Development of a nanoliter slow-MAS NMR metabolomics probe

描述(申请人提供)：代谢组学是一种强大的新的系统生物学工具，能够同时研究多个生物途径，检测和诊断疾病，并在早期阶段评估治疗的效果。核磁共振波谱是代谢组学的主要工具之一。组织代谢谱具有重要意义，因为疾病通常与局部组织/器官功能障碍有关。高分辨率魔角旋转(hr-MAS)技术已越来越多地被用于完整组织的代谢谱分析，并取得了显着的成功。然而，标准的hr-MAS核磁共振代谢组学分析需要10-30毫克或更多的组织质量，这在研究小鼠等小型实验动物时可能会出现问题，在这些动物中，需要牺牲动物以获得足够数量的组织用于后续分析。此外，10-30毫克的组织质量可能包含许多不同的细胞类型，在细胞生物化学方面需要研究较小的样本量。我们的研究目标是开发一种新的高分辨率、高灵敏度的核磁共振代谢组学研究方法，用于质量/体积小至300&lt；g/300nL的组织样品的研究。该项目的结果将使随着时间的推移，使用微创组织活检样本对单个小型实验动物进行持续调查成为可能。为了达到我们的目标，我们制定了两个具体目标。目的1：研制一种样品量小至300纳升的慢魔角样品旋转核磁共振探针。我们将使用磁化率匹配的微型射频线圈和毛细管样品管相结合的方法来获得最佳的灵敏度。通过整合我们建立的Slow-MAS 1H核磁共振方法，光谱分辨率将进一步提高，样品旋转速度约为80赫兹。采用Slow-MAS技术，消除了组织细胞内、样品和样品管之间由于磁化率梯度引起的谱线展宽，从而产生了高分辨率的~1H核磁共振谱。我们将扩大探头的适用范围，以便也可以研究从300nL到几毫升的组织样本。最初，将建造一个工作在7.05T磁场的探测器，随后将建造第二个工作在11.7T磁场的探测器，这样就可以探索Slow-MAS在更高磁场强度下的潜在优势。目的2：NL Slow-MAS探头的应用。我们将利用从大约6周到大约16周的正常和肥胖C57BL/6小鼠的微创活检肌肉样本(包括使用&lt；L或更少的血液和非侵入性尿样作为标准核磁共振代谢组学的补充方法)进行全面的核磁共振代谢组学研究，以确定可能与肥胖相关的代谢物生物标志物。每种动物的代谢物生物标志物的时间变化将被确定。结果将在同一组动物之间进行比较，从中将确定正常的生物变异，并将展示在一段时间内跟踪单一动物的优势。 与公共卫生相关：我们建议开发一种新型的慢魔角样品旋转探头，能够对生物样品，特别是组织样品进行高分辨率和高灵敏度的代谢图谱分析，体积小到300纳升(NL)，样品体积大到几毫升。NL的能力将使其有可能通过对单个小型实验动物的持续研究，并最终在患者身上，使用微创组织活检和血液样本，在很长一段时间内跟踪代谢变化。

DESCRIPTION (provided by applicant): Metabolomics is a powerful new systems biology tool that is capable of simultaneously investigating multiple biological pathways, detecting and diagnosing a disease and evaluating the efficacy of a therapy at an early stage. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the leading metabolomics tools. Tissue metabolic profiling is of significance because a disease is often associated with a localized tissue/organ malfunction. The technique of high resolution magic angle spinning (hr-MAS) has been increasingly used for metabolic profiling of intact tissues with notable successes. However, the requirement of tissue mass of 10- 30mg or more for standard hr-MAS NMR metabolomics analysis may present a problem in studying small laboratory animals such as mice, where animals need to be sacrificed to obtain adequate amount of tissue for subsequent analysis. Furthermore, a tissue mass of 10-30mg could encompass many different cell types and study on a smaller sample size would be desired in terms of cell biochemistry. The goal of our research is to develop a novel high resolution, high sensitivity method for NMR metabolomics investigations of tissue samples with mass/volume as small as 300<g/300nL. The outcome of the project will make it possible to carry out a continued investigation on a single small laboratory animal over time using minimally invasive tissue biopsy samples. To reach our goal, we have formulated two specific Aims. Aim 1: Development of a slow magic angle sample spinning NMR probe with sample volume as small as 300 nanoliters (nL). We will use a combination of micro-RF-coil with magnetic susceptibility matched wires, and capillary sample tube to obtain the optimal sensitivity. The spectral resolution will be further enhanced by integrating our established slow-MAS 1H NMR method applied at a sample spinning rate of about 80Hz. With slow-MAS technique, the line broadening due to magnetic susceptibility gradients within tissue cells, between the sample and the sample tube are eliminated, giving rise to a high resolution 1H NMR spectrum. We will expand the applicability of the probe such that tissue samples from 300nL to several mL can also be investigated. Initially, a probe will be built to operate at 7.05T field, followed by a second probe that will operate at 11.7 T field so the potential advantages of slow-MAS at higher magnetic field strengths can be explored. Aim 2: Application of the nL slow-MAS probe. We will carry out a comprehensive NMR metabolomics investigation using minimally invasive biopsy muscle samples of nL in volume (including the use of blood and non-invasive urine samples of <l or less as a supplementary method to standard NMR metabolomics) obtained from normal vs obese C57BL/6 mice from about 6 weeks to about 16 weeks of age to identify possible metabolite biomarkers that are related to obesity. The time variation of metabolite biomarkers for each animal will be determined. The results will be compared between the animals within the same group, from which the normal biological variations will be determined and the advantages of following a single animal over time period will be demonstrated. PUBLIC HEALTH RELEVANCE: We propose to develop a novel slow magic angle sample spinning probe that is capable of high resolution and high sensitivity metabolic profiling on biological samples, in particular tissue samples, with volume as small as 300 nanoliters (nL) to sample volume as large as a few milliliters. The nL capability will make it possible to follow the metabolic changes through a continued investigation on a single small laboratory animal, and ultimately on a patient, over a long period of time using minimally invasive tissue biopsy and blood samples.