Integrated microfluidic devices for preterm birth biomarker measurement

描述(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50万人早产。肺结核是新生儿死亡和新生儿疾病的主要原因。不幸的是，在宫缩前评估肺结核风险的能力在临床上是不可用的；这种能力将使预防或阻止分娩的治疗干预成为可能，潜在地减少肺结核和/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个集成的微流控平台，用于在宫缩发生前几周快速、灵敏和廉价地量化血清肺结核风险生物标记物(新发现的多肽和已知蛋白质)。因此，这项提议对人类健康产生了重大影响，潜在地降低了肺结核及其相关并发症的患病率。这一提议检验了这样一种假设，即进一步开发在单一平台上结合样品准备、分离和定量的微流控系统将能够直接测量血清中的结核杆菌生物标记物，使风险评估和预防措施能够在收缩开始之前很久就发生。提出的自动化微流控平台有三个主要组件：(1)多路免疫亲和提取模块，用于选择性地捕获未标记的结核病生物标记物；(2)纯化/标记模块，用于丰富结核病生物标记物，然后允许通过去除未反应标签进行荧光标记；以及(3)微芯片电泳模块，用于分离已标记的结核病生物标记物，使其能够定量。这种方法大大改进了目前仪器密集和人员密集的方法来确定结核分枝杆菌生物标记物，并且应该提供与夹心免疫分析相当的特异性，而夹心免疫分析在开发小肽靶标方面将是有问题的。这项更新提案的目标是进一步开发集成的微流控系统，用于方便、廉价、快速和定量地分析与肺结核风险相关的血清生物标记物。这一目标将通过三个具体目标来实现。在目标1中，将创建和测试电动操作的微芯片来量化结核分枝杆菌生物标记物，集免疫亲和提取、固相浓缩、荧光标记、电泳分离和生物标记物定量于一体。目标2将涉及开发PTB生物标记物分析微型设备，该设备使用芯片上阀门和泵的压力驱动来执行综合样品制备操作。然后将比较AIMS 1和AIMS 2中的设备的性能。在目标3中，这些微流控平台将被用来测量血液样本中的肺结核生物标志物，以确定微芯片方法在收缩发生前几周预测肺结核风险的可行性。重要的是，这项工作解决了在治疗干预仍然可行的情况下诊断肺结核风险的迫切需要；然而，该方法的一般性质应该远远适用于肺结核的生物标记物，进一步例证了这些研究对健康的重大影响。

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.