spFRET for Expression Profiling mRNA: Discovering Markers for Stroke Diagnosis

描述（由申请人提供）：中风是美国第三大杀手，也是每年超过795,000例成人残疾的主要原因。缺血性和出血性中风两大类型在临床上无法区分；30%出现中风样症状的患者根本没有中风。快速准确地诊断中风是必要的，因为缺血性和出血性中风需要不同的治疗方法，只有很短的时间窗口（3-6小时）才能有效。计算机断层扫描（CT）或磁共振成像（MRI）通常用于诊断，这会延迟治疗干预。不幸的是，目前还没有针对中风的分子诊断测试，主要原因是潜在的分子标记面板有待临床验证。这种验证过程在一定程度上受到用于分析的工具的阻碍，例如用于核酸标记物的反转录PCR （RT-PCR）或用于血清蛋白标记物的免疫测定，这是由于缺乏样品过程自动化和使用的冗长处理步骤。在此应用中，将开发一种高度创新的全自动系统，该系统具有精密的分析灵敏度，可监测不同分子标记表达水平的微小变化，周转时间（TAT）小于15分钟。该系统将有助于识别和验证新的分子标记物，用于快速、特异和敏感的脑卒中诊断。分子分析和新型硬件将用于评估信使RNA标记面板作为一种前所未有的基于血液的检测（使用外周血单核细胞，PBMC）用于中风诊断的潜力。两项技术将构成该系统的核心：(1)模块化流体生物处理器，由聚合物通过复制制成，包含所有样品处理步骤；(2)单对荧光共振能量转移（spFRET），消除了几个样品处理步骤。微复制将用于制造生物处理器，以保持低成本，并使这种消耗品适合临床诊断所需的单次使用。生物处理器将包含一个流体主板，其上连接着特定任务的模块，以提供设计灵活性。在处理器上准备的分子处理步骤将是：(1)PBMC分离；(2) PBMC裂解；(3) mRNA固相纯化；(4)逆转录为cDNA；(5)连接酶检测反应（LDRs），产生带有供体/受体荧光对的分子信标；(6) spFRET数字检测。使用spFRET的分子（即数字）计数将提供消除几个样品预处理步骤的能力，缩短分析TAT，并产生必要的分析灵敏度，以检测mRNA标记物表达水平的细微变化，这在中风事件发生后的早期可能是至关重要的。该系统可以潜在地用于任何需要mRNA表达谱分析的应用，但目前的项目将用于验证中风诊断的分子标记。

DESCRIPTION (provided by applicant): Stroke is the third leading killer in the US and the main cause for over 795,000 cases of adult disability each year. The two major types of stroke, ischemic and hemorrhagic, cannot be clinically differentiated; 30% of patients presenting stroke-like symptoms do not have a stroke at all. It is imperative that a stroke diagnosis be made quickly and accurately because ischemic and hemorrhagic strokes require different treatments that have only a small window of time to be effective (3-6 hours). Computed tomography (CT) or magnetic resonance imaging (MRI) is commonly used for the diagnosis, which delays therapeutic intervention. Unfortunately, a molecular diagnostic test does not currently exist for stroke due primarily to the fact that potential molecular marker panels are waiting to be clinically validated. This validation process is, in part, hampered by the tools used for their analysis, such as reverse-transcription PCR (RT-PCR) for nucleic acid-based markers or immunoassays for serum protein markers, due to the lack of sample process automation and the lengthy processing steps used. In this application, a highly innovative, fully-automated system will be developed that has exquisite analytical sensitivity to monitor minute changes in the expression levels of different molecular markers with a turn-around-time (TAT) of less than 15 minutes. The system will facilitate the identification and validation of new molecular markers for the rapid, specific, and sensitive diagnosis of stroke. The molecular assay and novel hardware will be used to evaluate the potential of a messenger RNA marker panel as an unprecedented blood-based test (using peripheral blood mononucleated cells, PBMC) for the diagnosis of stroke. Two technologies will form the core of the system: (1) modular fluidic bio-processor made from polymers via replication that contains all of the sample processing steps; and (2) single pair fluorescence resonance energy transfer (spFRET) that eliminates several sample processing steps. Micro-replication will be used for the fabrication of the bio-processor to keep cost low and make this consumable appropriate for single- use applications, as demanded by clinical diagnostics. The bio-processor will contain a fluidic motherboard with task-specific modules interconnected on it to provide design flexibility. The molecular processing steps poised on the processor will be: (1) PBMC isolation; (2) PBMC lysis; (3) mRNA solid-phase purification; (4) reverse transcription to cDNA; (5) ligase detection reactions (LDRs) to generate molecular beacons with donor/acceptor fluorescent pairs and; (6) spFRET digital detection. The molecular (i.e., digital) counting using spFRET will provide the ability to eliminate several sample pre-processing steps shortening the assay TAT and generate the necessary analytical sensitivity to detect subtle changes in expression levels of mRNA markers, which may be critical during early times following a stroke event. The system can potentially be used for any application requiring mRNA expression profiling, but will be used in the current project for validating molecular markers for the diagnosis of stroke. PUBLIC HEALTH RELEVANCE: Stroke is the third leading killer in the US and causes over 795,000 cases of adult disability each year. Unfortunately, in vitro diagnostic tests for stroke that can be administered to patients for effective and timely treatment are not currently available. The goal of this project is to develop an innovative, discovery-based technology for securing highly quantitative results quickly through full-process automation. The technology will assist in the validation of different molecular markers for stroke to deliver an unprecedented in vitro diagnostic blood-based test.