Raman probes to determine cellular redox potential

理解细胞——所有已知生物体的基本单位——的精妙复杂性，为创新研究提供了无与伦比的机会。为了研究细胞功能，实验生物学通常将分析限制在细胞“快照”上。这既忽略了发生在每个细胞及其细胞器中的时间依赖性变化，也忽略了其大分子通过生化途径的动态通量。这种生物学的“快照”方法不太可能提供对细胞生物学的真正深刻理解，因为细胞涉及一个综合的和暂时的网络。因此，现有的还原论方法可能会加剧现代实验生物学中的随机变异性，从而导致重大的“复制失败”问题。因此，通过建立先进的工具和技术，以了解细胞的分子电路如何以时空方式响应和适应环境变化，从而产生对细胞生物学的整体理解，提供了重要的机会。细胞内的氧化还原电位对细胞健康至关重要，并密切参与许多重要的过程，包括凋亡、分化、细胞周期和炎症。许多疾病如癌症、心血管疾病和神经退行性疾病的进展都与异常的细胞氧化还原电位有关。因此，用于监测和确定正常细胞功能和与疾病相关的氧化还原电位的工具和技术对于验证治疗干预的新靶点和未来药物的开发至关重要。在这个项目中，将设计和合成一套炔探针，以确定氧化还原电位、谷胱甘肽水平和活性氧浓度。这些将使用标准技术靶向特定细胞器，并在细胞背景下使用拉曼显微镜进行校准。这种分析技术提供了额外的丰富的、无标记的内源性生物分子分布信息，如脂质、核酸和蛋白质在不同的细胞区室（如线粒体、溶酶体、细胞核）内的分布。监测不同类型细胞响应外部信号和压力的时空变化将提供细胞器内部局部环境的时空洞察，并为细胞分析方法提供有效工具，以理解和监测细胞氧化还原环境。该项目的成功成果将提供一套新的分子，能够定量测量细胞内的氧化还原环境，并告知细胞通路受到外部刺激和疾病扰动的整体细胞效应，这将广泛应用于生命科学和化学生物学领域。

Understanding the exquisite complexity of a cell, the fundamental unit of all known organisms, provides unrivalled opportunity for innovation for research. In order to investigate cellular function, experimental biology has typically restricted analysis to cellular 'snapshots'. This ignores both the time-dependent changes occurring in each cell and its organelles, and also the dynamic flux of its macromolecules through biochemical pathways. This 'snapshot' approach to biology is unlikely to provide a truly deep understanding of cell biology as the cell involves an integrated and temporal network. As a result, existing reductionist approaches may exacerbate stochastic variability within modern experimental biology that can lead to major 'failure-to-replicate' problems. The generation of a holistic understanding of cellular biology by establishing advanced tools and technologies in order to understand how a cell's molecular circuitry responds and adapts to environmental change in a spatiotemporal manner therefore provides significant opportunity.Redox potential within a cell is essential to cellular health and is intimately involved in many important processes including apoptosis, differentiation, cell cycle and inflammation. The progression of many diseases such as cancer, cardiovascular disease and neurodegenerative disorders have been implicated in aberrant cellular redox potential. Therefore, tools and techniques with which to monitor and determine redox potential in both normal cell function and that associated with disease is critical in the validation of new targets for therapeutic intervention and the development of medicines of the future.Within this project a suite of alkyne probes to determine redox potential, glutathione levels and the concentration of reactive oxygen species will be designed and synthesised. These will be targeted to specific organelles using standard techniques and calibrated within a cellular context using Raman microscopy. This analytical technique offers additional rich, label-free information on the distribution of endogenous biomolecules such as lipids, nucleic acids, and proteins within different cellular compartments (e.g., mitochondria, lysosome, nucleus). Monitoring spatiotemporal changes in response to external signals and stresses within different types of cell will provide spatiotemporal insight into the internal localised environment within organelles and provide effective tools for a cellular analytical approach to understanding and monitoring the cellular redox environment.Successful outcome of this project will deliver a new suite of molecules able to quantitatively measure the redox environment within a cell and inform of the global cellular effect on the perturbation of cellular pathways to external stimuli and disease which will find broad application to those working within the life sciences and chemical biology communities.