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个强大且相对低成本的设备，可以使用预先存在的核磁共振探针和硬件对珍贵的核磁共振样品进行分析微尺度滴定。液态、高分辨率核磁共振波谱已被证明是生物医学科学中极有价值的工具，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，以及复杂生物标本（如人类血清、尿液和均质组织提取物）的分析。一个广泛使用的实验方案包括在二级化学或生物成分的逐步滴定过程中收集生物标本上的连续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特定位点的pKa值，用小分子或另一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，或在药物开发的治疗靶点筛选期间滴定化学混合物。然而，目前进行这些核磁共振滴定的方法要么对大多数学术机构来说过于昂贵，要么在实验努力或消耗通常非常珍贵的核磁共振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，用于使用单个核磁共振样品自动进行核磁共振滴定，该设备可以适用于任何现有的商业高分辨率核磁共振光谱仪。我们的设计适合两个电机驱动的非磁性注射器到标准核磁共振管支架的边缘（目前用于将样品插入高场磁铁的中心孔），并且将包括两个长度的小直径管连接到各自的注射器。在一个新的应用中，非磁性压电电机将控制注射器，一个包含滴定剂的浓缩溶液，另一个专用于样品混合。这种装置的可用性对各种各样的生物医学研究人员来说是非常有价值的，并将在基本上所有人类疾病的调查中得到应用。

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.