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

使用 TRAP 技术通过胎盘 miRNA 生物标志物无创监测妊娠健康

• 批准号: 10754097
• 负责人: Brian T. Cunningham
• 金额: $ 42.4万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10754097
• 关键词: Address; Adoption; Adult; Base Sequence; Biochemistry; Biological; Biological Assay; Biological Markers; Biosensor; Birth Weight; Blood; Blood specimen; Body Fluids; Cessation of life; Circulation; Clinical; Conceptus; Coupling; Data; Detection; Development; Diagnosis; Disease; Electromagnetics; Embryonic and Fetal Development; Engineering; Fetal Growth; Fetal Growth Retardation; Fetal health; Fetus; Fluorescent Dyes; Functional disorder; Gestational Age; Growth; Health; Image; Individual; Infant; Laboratories; Lighting; Lipids; Liquid substance; Maternal Health; Measures; Methods; MicroRNAs; Microfluidics; Molecular; Monitor; Morbidity - disease rate; Mothers; Newborn Infant; Noise; Non-Invasive Detection; Nucleic Acid Probes; Nucleic Acids; Organ; Outcome; Pathologic; Pathology; Performance; Physiological; Placenta; Plasma; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Pregnant Women; Premature Birth; Premature Labor; Process; Proteins; Publications; RNA; Reagent; Recycling; Reporting; Reproducibility; Role; Sampling; Serum; Signal Transduction; Surface; Symptoms; System; Technology; Testing; Time; Tissues; Transcript; Urine; Variant; Woman; biomarker identification; clinical application; clinical translation; cost; design; detection limit; detection sensitivity; diagnostic platform; digital; exosome; extracellular vesicles; feasibility testing; genetic variant; genomic biomarker; infant death; innovation; insight; instrument; invention; maternal morbidity; microRNA biomarkers; minimally invasive; mortality; myometrium; nanoGold; nanoparticle; neonatal morbidity; next generation sequencing; non-invasive monitor; novel; nucleic acid detection; obstetrical syndromes; operation; photonics; portability; potential biomarker; pregnant; sensor; specific biomarkers; stillbirth; success; tool; transcriptome; transcriptome sequencing; urinary

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.

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） 死产是造成产妇和新生儿发病率和死亡率的重要因素。超过1500万早产儿 每年有100多万婴儿死亡。这些怀孕的共同点是 并发症是胎盘功能障碍。胎盘是妊娠期不可或缺的器官， 胚胎发育和胎儿生长之间的直接物理界面， 胎儿许多产科综合征的病理生理开始于妊娠早期，但症状往往 出现在后期阶段。在血液中循环的细胞外囊泡（EV）来源于多种组织 类型，包括胎盘，并代表了越来越多地用于诊断的生物标志物的宝库 生理失调血液样本可以在整个怀孕期间相对非侵入性地获得， 可以潜在地用于鉴定与胎盘功能障碍相关的生物标志物。虽然下一代 测序（NGS），如RNA-seq，可以鉴定可以提供生物学和临床应用的基因组变体。 但是，与执行NGS相关的成本和复杂性的结合阻止了其广泛采用 作为常规临床应用的工具。目前可用于定量miRNA的实验室方法 缺乏定量、灵敏度和选择性以满足临床需要。为了弥补这些差距，我们 寻求应用一种新的测定方法，其中金纳米颗粒用工程核酸官能化 “立足点探针”，其在靶miRNA序列被释放时通过释放保护序列而被激活。 遇到液体。激活揭示了一种新的核酸序列，使纳米颗粒能够被 捕获在光子晶体（PC）生物传感器表面上。当金纳米粒子的等离子体共振 波长被选择为与PC谐振反射波长相匹配，发生强电磁耦合 这导致PC反射强度的强烈和高度局部化的降低-使得能够对 纳米粒子“目标再循环扩增过程”（TRAP）技术是一种单步测定， 在<50 µl的测试样品中能够达到100 aM的检测限，采用低强度LED照明， 图像传感器，无酶放大，无荧光染料。在这个项目中，我们开发了 同时定量从正常人血清外泌体提取的特异性miRNA序列， 健康孕妇和早产妇女产生四种特异性 在怀孕期间的生物标志物miRNAs。