Biospecimen preparation technologies to enable high throughput and highly sensiti

生物样本制备技术可实现高通量和高灵敏度

• 批准号: 8471943
• 负责人: James Lai
• 金额: $ 21.08万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471943
• 关键词: Address; Anterior; Antibodies; Automation; Binding; Biological; Biological Assay; Cancer Patient; Characteristics; Chromatography; Clinical; Data; Detection; Diffuse; Digestion; Disease; Disease Progression; Enzyme-Linked Immunosorbent Assay; Evaluation; Exhibits; Future; HIV Core Protein p24; Heating; High Pressure Liquid Chromatography; Immunoassay; Individual; Liquid substance; Magnetism; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Medical; Micelles; Molecular; Molecular Profiling; Monitor; Monoclonal Antibodies; Outcome; Performance; Phase; Polymers; Preparation; Process; Protein Microchips; Proteins; Proteomics; Reaction; Reagent; Sampling; Serum; Solid; Solubility; Solutions; Specimen; Stimulus; Stromal Cells; Surface; System; Technology; Temperature; Time; Tissues; Urine; Variant; analytical tool; antibody conjugate; cancer diagnosis; cancer proteomics; improved; innovation; magnetic field; multiple reaction monitoring; nanoparticle; neoplastic cell; novel; particle; pre-clinical; protein expression; public health relevance; research clinical testing; screening; success

DESCRIPTION (provided by applicant): Many diseases result in specific and characteristic changes in the molecular profiles of biological fluids (e.g., urine) and tissues. For cancer, thes changes can be utilized for screening, or as indicators of disease progression or of treatment-associated changes, which can significantly improve the medical outcome. Mass spectrometry (MS), a highly sensitive analytical technology with multiplex analysis capability, has been utilized for detecting changes in protein expression. The sensitivity of MS is inherently dependent on the available analyte concentration and the level of background interference, so specimens such as urine require extensive preparation (i.e., HPLC) prior to the analytical phase. These preparatory processes can result in significant loss of the analyte and are cumbersome, time consuming, not amenable to automation, and more susceptible to contaminations, which significantly impact the overall assay performance such as limit of detection (LOD). To address the need, this project aims to develop a novel biospecimen preparation technology that can process entire available urine samples rapidly (d 30 minutes) by concentrating multiple target analytes (e 102- fold) to enable high throughput multiplex targeted cancer proteomics with significantly lower LOD. The proposed sample preparation utilizes the stimuli-responsive binary reagent system for effective and rapid chromatography without solid phase. The key innovation lies in the combination of stimuli-responsive reagents, including magnetic nanoparticles (mNPs) and polymer-antibody conjugates to maximize the analyte binding efficiency while maintaining effective magnetic separation. This binary reagent system can achieve significantly higher analyte binding then the existing magnetic microparticle by simply applying a larger amount of the antibody conjugates without increasing the mass of magnetic nanoparticles because the antibodies are not attached to the particle surfaces. When a stimulus (e.g., heating) is applied to the solution, both stimuli- responsive reagents, including mNPs and polymer-antibody conjugates, form large aggregates that can be separated using a modest magnetic field to concentrate the bound analytes. We will develop a temperature- responsive separation module for isolating multiple analytes simultaneously in urine. The separation module will be optimized for processing the entire available urine (ca. 200 ml) and interfacing with MS for target protein quantification. Additionally, we will utilize clinical urine samples to evaluate the multiplex capability of the proposed specimen preparation process in conjunction with the MS assay improvement. The success of the project will result in a biospecimen preparation technology that can rapidly (d 30 minutes) process the entire available urine sample to produce multiple concentrated target analytes (e 102-fold) to enable high throughput multiplex target proteomics via MS with significantly lower assay LOD. The technology is applicable to different specimens (e.g., serum) for different analyte panels, which will significantly accelerate cancer proteomics and facilitate high quality cancer diagnosis in the future.

描述（由申请人提供）：许多疾病会导致生物液（例如尿液）和组织的分子特征的特异性和特征变化。 对于癌症，可以将这些变化用于筛查，也可以用作疾病进展的指标或与治疗相关的变化，这可以显着改善医疗结果。 质谱（MS）是一种具有多重分析能力的高度敏感的分析技术，已用于检测蛋白质表达的变化。 MS的敏感性固有地取决于可用的分析物浓度和背景干扰水平，因此诸如尿液之类的标本需要在分析阶段进行大量准备（即HPLC）。 这些准备过程可能会导致分析物的大量损失，并且繁琐，耗时，不适合自动化，并且更容易受到污染，从而显着影响整体测定性能，例如检测限（LOD）。 为了满足需求，该项目旨在开发一种新型的生物测量制备技术，该技术可以通过集中多个目标分析物（E 102-倍）来迅速处理整个可用的尿液样品（D 30分钟），以实现高通量多重目标癌组学，而LOD明显较低。 提出的样品制备利用刺激响应性二进制试剂系统进行无固相的有效和快速色谱法。 关键创新在于刺激反应性试剂（包括磁性纳米颗粒（MNP）和聚合物 - 抗体结合物）的组合，以最大程度地提高分析物结合效率，同时保持有效的磁分离。 该二进制试剂系统可以通过简单地应用大量的抗体偶联物而不会增加磁性纳米颗粒的质量，从而获得比现有的磁性微粒的更高分析物结合，因为抗体未连接到颗粒表面上。 当将刺激（例如，加热）应用于溶液时，两种刺激性反应的试剂（包括MNP和聚合物 - 抗体结合物）形成可以使用适度的磁场分离的大骨料，以浓缩结合分析物。 我们将开发一个温度响应分离模块，用于在尿液中同时隔离多个分析物。 分离模块将被优化，以处理整个可用的尿液（约200 mL），并与MS接口进行目标蛋白质定量。 此外，我们将利用临床尿液样品与MS分析改进一起评估所提出的样品制备过程的多重能力。 该项目的成功将导致生物测量制备技术，该技术可以迅速（D 30分钟）处理整个可用的尿液样品，从而产生多个集中靶向分析物（E 102倍），以通过MS通过具有明显较低的测定LOD的MS实现高通量多重目标蛋白质组学。 该技术适用于不同分析物面板的不同标本（例如血清），这将显着加速癌症蛋白质组学并促进未来的高质量癌症诊断。