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

用于研究金黄色葡萄球菌在活细胞中存活的 SERS 纳米针系统

• 批准号: 10025173
• 负责人: John Goertz
• 金额: $ 5.31万
• 依托单位: IMPERIAL COLLEGE OF SCIENCE, TECHNOLOGY AND MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-23 至 2022-09-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10025173
• 关键词: Address; Antibiotic Resistance; Antibiotics; Architecture; Attention; Bacteria; Behavior; Binding; Biochemical; Biological Sciences; Biomedical Research; Cell Compartmentation; Cells; Characteristics; Chemicals; Communication; Complex; Detection; Development; Diagnosis; Diagnostic; Diffuse; Disease; Dyes; Engineering; Ensure; Equilibrium; Event; Exhibits; Fellowship; Fluorescence; Future; Genetic Transcription; Goals; Gold; Grant; Heterogeneity; Hybrids; Image; Immune response; Immunotherapy; Individual; Infection; Inflammation; Investigation; Kinetics; Label; Lead; Literature; Maps; Mediating; Medical; Mentorship; Metabolic; MicroRNAs; Molecular; Monitor; Nanostructures; Nitrogen; Oligonucleotides; Oxygen; Phagocytes; Phagocytosis; Phenotype; Play; Raman Spectrum Analysis; Reaction; Real-Time Systems; Recurrent disease; Reporter; Research; Research Personnel; Signal Transduction; Silicon; Silver; Small Interfering RNA; Staphylococcus aureus; Staphylococcus aureus infection; Stimulus; Structure; Surface; System; TNF gene; Techniques; Technology; Time; Training; Untranslated RNA; Virulent; Writing; career; chemical conjugate; chronic infection; cytokine; design; disease diagnosis; frontier; imaging system; improved; macrophage; metabolomics; minimally invasive; nanoneedle; novel; pathogen; plasmonics; pressure; prototype; real time monitoring; response; tool; transcriptomics

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.

项目摘要 我们提出了一个设计导向的工程解决方案，以解决在活的，单细胞调查的挑战， 为生物医学研究提供新的机会。了解单个细胞的动力学对于 揭示了复杂疾病状态中涉及的特异质行为，但传统技术未能 实时捕捉这些异质性。这些活的、单细胞的研究对于以下方面特别必要： 由可在宿主体内存活的持续性细胞内细菌（如金黄色葡萄球菌（SA））引起的感染 细胞长时间，导致复发性疾病，抵抗治疗。去调查那个 涉及这种宿主-病原体相互作用的生物化学库，使得能够进行多重分析的工具， 迫切需要与动态转录组学相结合的细胞内代谢物。 我们提出了用于表面增强拉曼的等离子体纳米针基底的发展 将用于描绘个体细胞内miRNA和代谢产物景观的SERS技术。 巨噬细胞吞噬SA期间真实的时间。通过在硅上制造银和金纳米结构 纳米针，我们将创建一个等离子体基片能够询问活细胞。拉曼成像 用化学探针功能化的基质将用于SA表型的实时表征 转换、局部抗生素浓度的定量、确定细菌对细胞内pH的改变， 以及研究活性氧和氮的相互作用。此外，我们会实施一项 一种新的动态调节的miRNA探针结构， 监测miRNA的表达。这种共轭拉曼染料的Sers光谱特征丰富， 修饰纳米针表面的寡核苷酸信标将能够灵敏地检测特定的miRNA 序列在SA感染的巨噬细胞在高复用能力，使我们能够调查 这些巨噬细胞之间的特异质差异，这些巨噬细胞有效地破坏SA后立即 吞噬作用和那些在TNF-α刺激后仅破坏SA或根本不破坏SA的细胞。 除了在该研究金期间提供的技术培训外，还将为研究员提供 专业发展机会，以扩大他在实验室管理，赠款写作，科学 沟通，和导师，这将为他的独立学术生涯，他是专门准备。 这里提出的研究将产生一个更好的理解时间宿主细胞响应动态 感染事件。实时观察miRNA的表达可以识别siRNA介导的候选基因。 免疫治疗，并将提供一个必要的工具，为未来的转录反应的特点， 各种刺激。在我们的目标完成后，研究人员将能够对 在广泛的背景下，化学，转录和表型反应的细胞库。