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PEGylated Red-Emitting Calcium Probe with Improved Sensing Properties for Neuroscience
ACS Sensors ( IF 8.2 ) Pub Date : 2017-10-24 00:00:00 , DOI: 10.1021/acssensors.7b00665 Flavien Ponsot 1 , Weida Shen 2, 3 , Pichandi Ashokkumar 1 , Etienne Audinat 2, 3 , Andrey S. Klymchenko 1 , Mayeul Collot 1
ACS Sensors ( IF 8.2 ) Pub Date : 2017-10-24 00:00:00 , DOI: 10.1021/acssensors.7b00665 Flavien Ponsot 1 , Weida Shen 2, 3 , Pichandi Ashokkumar 1 , Etienne Audinat 2, 3 , Andrey S. Klymchenko 1 , Mayeul Collot 1
Affiliation
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Monitoring calcium concentration in the cytosol is of main importance as this ion drives many biological cascades within the cell. To this end, molecular calcium probes are widely used. Most of them, especially the red emitting probes, suffer from nonspecific interactions with inner membranes due to the hydrophobic nature of their fluorophore. To circumvent this issue, calcium probes conjugated to dextran can be used to enhance the hydrophilicity and reduce the nonspecific interaction and compartmentalization. However, dextran conjugates also feature important drawbacks including lower affinity, lower dynamic range, and slow diffusion. Herein, we combined the advantage of molecular probes and dextran conjugate without their drawbacks by designing a new red emitting turn-on calcium probe based on PET quenching, Rhod-PEG, in which the rhodamine fluorophore bears four PEG4 units. This modification led to a high affinity calcium probe (Kd = 748 nM) with reduced nonspecific interactions, enhanced photostability, two-photon absorbance, and brightness compared to the commercially available Rhod-2. After spectral characterizations, we showed that Rhod-PEG quickly and efficiently diffused through the dendrites of pyramidal neurons with an enhanced sensitivity (ΔF/F0) at shorter time after patching compared to Rhod-2.
中文翻译：
具有改善的神经科学传感特性的聚乙二醇化的红色发光钙探针
监测细胞质中钙的浓度非常重要，因为该离子会驱动细胞内的许多生物级联反应。为此，分子钙探针被广泛使用。它们中的大多数，特别是红色发射探针，由于其荧光团的疏水性而遭受与内膜的非特异性相互作用。为了解决这个问题，可以使用与葡聚糖偶联的钙探针增强亲水性并减少非特异性相互作用和分隔。然而，右旋糖酐共轭物还具有重要的缺点，包括较低的亲和力，较低的动态范围和缓慢的扩散。在此，我们通过设计一种基于PET猝灭的新型红色发射开启钙探针，Rhod-PEG，4个单位。与市售的Rhod-2相比，这种修饰导致了高亲和力的钙探针（K d = 748 nM），具有减少的非特异性相互作用，增强的光稳定性，两光子吸收率和亮度。光谱表征后，我们发现与Rhod-2相比，Rhod-PEG在贴片后的更短时间内以增强的灵敏度（ΔF / F 0）快速有效地扩散通过锥体神经元的树突。
更新日期：2017-10-25
中文翻译：
具有改善的神经科学传感特性的聚乙二醇化的红色发光钙探针
监测细胞质中钙的浓度非常重要，因为该离子会驱动细胞内的许多生物级联反应。为此，分子钙探针被广泛使用。它们中的大多数，特别是红色发射探针，由于其荧光团的疏水性而遭受与内膜的非特异性相互作用。为了解决这个问题，可以使用与葡聚糖偶联的钙探针增强亲水性并减少非特异性相互作用和分隔。然而，右旋糖酐共轭物还具有重要的缺点，包括较低的亲和力，较低的动态范围和缓慢的扩散。在此，我们通过设计一种基于PET猝灭的新型红色发射开启钙探针，Rhod-PEG，4个单位。与市售的Rhod-2相比，这种修饰导致了高亲和力的钙探针（K d = 748 nM），具有减少的非特异性相互作用，增强的光稳定性，两光子吸收率和亮度。光谱表征后，我们发现与Rhod-2相比，Rhod-PEG在贴片后的更短时间内以增强的灵敏度（ΔF / F 0）快速有效地扩散通过锥体神经元的树突。
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