Graphene-based optoelectrochemical sensor for the simultaneous monitoring of the electrical and chemical activity of single cells

对神经元通讯分子原理的进一步了解，使我们对神经退行性疾病有了新的认识，并可能找到新的治疗靶点。然而，目前还缺乏能够同时监测单个细胞电信号和化学信号的工具。因此，我们建议开发一种新型石墨烯增强传感器，允许在细胞培养和器官型组织切片中以单细胞分辨率同时监测这两种信号。该传感器将基于表面等离子体共振成像（SPRi）及其为电化学测量提供优异空间分辨率的能力。更重要的是，通过集成石墨烯，SPR检测的灵敏度和电化学测量的电流密度都将得到提高，同时生物相容性也将得到增强。除了产生关于活细胞电信号和化学信号相互作用的新知识外，预期传感器的开发将是迈向基于与活细胞双向通信的新型假体的重要一步。传感器的核心将是一个携带圆盘微电极的细胞芯片，细胞附着在圆盘微电极上，周围是无细胞的环形微电极。一旦细胞粘附在圆盘微电极上，环形微电极（用酶修饰或未修饰）就会极化到允许细胞分泌的信号分子氧化或减少的电位。随后，在对细胞施加物理或化学刺激的同时，以高帧速率（~ 10000 fps）记录细胞芯片的高分辨率SPR图像。覆盖细胞的圆盘微电极的SPR图像通过细胞的细胞外场电位的变化来调节，例如能够监测动作电位的传播。环形微电极的SPR图像将被信号分子局部浓度变化引起的局部电流密度变化所改变，并将用于观察来自细胞的化学信号。神经细胞在低毫伏范围内改变其细胞外场电位，只释放少量的信号分子。因此，为了能够同时记录来自细胞的电信号和化学信号，必须提高SPRi的灵敏度。石墨烯已经被证明可以提高SPR和电化学检测的灵敏度。因此，石墨烯将用于信号放大的研究。我们的测量细胞外场电位的方法将取代电压敏感染料的应用，同时它将至少保持光学显微镜的空间分辨率，这优于电询问微电极阵列提供的空间分辨率。该方法还将为细胞化学信号的电化学监测提供前所未有的空间分辨率和干扰消除。

Improved understanding of molecular principles of neuronal communication allows new insights into neurodegenerative diseases and possibly to new therapeutic targets. However, there is a lack of tools able to monitor simultaneously electrical and chemicals signals of single cells. Therefore, we propose the development of a novel graphene-enhanced sensor allowing simultaneous monitoring of these two signals with single cell resolution both in cell cultures and organotypic tissue slices. The sensor will be based on surface plasmon resonance imaging (SPRi) and its ability to provide excellent spatial resolution also to electrochemical measurements. More importantly, by integrating graphene both the sensitivity of SPR detection and the current densities of the electrochemical measurement will be enhanced with concomitantly enhanced biocompatibility. In addition to generating new knowledge about the interplay of electrical and chemical signals of living cells, the development of the anticipated sensors will be an important step towards novel prostheses based on the bidirectional communication with living cells. The core of the sensor will be a cell chip carrying disk microelectrodes, to which cells adhere, surrounded by cell-free ring microelectrodes. Once cells have adhered at the disk microelectrodes, the ring microelectrodes (modified with enzymes or unmodified) are polarized to a potential that allows oxidation or reduction of signaling molecules secreted by the cells. Subsequently, high-resolution SPR images of the cell chip are recorded at high frame rates (~ 10000 fps) while a physical or chemical stimulus is applied to the cells. SPR images of cell-covered disk microelectrodes are modulated by changes in the extracellular field potential of the cells which e.g. enables to monitor the propagation of action potentials. SPR images of the ring microelectrodes will be altered by changes in local current densities invoked by variations in the local concentrations of signaling molecules and will be used to observe chemical signals from the cells. Neuronal cells change their extracellular field potential within the low millivolts range and release only tiny amounts of signaling molecules. Therefore, the sensitivity of SPRi has to be improved in order to be able to record electrical and chemical signals from cells simultaneously. Graphene has already been proved to enhance the sensitivity of both SPR and electrochemical detection. Hence, graphene will be investigated for signal amplification. Our approach to measure extracellular field potentials will replace the application of voltage sensitive dyes while it will at least maintain the spatial resolution of optical microscopy which is superior to the spatial resolution provided by the electrically interrogated microelectrode array. The approach will additionally provide unprecedented spatial resolution and interference elimination to electrochemical monitoring of chemical signals from cells.