Multispectral near Infrared Imaging of Catecholamine Neurotransmitters with Fluorescent Nanosensors

该项目的目的是创建神经递质的近红外(NIR)荧光纳米传感器，测量和了解它们的动力学/选择性，并将它们用于细胞间化学通讯的多光谱成像。这个项目的动机是多细胞生物体中化学交流的重要性。对于这一过程的复杂性，人类大脑中的神经网络是最耐人寻味的例子。到目前为止，还没有太多的分析工具可以在复杂的生物环境中对小分子，如小长度尺度和快时间尺度上的神经递质进行成像。因此，细胞之间的快速化学交流，尤其是神经网络中神经递质的释放和扩散，在很大程度上仍未被探索。我们最近推出了半导体单壁碳纳米管(SWCNTs)，作为(分子)传感器或探测器的多功能构建块。单壁碳纳米管是由一个原子厚度的碳片组成的中空圆柱体。它们具有固有的与尺寸相关的带隙，这导致了近红外(NIR)荧光(900 nm-1700 nm)的非漂白，这是一个非常有利于光学成像的光谱区域，因为减少了散射和背景。我们以前的研究结果表明，与某些单链DNA序列非共价功能化的SWCNTs能够检测到包括多巴胺在内的重要的儿茶酚胺类神经递质。单壁碳纳米管周围的有机DNA相(光晕)与多巴胺相互作用，从而影响和改变其荧光。该项目的目标是通过改变单壁碳纳米管周围的固定DNA序列来创建不同的儿茶酚胺传感器。最重要的是，我们将在单分子实验中测量传感器动力学(开启和关闭速率)。为此，我们将使用近红外显微镜，它可以为不同发射波长(“颜色”)的单个纳米传感器成像。我们的理论模拟表明，只有具有某些动力学性质的传感器才能用于快速过程的成像，例如神经递质的释放。计划中的实验将提供对SWCNT光物理以及日冕(DNA序列)如何影响速率常数和对结构非常相似的儿茶酚胺神经递质(多巴胺、肾上腺素、去甲肾上腺素)的选择性的基本见解。此外，这些实验将揭示具有适合于不同设想的传感应用的候选特性，并使用动力学模拟来预测其时空分辨率。该项目的主要思想是组合多个传感器以提高灵敏度或选择性。为此，不同颜色(发射波长)和不同动力学/选择性的单壁碳纳米管将以多路复用法(多光谱成像)同时成像。最后，我们希望通过解决细胞中儿茶酚胺的释放来证明这种方法的多功能性。

The aim of this project is to create near infrared (nIR) fluorescent nanosensors for neurotransmitters, measure and understand their kinetics/selectivity and use them for multispectral imaging of chemical communication between cells. This project is motivated by the importance of chemical communication in multicellular organisms. Neuronal networks such as those in the human brain are the most intriguing example for the complexity of this process. So far, there are not many analytical tools available to image small molecules such as neurotransmitters on small length scales and fast time scales in complex biological environments. For that reason, fast chemical communication between cells and most notably neurotransmitter release and diffusion in neural networks remains largely unexplored. We have recently introduced semiconducting single-walled carbon nanotubes (SWCNTs) as versatile building blocks for (molecular) sensors or probes. SWCNTs are hollow cylinders of one-atom-thick sheets of carbon. They possess an intrinsic size-dependent bandgap, which results in non-bleaching near infrared (nIR) fluorescence (900 nm - 1700 nm), a very beneficial spectral region for optical imaging because of reduced scattering and background. Our previous results indicate that SWCNTs functionalized non-covalently with certain single stranded DNA sequences are able to detect important catecholamine neurotransmitters including dopamine. The organic DNA phase (corona) around the SWCNTs interacts with dopamine, which affects and changes their fluorescence. The goal of this project is to create different sensors for catecholamines by changing the immobilized DNA sequence around SWCNTs. Most importantly, we will measure sensor kinetics in single-molecule experiments (on- and off-rates). For that purpose, we will make use of a nIR microscope that can image single nanosensors of different emission wavelength ("color"). Our theoretical simulations imply that only sensors with certain kinetic properties are useful for imaging of fast processes such as neurotransmitter release. The planned experiments will provide fundamental insights into SWCNT photophysics and how the corona (DNA sequence) affects rate constants and selectivity to structurally very similar catecholamine neurotransmitters (dopamine, epinephrine, norepinephrine). Furthermore, these experiments will reveal candidates with suitable properties for different envisioned sensing applications and kinetic simulations are used to predict their spatiotemporal resolution. The major idea of this project is that combining multiple sensors enhances sensitivity or selectivity. To this end SWCNTs of different color (emission wavelength) and different kinetics/selectivity will be imaged at the same time in a multiplexing approach (multispectral imaging). Finally, we want to demonstrate the versatility of this approach by resolving catecholamine release from cells.