A Device for Automating Titration Experiments in High-Resolution NMR Spectroscopy

描述（由申请人提供）：拟议项目的总体目标是开发一种稳健且成本相对较低的设备，该设备可以使用预先存在的NMR探针和硬件对珍贵的NMR样品进行分析微量滴定。液态，高分辨率NMR光谱已被证明是生物医学科学中一个非常有价值的工具，其应用包括生物大分子的结构和生物物理表征，小分子的物理化学表征，以及复杂生物样品（如人血清，尿液和均质组织提取物）的分析。广泛使用的实验方案涉及在二级化学或生物组分的逐步滴定期间收集生物样品上的连续NMR光谱。常见的例子包括在pH滴定中滴定酸或碱以测量大分子中的位点特异性pKa值，用小分子或另一种大分子滴定标记的生物分子（例如蛋白质）以进行配体结合的生物物理表征，或在筛选药物开发的治疗靶点期间滴定化学混合物。然而，目前用于执行这些NMR滴定的方法对于大多数学术环境来说成本过高，或者在实验努力或消耗通常非常珍贵的NMR样品材料方面效率低下。作为回应，我们建议开发一种低成本的设备，用于使用单个NMR样品自动进行NMR滴定，该设备可以适用于任何现有的商业高分辨率NMR光谱仪。我们的设计将两个电机驱动的非磁性注射器安装到标准NMR管保持器（目前用于将样品插入高场磁体的中心孔）的边缘，并将包括连接到其各自注射器的两段小直径管。在一个新的应用中，非磁性压电电机将控制注射器，一个含有滴定剂的浓缩溶液，另一个专用于样品混合。这种设备的可用性将对各种生物医学研究人员非常有价值，并将应用于基本上所有人类疾病的调查。 公共卫生相关性（由申请人提供）：高分辨率NMR光谱学是生物医学研究中广泛使用的实验工具。其主要用途之一是研究与人类疾病发展相关的生物大分子。NMR光谱描述了这些大分子在溶液中的三维结构，以及它们的动态行为。NMR光谱也用于分析人体体液或均质化组织提取物。这些研究有助于确定与许多疾病相关的代谢变化，有助于诊断测试的发展，并更好地了解潜在的疾病机制。在所有上述应用中，研究人员通常希望了解靶分子对第二分子浓度增加的响应。被称为滴定，这类实验可以帮助识别复杂混合物中的特定成分，定义两种分子如何相互作用，并在开发新的治疗药物中发挥关键作用。目前用于进行这种NMR滴定的仪器和方法特别麻烦，浪费了大量的时间和资源。我们建议建立一个设备，将自动化这样的核磁共振滴定，而不浪费任何珍贵的样品进行分析，并将适应任何目前的核磁共振仪器。该设备设计简单，坚固耐用，应该大大提高这些关键实验的速度，这对当前生物医学研究的几乎所有领域都有影响。

DESCRIPTION (provided by applicant): The overall goal of the proposed project is to develop a robust and relatively low-cost device that can perform analytical micro-scale titrations on precious NMR samples using pre-existing NMR probes and hardware. Liquid-state, high resolution NMR spectroscopy has proven to be an extremely valuable tool in the biomedical sciences, with applications including the structural and biophysical characterization of biologic macromolecules, physicochemical characterization of small molecules, and the analysis of complex biologic specimens such as human serum, urine and homogenized tissue extracts. A widely utilized experimental protocol involves the collection of successive NMR spectra on a biological specimen during a stepwise titration of a secondary chemical or biological component. Common examples include titration of acid or base in a pH titration to measure site-specific pKa values in macromolecules, titration of a labeled biomolecule (e.g. protein) with a small molecule or another macromolecule for biophysical characterization of ligand-binding, or titrations of chemical mixtures during screening of therapeutic targets for drug development. However, current methodologies for performing these NMR titrations are either prohibitively costly for most academic settings or inefficient in terms of experimental effort or the consumption of often highly precious NMR sample material. In response, we are proposing to develop a low-cost device for automating NMR titrations using a single NMR sample, which could be adapted to any existing, commercial high-resolution NMR spectrometer. Our design fits two motor-driven, non-magnetic syringes to the edges of a standard NMR tube holder (currently used to insert the sample into central bore of the high field magnet), and will include two lengths of small diameter tubing connected to their respective syringes. In a novel application, non-magnetic Piezo-electric motorswill control the syringes, one containing a concentrated solution of titrant and the other dedicated to sample mixing. The availability of such a device would be highly valuable to a wide diversity of biomedical researchers and would have applications in the investigation of essentially all human diseases. PUBLIC HEALTH RELEVANCE (provided by applicant): High-resolution NMR spectroscopy is a widely utilized experimental tool in biomedical research. One of its primary uses is the investigation of biological macromolecules involved in the development of human diseases. NMR spectroscopy describes the three-dimensional structures of these macromolecules in solution, as well as, their dynamic behavior. NMR spectroscopy is also used in the analysis of human bodily fluids or homogenized tissue extracts. Such studies help identify metabolic changes associated with many diseases, contributing both to the development of diagnostic tests and to better understand the underlying disease mechanism. In all of the above applications, researchers often desire to understand the response of a target molecule to increasing concentrations of a second molecule. Known as a titration, these sorts of experiment can help in the identification of specific components in complex mixtures, define how two molecules interact, and serve a critical role in the development of new therapeutic agents. Current instrumentation and methodology for the performance of such NMR titrations are particularly troublesome, wasting significant time and resources. We are proposing to build a device that will automate such NMR titrations, without wasting any of the precious specimen being analyzed, and will be adaptable to any of the current NMR instrumentation. The device is designed to be simple and robust and should greatly enhance the pace of these critical experiments, which have implications for nearly all areas of current biomedical research.