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系统生物学工具，能够同时研究多种生物学途径，检测和诊断疾病，并在早期阶段评估治疗效果。核磁共振（NMR）光谱是领先的代谢组学工具之一。组织代谢谱是重要的，因为疾病通常与局部组织/器官功能障碍相关。高分辨率魔角旋转（hr-MAS）技术已越来越多地用于完整组织的代谢分析，取得了显着的成功。然而，标准hr-MAS NMR代谢组学分析需要10- 30 mg或更多的组织质量，这可能会在研究小实验室动物如小鼠时出现问题，其中需要处死动物以获得足够量的组织用于后续分析。此外，10- 30 mg的组织块可能包含许多不同的细胞类型，并且在细胞生物化学方面需要对较小的样本量进行研究。我们的研究目标是开发一种新的高分辨率，高灵敏度的NMR代谢组学研究的组织样品的质量/体积小到300<g/300 nL的方法。该项目的成果将使人们有可能在一段时间内使用微创组织活检样本对单个小型实验室动物进行持续研究。为了实现我们的目标，我们制定了两个具体目标。目标1：开发一种样品体积小至300纳升（nL）的慢魔角样品旋转NMR探针。我们将使用微RF线圈与磁化率匹配的导线，和毛细管样品管的组合，以获得最佳的灵敏度。结合我们建立的慢MAS ~ 1H NMR方法，在约80 Hz的样品旋转速率下应用，将进一步提高光谱分辨率。利用慢MAS技术，消除了由于组织细胞内、样品和样品管之间的磁化率梯度而导致的谱线展宽，从而产生高分辨率的1H NMR谱。我们将扩展探针的适用性，以便也可以研究从300 nL到几mL的组织样本。最初，将构建一个在7.05 T磁场下运行的探头，然后构建第二个在11.7 T磁场下运行的探头，以便探索在更高磁场强度下慢MAS的潜在优势。目的2：nL慢MAS探针的应用。我们将使用从约6周龄至约16周龄的正常与肥胖C57 BL/6小鼠获得的nL体积的微创活检肌肉样品（包括使用<1或更少的血液和非侵入性尿液样品作为标准NMR代谢组学的补充方法）进行全面的NMR代谢组学研究，以鉴定与肥胖相关的可能的代谢物生物标志物。将确定每只动物代谢物生物标志物的时间变化。将在同一组内的动物之间比较结果，从中确定正常的生物学变异，并证明随时间推移跟踪单个动物的优势。 公共卫生相关性：我们建议开发一种新型的慢魔角样本旋转探针，能够对生物样本（特别是组织样本）进行高分辨率和高灵敏度的代谢分析，其体积小至300纳升（nL），样本体积大至几毫升。nL能力将使人们有可能通过对单个小型实验室动物的持续研究，并最终对患者进行长期的微创组织活检和血液样本，来跟踪代谢变化。

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.