Integrated microfluidic devices for preterm birth biomarker measurement

描述（由申请人提供）：早产（PTB）是最常见的妊娠并发症，在美国每年有超过50万例早产。肺结核是造成大多数新生儿死亡和新生儿疾病的原因。不幸的是，在宫缩之前评估PTB风险的能力在临床上是不可用的;这种能力将使得能够进行预防或阻止分娩的治疗干预，从而潜在地降低PTB和/或新生儿并发症的严重程度。这项更新提案的重点是开发一种集成的微流体平台，用于在收缩发生前几周快速，灵敏和廉价地定量血清PTB风险生物标志物（新发现的肽和已知蛋白质）。因此，这一提议对人类健康产生了重大影响，可能会降低PTB及其相关并发症的患病率。 该提案测试了以下假设：微流体系统的进一步发展，其在单个平台中组合联合收割机样品制备、分离和定量，将使得能够直接测量血清中的PTB生物标志物，从而允许在收缩开始之前进行风险评估和预防措施。所提出的自动化微流体平台具有三个主要组件：（1）用于选择性捕获未标记的PTB生物标志物的多重免疫亲和提取模块;（2）用于富集PTB生物标志物并且然后允许荧光标记并去除未反应的标签的纯化/标记模块;以及（3）用于分离标记的PTB生物标志物的微芯片电泳模块，使得能够对其进行定量。这种方法显著改善了目前的仪器和人员密集型方法来确定PTB生物标志物，并应提供与夹心免疫测定法相当的特异性，夹心免疫测定法对于开发小肽靶标是有问题的。 该更新提案的目标是进一步开发集成的微流体系统，用于方便，廉价，快速和定量分析与PTB风险相关的血清生物标志物。这一目标将通过三个具体目标来实现。在目标1中，将创建和测试用于定量PTB生物标志物的电动微芯片，其集成了免疫亲和提取、固相富集、荧光标记、电泳分离和生物标志物定量。目标2将涉及开发PTB生物标志物分析微器件，该微器件使用片上阀和泵的压力驱动来进行集成的样品制备操作。然后将比较目标1和2中器械的性能。在目标3中，这些微流体平台将用于测量血液样品中的PTB生物标志物，以建立微芯片方法在收缩发生前几周预测PTB风险的可行性。 重要的是，这项工作解决了在治疗干预仍然可行时诊断PTB风险的迫切需要;然而，该方法的一般性质应适用于PTB的生物标志物之外，进一步举例说明这些研究的巨大而重要的健康影响。

DESCRIPTION (provided by applicant): Preterm birth (PTB) is the most common complication of pregnancy, with over 500,000 early births annually in the United States. PTB is responsible for the majority of neonatal deaths and newborn illnesses. Unfortunately, the ability to assess PTB risk prior to contractions is not available clinically; this capability would enable therapeutic interventions that prevent or forestall delivery, potentially decreasing PTBs and/or the severity of neonatal complications. This renewal proposal focuses on the development of an integrated microfluidic platform for rapid, sensitive and inexpensive quantitation of serum PTB risk biomarkers (newly discovered peptides and known proteins), weeks before contractions occur. This proposal thus offers a major human health impact, potentially decreasing the prevalence of PTBs and their associated complications. This proposal tests the hypothesis that further development of microfluidic systems, which combine sample preparation, separation and quantitation in a single platform, will enable the direct measurement of PTB biomarkers in serum, allowing risk assessment and preventative measures to occur well before contractions begin. The proposed automated microfluidic platform has three main components: (1) a multiplexed immunoaffinity extraction module to selectively capture unlabeled PTB biomarkers; (2) a purification/labeling module to enrich PTB biomarkers and then allow fluorescence labeling with removal of unreacted tag; and (3) a microchip electrophoresis module to separate the labeled PTB biomarkers, enabling their quantitation. This approach improves significantly over present instrument- and personnel-intensive methods to determine PTB biomarkers, and should offer specificity comparable to sandwich immunoassays that would be problematic to develop for small peptide targets. The goal of this renewal proposal is to further develop integrated microfluidic systems for convenient, inexpensive, rapid and quantitative analysis of serum biomarkers with PTB risk correlation. This objective will be accomplished via three specific aims. In Aim 1 electrokinetically operated microchips to quantify PTB biomarkers will be created and tested, integrating immunoaffinity extraction, solid-phase enrichment, fluorescence labeling, electrophoretic separation and biomarker quantitation. Aim 2 will involve developing PTB biomarker analysis microdevices that use pressure actuation of on-chip valves and pumps to carry out integrated sample preparation operations. Performance of the devices from Aims 1 and 2 will then be compared. In Aim 3 these microfluidic platforms will be used to measure PTB biomarkers in blood samples to establish the feasibility of microchip methods in predicting PTB risk weeks before contractions occur. Importantly, this work addresses the urgent need to diagnose PTB risk when therapeutic intervention is still feasible; however, the general nature of the approach should be applicable well beyond biomarkers for PTB, further exemplifying the large and important health impact of these studies.