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

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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.