Non-invasive monitoring of gestational health via placental miRNA biomarkers using TRAP technology

摘要 常见的妊娠并发症包括早产、先兆子痫、宫内生长受限(IUGR) 和死产是孕产妇和新生儿发病率和死亡率的重要因素。超过1500万早产 每年都有婴儿出生，导致100多万婴儿死亡。这些怀孕的一个共同点 并发症是胎盘功能障碍。胎盘是妊娠特有的器官，对于 胚胎发育和胎儿生长，是母亲和胎儿生长之间的直接物理接口 胎儿。许多产科综合征的病理生理在妊娠早期就开始了，但症状通常 在以后的阶段出现。在血液中循环的细胞外小泡(EV)来自多种组织 类型，包括胎盘，代表了生物标志物的宝库，越来越多地被用于诊断 生理障碍。在怀孕期间可以相对非侵入性地获取血液样本，并且 可能被用来确定与胎盘功能障碍相关的生物标志物。而下一代 测序(Ngs)，如rna-seq，可以识别可能提供生物学和临床信息的基因组变异。 洞察力，与执行NGS相关的成本和复杂性的结合阻碍了它的广泛采用 作为常规临床应用的工具。目前可用的基于实验室的miRNA定量方法 体液中缺乏定量、灵敏度和选择性，无法满足临床需要。为了解决这些差距，我们 寻求应用一种新的检测方法，其中金纳米颗粒被工程核酸功能化 当目标miRNA序列为 在液体中遇到。这种激活揭示了一种新的核酸序列，使纳米颗粒能够 在光子晶体(PC)生物传感器表面捕获。当金纳米粒子的等离子激元共振 选择与PC谐振反射波长匹配的波长，发生强电磁耦合 这导致了PC反射强度的强烈和高度局部化的减少-使得能够对 纳米粒子。“靶循环扩增过程”(TRAP)技术是一种一步法，即 在&lt；50微米L测试样品中能够达到100 am的检测极限，采用低强度LED照明，价格低廉 图像传感器，没有酶放大，也没有荧光染料。在这个项目中，我们开发了用于 同时定量从正常人血清外切体中提取的特定miRNA序列， 健康孕妇和早产妇女生成四个特定的表达谱 怀孕期间的生物标记物miRNAs。

ABSTRACT Common pregnancy complications including preterm birth, preeclampsia, intra-uterine growth restriction (IUGR) and stillbirth are significant contributors to maternal and neonatal morbidity and mortality. Over 15 million preterm births occur annually resulting in more than one million infant deaths. A common denominator to these pregnancy complications is the dysfunction of the placenta. The placenta is a pregnancy-specific organ indispensable for embryonic development and fetal growth and is the direct physical interface between the mother and the growing fetus. The pathophysiology of many obstetrical syndromes is initiated early in gestation, but symptoms often emerge at later stages. Extracellular vesicles (EVs) circulating in the blood are derived from multiple tissue types, including placenta, and represent a trove of biomarkers that are increasingly being utilized to diagnose physiological disorders. Blood samples can be obtained throughout pregnancy relatively non-invasively and could potentially be utilized to identify biomarkers related to placental dysfunction. While next-generation sequencing (NGS), such as RNA-seq, can identify genomic variants that may provide biological and clinical insights, the combination of cost and complexity associated with performing NGS precludes its broad adoption as a tool for routine clinical applications. Currently available laboratory-based methods for quantifying miRNA in bodily fluids lack quantitation, sensitivity, and selectivity to meet clinical needs. To address these gaps, we seek to apply a novel assay method in which gold nanoparticles are functionalized with engineered nucleic acid “toehold probes” that are activated by release of a protector sequence when the target miRNA sequence is encountered in liquid. The activation reveals a new nucleic acid sequence that enables the nanoparticle to be captured on a photonic crystal (PC) biosensor surface. When the gold nanoparticle’s plasmon resonant wavelength is selected to match the PC resonant reflection wavelength, strong electromagnetic coupling occurs that results in strong and highly localized reduction of the PC reflected intensity – enabling digital counting of the nanoparticles. The “Target Recycling Amplification Process” (TRAP) technology is a single-step assay that is capable of 100 aM detection limits in a <50 µl test sample, with low intensity LED illumination, an inexpensive image sensor, no enzymatic amplification, and no fluorescent dyes. In this project, we develop assays for simultaneous quantification of specific miRNA sequences extracted from blood serum exosomes of normal, healthy pregnant women and women with preterm birth to generate expression profiles for four specific biomarker miRNAs across pregnancy.