A Mass Spectrometry Probe for Transient Imaging of In-Solution Biochemistry

描述（由申请人提供）：在生物医学研究中，经常需要检测和监测界面附近溶液中的动态化学物质。例如，许多研究集中于检测从培养的组织或细胞到周围介质中的化学分泌物。通常，这种应用对检测方法所需的空间和时间分辨率提出了具体要求。当监测分泌物时，这可能是由于细胞类型和行为的异质性，以及细胞活动随时间的变化。标记，例如，荧光标记，放射性标记，或使用抗原/抗体附着，已经非常成功地作为成像动态生物化学的基础，但有关标记的分析物的行为和非特异性结合的改变的担忧不能消除。此外，所有有针对性的方法，包括那些基于标记的方法，在其发现潜力方面固有地受到限制，因为人们无法找到他们不寻找的东西。这项研究的目的是克服目前生化成像技术的固有局限性。这将通过电喷雾离子源的发展，可以作为质谱探针（MSP）的高分辨率生化检测从邻近生物界面的微环境。该研究小组在成功发明新型质谱离子源方面有着悠久的历史，并提出，对于该项目，要完成将样品收集，处理和电离的所有先决条件结合到微量取样毛细管中的雄心勃勃的任务。这种“尖端实验室”将包括在线微透析，以去除盐和交换溶剂，以及集成的胰蛋白酶消化微反应器。该研究团队将通过一种成熟的多方面方法开发，优化和演示MSP，该方法结合了实验（包括光学和质谱表征），分析和模拟（第一原理物理模型和计算流体动力学）以及最先进的制造（微加工）。MSP将在生物学研究中作为假设生成器发挥重要作用，并将成为改善再生医学应用生物反应器开发的关键工具。成功的结果有可能为广泛的研究应用带来变革性的好处，包括生物标志物的发现，提高对健康和患病细胞生物学的理解，生物传感器的开发以及生物制造过程的分析和控制。除了在会议上介绍和在档案期刊上发表，MSP技术在生物学问题上的应用将通过在佐治亚州举办的教育讲习班传播。Tech.此外，该探针将与TOF质谱仪耦合，该质谱仪是NSF支持的国家纳米技术基础设施网络（NNIN）的一部分，因此可供工业和学术机构的用户使用。 公共卫生相关性（由申请人提供）：在本申请中，我们提出开发一种环境质谱探针（MSP），其在生理条件下直接从液体中被动采样并软电离大生物分子，从而使得在复杂液体环境中精确控制位置的瞬态质谱监测成为可能，MS光谱作为时间的函数收集。由此产生的在生理相关溶液条件下直接监测生物化学的能力将使生物学家、生物化学家和生物工程师能够以高精度在空间上关联化学数据，不仅用于生成体外培养物的清晰化学“图像”，而且用于研究异质性和梯度在许多重要应用中的作用，包括用于再生医学的生物反应器，生物工程材料的活性和行为，以及生物传感器表征。MSP将在生物学研究中作为假设生成器发挥重要作用，并有可能为广泛的研究工作带来转型效益，包括生物标志物发现，健康和患病细胞生物学研究，生物传感器开发以及生物制造过程分析和控制。

DESCRIPTION (provided by applicant): Quite frequently in biomedical research there is a need to detect and monitor dynamic chemistries in solution in the vicinity of an interface. For instance, many studies focus on detection of chemical secretions from cultured tissues or cells into the surrounding medium. Often, such applications place specific demands on the required spatial and temporal resolution of the detection method. When monitoring secretions this would be due to heterogeneity in the cell types and behavior, and also variation in cellular activities with time. Labeling, with, for instance, a fluorescent marker, a radioactive marker, or using antigen/ antibody attachment, has been spectacularly successful as the foundation for imaging dynamic biochemistry, but concerns about the altering of labeled analyte behavior and non-specific binding cannot be eliminated. Furthermore, all targeted methods, including those based on labeling, are inherently limited in their discovery potential, as one cannot find what one is not looking for. The purpose of the proposed research is to overcome the inherent limitations of current biochemical imaging technologies. This will be done through the development of electrospray ion sources that can serve as mass spectrometry probes (MSP) for highly resolved biochemical detection from the microenvironment adjacent to biological interfaces. The research team has a demonstrated history of success inventing novel mass spectrometry ion sources, and proposes, for this project, to accomplish the ambitious task of combining all prerequisite capabilities for sample collection, processing, and ionization into a micro- sampling capillary. This "lab-on-a-tip" will include in-line microdialysis to remove salts and exchange solvent, as well as an integrated tryptic digestion micro-reactor. The research team will develop, optimize and demonstrate MSP through an established multifaceted approach combining experiment (including optical and mass spec characterization), analysis and simulation (first principles physical models and computational fluid dynamics), and state of the art manufacturing (microfabrication). MSP will assume an important role in biological research as a hypothesis generator, and will become a key tool in improving development of bioreactors for regenerative medicine applications. Successful results have potential for transformational benefits to a wide range of research applications, including biomarker discovery, improved understanding of healthy and diseased cell biology, biosensor development, and bio-manufacturing process analysis and control. In addition to presentation at conferences and publication in archival journals, the application of MSP technology to biological problems will be disseminated through an educational workshop hosted at Ga. Tech. Furthermore, the probe will be coupled to a TOF mass spectrometer that is part of the NSF supported National Nanotechnology Infrastructure Network (NNIN), and therefore available to users from industry and academic institutions alike. PUBLIC HEALTH RELEVANCE (provided by applicant): In this application, we propose to develop an ambient Mass Spectrometry Probe (MSP), which passively samples and softly ionizes large biomolecules directly from liquid at physiological conditions, and thus makes possible transient mass spectrometric monitoring at precisely controlled locations in a complex liquid environment, with MS spectra collected as a function of time. The resulting capability to directly monitor biochemistry in physiologically relevant solution conditions will enable biologists, biochemists, and bioengineers to spatially correlate chemical data with high precision, not only for the generation of clear chemical "images" of in vitro cultures, but also for the investigation of the role of heterogeneity and gradients in numerous important applications, including bio reactors for regenerative medicine, activity and behavior of bioengineered materials, and biosensor characterization. MSP will assume an important role in biological research as a hypothesis generator, and has the potential for transformational benefits to a wide range of research endeavors, including biomarker discovery, investigations of healthy and diseased cell biology, biosensor development, and bio-manufacturing process analysis and control.