to enable in vivo measurements. Proc Natl Acad Sci U S A 108:2656-61|
|Cash, Kevin J; Clark, Heather A (2010) Nanosensors and nanomaterials for monitoring glucose in diabetes. Trends Mol Med 16:584-93|
|Billingsley, Kelvin; Balaconis, Mary K; Dubach, J Matthew et al. (2010) Fluorescent nano-optodes for glucose detection. Anal Chem 82:3707-13|
|Dubach, J Matthew; Das, Saumya; Rosenzweig, Anthony et al. (2009) Visualizing sodium dynamics in isolated cardiomyocytes using fluorescent nanosensors. Proc Natl Acad Sci U S A 106:16145-50|