We have developed a fluorescence-based turn-on molecular sensor (NeuroSensor 521) based on a coumarin aldehyde scaffold that allows for the selective recognition and sensing of norepinephrine in live and fixed chromaffin cells. Based on NeuroSensor 521, we have now developed Miz-2: a promising member of a new series of catecholamine-selective sensors, which has both a functional aldehyde group to bind to primary amines, and other recognition elements to improve selectivity for binding catecholamine over other amines. The excitation maximum of Miz-2 is near 488 nm. Fluorescence is quenched when the sensor interacts with epinephrine and dopamine whereas the fluorescence increases upon binding with norepinephrine. We are using adrenal chromaffin cells as a model cell to test fluorescent catecholamine sensors and sub-populations of cells enriched in epinephrine versus norepinephrine can be isolated upon gradient centrifugation. Norepinephrine-enriched chromaffin cells exhibit distinct fluorescence punctae by total internal reflection fluorescence (TIRF) microscopy when loaded with Miz-2 that are consistent with labeling of granules. Upon stimulation with a high-K+ solution, punctae abruptly disappear during TIRF imaging, consistent with granule fusion and loss of dye via exocytosis. Using underlying transparent electrodes, we recorded amperometric spikes consistent with quantal exocytosis of catecholamine in cells loaded with the sensor. Experiments are in progress to combine TIRF imaging with amperometric measurements in underlying transparent electrodes to resolve features of norepinephrine release from individual granule. Supported by NIH R01EB020415.

中文翻译：

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Miz-2，一种用于可视化去甲肾上腺素的新型儿茶酚胺选择性荧光传感器

我们开发了一种基于荧光的开启分子传感器 (NeuroSensor 521)，它基于香豆素醛支架，可选择性识别和感知活细胞和固定嗜铬细胞中的去甲肾上腺素。基于 NeuroSensor 521，我们现在开发了 Miz-2：一个新的儿茶酚胺选择性传感器系列中的一个很有前途的成员，它具有与伯胺结合的功能性醛基和其他识别元件，以提高结合儿茶酚胺的选择性。其他胺类。Miz-2 的激发最大值接近 488 nm。当传感器与肾上腺素和多巴胺相互作用时荧光被淬灭，而在与去甲肾上腺素结合时荧光增加。我们使用肾上腺嗜铬细胞作为模型细胞来测试荧光儿茶酚胺传感器，并且可以通过梯度离心分离富含肾上腺素和去甲肾上腺素的细胞亚群。当装载与颗粒标记一致的 Miz-2 时，富含去甲肾上腺素的嗜铬细胞通过全内反射荧光 (TIRF) 显微镜显示出明显的荧光点。在用高 K+ 溶液刺激后，点状细胞在 TIRF 成像过程中突然消失，这与颗粒融合和通过胞吐作用丢失染料一致。使用下面的透明电极，我们记录了与加载有传感器的细胞中儿茶酚胺的量子胞吐作用一致的电流峰值。正在进行实验以将 TIRF 成像与底层透明电极中的电流测量相结合，以解决单个颗粒释放去甲肾上腺素的特征。由 NIH R01EB020415 支持。