A SERS nanoneedle system for studying Staphylococcus aureus survival in live cells

项目摘要 我们提出了一种设计导向的工程解决方案，以应对活的单细胞研究中的挑战，因此 为生物医学研究创造新的机会。了解单个细胞的动态对于 揭开复杂疾病状态中涉及的特殊行为，然而传统技术无法 实时捕捉这些异质性。这些活的、单细胞的研究对于 可在宿主内存活的持久性细胞内细菌的感染，如金黄色葡萄球菌(SA) 细胞周期延长，导致抗拒治疗的复发疾病。为了调查广泛的 参与这种宿主-病原体相互作用的生化谱系，使多重分析成为可能的工具 细胞内代谢产物和动态转录产物是迫切需要的。 提出了表面增强拉曼等离子体纳米针衬底的发展方向 光谱(SERS)将被用来描述个体的细胞内miRNA和代谢物景观 巨噬细胞在SA吞噬过程中的实时变化。通过在硅上制造银和金纳米结构 纳米针，我们将创造一种能够询问活细胞的等离子底物。拉曼成像技术 用化学探针功能化的底物将用于SA表型的实时表征 切换，局部抗生素浓度的量化，细菌对细胞内pH的改变的确定， 并研究了活性氧和氮物种之间的相互作用。此外，我们将实现一个 专为实时序列特异性定制的新型动力学调控的miRNA探针结构 监测miRNA的表达。与此偶联的拉曼染料的特征丰富的SERS光谱 修饰纳米针表面的寡核苷酸信标将使特定miRNA的灵敏检测成为可能 在金黄色葡萄球菌感染的巨噬细胞内具有高多路复用能力的序列，使我们能够研究 立即有效地破坏SA的巨噬细胞之间的特异性差异 吞噬作用和在肿瘤坏死因子-α刺激后仅破坏SA或根本不破坏SA的作用。 除了在此奖学金期间提供的技术培训外，还将为该研究员提供 有专业发展机会，扩大他在实验室管理、拨款撰写、科学研究方面的专长 交流和指导，这将为他致力于独立的学术生涯做好准备。 本文提出的研究将对寄主细胞对动态的时间响应有更好的理解 感染事件。对miRNA表达的实时观察可能确定siRNA介导的候选 免疫疗法，并将为未来对转录反应的表征提供必要的工具 各种刺激。在我们的目标完成后，研究人员将能够非常详细地探索 在广泛的背景下化学、转录和表型反应的细胞谱系。

Project Summary We propose a design-directed engineering solution to address challenges in live, single-cell investigations, thus enabling new opportunities for biomedical research. Understanding the dynamics of individual cells is crucial to unraveling the idiosyncratic behaviors involved in complex disease states, yet traditional techniques fail to capture these heterogeneities in real-time. These live, single-cell investigations are particularly necessary for infections by persistent intracellular bacteria such as Staphylococcus aureus (SA) that can survive within host cells for prolonged periods, leading to relapsing disease that resists treatment. To investigate the broad biochemical repertoire involved in such host-pathogen interactions, tools that enable multiplexed profiling of intracellular metabolites in tandem with dynamic transcriptomics are urgently needed. We propose the development of plasmonic nanoneedle substrates for Surface-Enhanced Raman Spectroscopy (SERS) that will be used to profile the intracellular miRNA and metabolite landscape of individual macrophages in real time during phagocytosis of SA. By fabricating silver and gold nanostructures onto silicon nanoneedles, we will create a plasmonic substrate capable of interrogating live cells. Raman imaging of substrates functionalized with chemical probes will be used for real-time characterization of SA phenotype switching, quantification of local antibiotic concentration, determination of bacterial alterations to intracellular pH, and investigation of the interplay of reactive oxygen and nitrogen species. Furthermore, we will implement a novel kinetically-regulated miRNA probe architecture specifically tailored to real-time sequence-specific monitoring of miRNA expression. The feature-rich SERS spectra of Raman dyes conjugated to such oligonucleotide beacons decorating the nanoneedle surface will enable sensitive detection of specific miRNA sequences within SA-infected macrophages at high multiplexing capacity, enabling us to investigate the idiosyncratic differences between those macrophages which effectively destroy SA immediately upon phagocytosis and those which only destroy SA after TNF-α stimulation, or not at all. In addition to the technical training provided over the course of this fellowship, the Fellow will be provided with professional development opportunities to expand his expertise in lab management, grant writing, scientific communication, and mentorship that will prepare him for the independent academic career he is dedicated to. The research proposed here will produce a greater understanding of the temporal host-cell response to dynamic infection events. Real-time observations of miRNA expression may identify candidates for siRNA-mediated immunotherapy and will provide an essential tool for future characterizations of transcriptional responses to various stimuli. Upon completion of our aims, researchers will be capable of highly detailed explorations into the cellular repertoire of chemical, transcriptional, and phenotypic responses in a broad range of contexts.